Knockout of Twi2 (or Rdn2) increased the accumulation of sRNAs enriched by Twi7 and Twi8. TWI2 mRNA also increased in a strain lacking Rdn2. Rdf2KO cells have reduced accumulation of 23- to 24-nt sRNAs Loss of Rdf1 increased the accumulation of telo-sRNAs.

Knockout of Twi2 (or Rdn2) increased the accumulation of sRNAs enriched by Twi7 and Twi8. TWI2 mRNA also increased in a strain lacking Rdn2. Rdf2KO cells have reduced accumulation of 23- to 24-nt sRNAs Loss of Rdf1 increased the accumulation of telo-sRNAs.

Knockout of Twi2 (or Rdn2) increased the accumulation of sRNAs enriched by Twi7 and Twi8. TWI2 mRNA also increased in a strain lacking Rdn2. Rdf2KO cells have reduced accumulation of 23- to 24-nt sRNAs Loss of Rdf1 increased the accumulation of telo-sRNAs.

The first 25-nt sequences of scnRNAs (26–32 nt) from TWI1 knockout cells at 3 h post-mixing were mapped to the annotated Mic genome. Among the scnRNAs from TWI1 knockout cells, 67.0% mapped to the annotated Mic genome without any mismatch. Of these matched scnRNAs, only 15.3% were exclusively complementary to MDSs, while 80.1% mapped exclusively to IESs. The densities of TWI1 knockout scnRNAs mapped to different MDS fragments is variable. Among the scnRNAs from TWI1 knockout cells at 3 h post-mixing, 52.2%, 29.1%, and 18.6% were complementary to Unique, Mid-Rep, and High-Rep sequences, respectively, indicating that scnRNAs are indeed produced from repeated sequences more frequently. scnRNAs complementary to MDSs remained at high levels throughout conjugation in EMA1 knockout cells.

The first 25-nt sequences of scnRNAs (26–32 nt) from TWI1 knockout cells at 3 h post-mixing were mapped to the annotated Mic genome. Among the scnRNAs from TWI1 knockout cells, 67.0% mapped to the annotated Mic genome without any mismatch. Of these matched scnRNAs, only 15.3% were exclusively complementary to MDSs, while 80.1% mapped exclusively to IESs. The densities of TWI1 knockout scnRNAs mapped to different MDS fragments is variable. Among the scnRNAs from TWI1 knockout cells at 3 h post-mixing, 52.2%, 29.1%, and 18.6% were complementary to Unique, Mid-Rep, and High-Rep sequences, respectively, indicating that scnRNAs are indeed produced from repeated sequences more frequently. scnRNAs complementary to MDSs remained at high levels throughout conjugation in EMA1 knockout cells.

The first 25-nt sequences of scnRNAs (26–32 nt) from TWI1 knockout cells at 3 h post-mixing were mapped to the annotated Mic genome. Among the scnRNAs from TWI1 knockout cells, 67.0% mapped to the annotated Mic genome without any mismatch. Of these matched scnRNAs, only 15.3% were exclusively complementary to MDSs, while 80.1% mapped exclusively to IESs. The densities of TWI1 knockout scnRNAs mapped to different MDS fragments is variable. Among the scnRNAs from TWI1 knockout cells at 3 h post-mixing, 52.2%, 29.1%, and 18.6% were complementary to Unique, Mid-Rep, and High-Rep sequences, respectively, indicating that scnRNAs are indeed produced from repeated sequences more frequently. scnRNAs complementary to MDSs remained at high levels throughout conjugation in EMA1 knockout cells.

The first 25-nt sequences of scnRNAs (26–32 nt) from TWI1 knockout cells at 3 h post-mixing were mapped to the annotated Mic genome. Among the scnRNAs from TWI1 knockout cells, 67.0% mapped to the annotated Mic genome without any mismatch. Of these matched scnRNAs, only 15.3% were exclusively complementary to MDSs, while 80.1% mapped exclusively to IESs. The densities of TWI1 knockout scnRNAs mapped to different MDS fragments is variable. Among the scnRNAs from TWI1 knockout cells at 3 h post-mixing, 52.2%, 29.1%, and 18.6% were complementary to Unique, Mid-Rep, and High-Rep sequences, respectively, indicating that scnRNAs are indeed produced from repeated sequences more frequently. scnRNAs complementary to MDSs remained at high levels throughout conjugation in EMA1 knockout cells.

The first 25-nt sequences of scnRNAs (26–32 nt) from TWI1 knockout cells at 3 h post-mixing were mapped to the annotated Mic genome. Among the scnRNAs from TWI1 knockout cells, 67.0% mapped to the annotated Mic genome without any mismatch. Of these matched scnRNAs, only 15.3% were exclusively complementary to MDSs, while 80.1% mapped exclusively to IESs. The densities of TWI1 knockout scnRNAs mapped to different MDS fragments is variable. Among the scnRNAs from TWI1 knockout cells at 3 h post-mixing, 52.2%, 29.1%, and 18.6% were complementary to Unique, Mid-Rep, and High-Rep sequences, respectively, indicating that scnRNAs are indeed produced from repeated sequences more frequently. scnRNAs complementary to MDSs remained at high levels throughout conjugation in EMA1 knockout cells.

Twenty-five-nucleotide sRNAs are substantially depleted by both DCL2 (DCL2-KD) and DCL3 silencing (DCL3-KD), suggesting that both of these Dicer-like proteins contribute to scnRNA production. Independent DCL2 and DCL3 silencing had no lethal effect on the progeny of the silenced cells, whereas DCL2/3 cosilencing was 100% lethal. Only the most sensitive epiIES that we examined were retained in these independent silencings, whereas combined DCL2/3 silencing led to retention of most epiIESs (5/7) but no retention of non-epiIESs. Silencing of the PiggyMac gene (PGM) was previously used to identify all known Paramecium IESs and has an average IES retention of ~67%. Compared to PGM silencing, most IESs are weakly or not retained after DCL2/3 cosilencing, with a mean of 3.0% retention for all IESs (compared to 0.4% for the control). In contrast to short IESs, both Sardine transposons and long IESs, including Anchois transposon-derived IESs, are strongly retained after DCL2/3 cosilencing. Both DCL2/3 cosilencing and PGM silencing substantially reduce iesRNA levels. PGM silencing also appears to reduce scnRNA levels. Knockdown of DCL5 effectively eliminates iesRNA production but not scnRNAs. Like the individual silencings of DCL2 and DLC3, DCL5 silencing did not result in lethality of the progeny. Like the DCL2/3 cosilencing, DCL5 silencing leads to relatively low levels of IES retention. The most striking difference between the IESs affected by DCL5 silencing compared to DCL2/3 silencing is that the DCL5 silencing does not result in pronounced retention of long IESs (including Anchois IESs) or Sardine transposons beyond that of IESs in general, whereas DCL2/3 cosilencing certainly does.

Twenty-five-nucleotide sRNAs are substantially depleted by both DCL2 (DCL2-KD) and DCL3 silencing (DCL3-KD), suggesting that both of these Dicer-like proteins contribute to scnRNA production. Independent DCL2 and DCL3 silencing had no lethal effect on the progeny of the silenced cells, whereas DCL2/3 cosilencing was 100% lethal. Only the most sensitive epiIES that we examined were retained in these independent silencings, whereas combined DCL2/3 silencing led to retention of most epiIESs (5/7) but no retention of non-epiIESs. Silencing of the PiggyMac gene (PGM) was previously used to identify all known Paramecium IESs and has an average IES retention of ~67%. Compared to PGM silencing, most IESs are weakly or not retained after DCL2/3 cosilencing, with a mean of 3.0% retention for all IESs (compared to 0.4% for the control). In contrast to short IESs, both Sardine transposons and long IESs, including Anchois transposon-derived IESs, are strongly retained after DCL2/3 cosilencing. Both DCL2/3 cosilencing and PGM silencing substantially reduce iesRNA levels. PGM silencing also appears to reduce scnRNA levels. Knockdown of DCL5 effectively eliminates iesRNA production but not scnRNAs. Like the individual silencings of DCL2 and DLC3, DCL5 silencing did not result in lethality of the progeny. Like the DCL2/3 cosilencing, DCL5 silencing leads to relatively low levels of IES retention. The most striking difference between the IESs affected by DCL5 silencing compared to DCL2/3 silencing is that the DCL5 silencing does not result in pronounced retention of long IESs (including Anchois IESs) or Sardine transposons beyond that of IESs in general, whereas DCL2/3 cosilencing certainly does.

Twenty-five-nucleotide sRNAs are substantially depleted by both DCL2 (DCL2-KD) and DCL3 silencing (DCL3-KD), suggesting that both of these Dicer-like proteins contribute to scnRNA production. Independent DCL2 and DCL3 silencing had no lethal effect on the progeny of the silenced cells, whereas DCL2/3 cosilencing was 100% lethal. Only the most sensitive epiIES that we examined were retained in these independent silencings, whereas combined DCL2/3 silencing led to retention of most epiIESs (5/7) but no retention of non-epiIESs. Silencing of the PiggyMac gene (PGM) was previously used to identify all known Paramecium IESs and has an average IES retention of ~67%. Compared to PGM silencing, most IESs are weakly or not retained after DCL2/3 cosilencing, with a mean of 3.0% retention for all IESs (compared to 0.4% for the control). In contrast to short IESs, both Sardine transposons and long IESs, including Anchois transposon-derived IESs, are strongly retained after DCL2/3 cosilencing. Both DCL2/3 cosilencing and PGM silencing substantially reduce iesRNA levels. PGM silencing also appears to reduce scnRNA levels. Knockdown of DCL5 effectively eliminates iesRNA production but not scnRNAs. Like the individual silencings of DCL2 and DLC3, DCL5 silencing did not result in lethality of the progeny. Like the DCL2/3 cosilencing, DCL5 silencing leads to relatively low levels of IES retention. The most striking difference between the IESs affected by DCL5 silencing compared to DCL2/3 silencing is that the DCL5 silencing does not result in pronounced retention of long IESs (including Anchois IESs) or Sardine transposons beyond that of IESs in general, whereas DCL2/3 cosilencing certainly does.

Twenty-five-nucleotide sRNAs are substantially depleted by both DCL2 (DCL2-KD) and DCL3 silencing (DCL3-KD), suggesting that both of these Dicer-like proteins contribute to scnRNA production. Independent DCL2 and DCL3 silencing had no lethal effect on the progeny of the silenced cells, whereas DCL2/3 cosilencing was 100% lethal. Only the most sensitive epiIES that we examined were retained in these independent silencings, whereas combined DCL2/3 silencing led to retention of most epiIESs (5/7) but no retention of non-epiIESs. Silencing of the PiggyMac gene (PGM) was previously used to identify all known Paramecium IESs and has an average IES retention of ~67%. Compared to PGM silencing, most IESs are weakly or not retained after DCL2/3 cosilencing, with a mean of 3.0% retention for all IESs (compared to 0.4% for the control). In contrast to short IESs, both Sardine transposons and long IESs, including Anchois transposon-derived IESs, are strongly retained after DCL2/3 cosilencing. Both DCL2/3 cosilencing and PGM silencing substantially reduce iesRNA levels. PGM silencing also appears to reduce scnRNA levels. Knockdown of DCL5 effectively eliminates iesRNA production but not scnRNAs. Like the individual silencings of DCL2 and DLC3, DCL5 silencing did not result in lethality of the progeny. Like the DCL2/3 cosilencing, DCL5 silencing leads to relatively low levels of IES retention. The most striking difference between the IESs affected by DCL5 silencing compared to DCL2/3 silencing is that the DCL5 silencing does not result in pronounced retention of long IESs (including Anchois IESs) or Sardine transposons beyond that of IESs in general, whereas DCL2/3 cosilencing certainly does.

Twenty-five-nucleotide sRNAs are substantially depleted by both DCL2 (DCL2-KD) and DCL3 silencing (DCL3-KD), suggesting that both of these Dicer-like proteins contribute to scnRNA production. Independent DCL2 and DCL3 silencing had no lethal effect on the progeny of the silenced cells, whereas DCL2/3 cosilencing was 100% lethal. Only the most sensitive epiIES that we examined were retained in these independent silencings, whereas combined DCL2/3 silencing led to retention of most epiIESs (5/7) but no retention of non-epiIESs. Silencing of the PiggyMac gene (PGM) was previously used to identify all known Paramecium IESs and has an average IES retention of ~67%. Compared to PGM silencing, most IESs are weakly or not retained after DCL2/3 cosilencing, with a mean of 3.0% retention for all IESs (compared to 0.4% for the control). In contrast to short IESs, both Sardine transposons and long IESs, including Anchois transposon-derived IESs, are strongly retained after DCL2/3 cosilencing. Both DCL2/3 cosilencing and PGM silencing substantially reduce iesRNA levels. PGM silencing also appears to reduce scnRNA levels. Knockdown of DCL5 effectively eliminates iesRNA production but not scnRNAs. Like the individual silencings of DCL2 and DLC3, DCL5 silencing did not result in lethality of the progeny. Like the DCL2/3 cosilencing, DCL5 silencing leads to relatively low levels of IES retention. The most striking difference between the IESs affected by DCL5 silencing compared to DCL2/3 silencing is that the DCL5 silencing does not result in pronounced retention of long IESs (including Anchois IESs) or Sardine transposons beyond that of IESs in general, whereas DCL2/3 cosilencing certainly does.

Twenty-five-nucleotide sRNAs are substantially depleted by both DCL2 (DCL2-KD) and DCL3 silencing (DCL3-KD), suggesting that both of these Dicer-like proteins contribute to scnRNA production. Independent DCL2 and DCL3 silencing had no lethal effect on the progeny of the silenced cells, whereas DCL2/3 cosilencing was 100% lethal. Only the most sensitive epiIES that we examined were retained in these independent silencings, whereas combined DCL2/3 silencing led to retention of most epiIESs (5/7) but no retention of non-epiIESs. Silencing of the PiggyMac gene (PGM) was previously used to identify all known Paramecium IESs and has an average IES retention of ~67%. Compared to PGM silencing, most IESs are weakly or not retained after DCL2/3 cosilencing, with a mean of 3.0% retention for all IESs (compared to 0.4% for the control). In contrast to short IESs, both Sardine transposons and long IESs, including Anchois transposon-derived IESs, are strongly retained after DCL2/3 cosilencing. Both DCL2/3 cosilencing and PGM silencing substantially reduce iesRNA levels. PGM silencing also appears to reduce scnRNA levels. Knockdown of DCL5 effectively eliminates iesRNA production but not scnRNAs. Like the individual silencings of DCL2 and DLC3, DCL5 silencing did not result in lethality of the progeny. Like the DCL2/3 cosilencing, DCL5 silencing leads to relatively low levels of IES retention. The most striking difference between the IESs affected by DCL5 silencing compared to DCL2/3 silencing is that the DCL5 silencing does not result in pronounced retention of long IESs (including Anchois IESs) or Sardine transposons beyond that of IESs in general, whereas DCL2/3 cosilencing certainly does.

Twenty-five-nucleotide sRNAs are substantially depleted by both DCL2 (DCL2-KD) and DCL3 silencing (DCL3-KD), suggesting that both of these Dicer-like proteins contribute to scnRNA production. Independent DCL2 and DCL3 silencing had no lethal effect on the progeny of the silenced cells, whereas DCL2/3 cosilencing was 100% lethal. Only the most sensitive epiIES that we examined were retained in these independent silencings, whereas combined DCL2/3 silencing led to retention of most epiIESs (5/7) but no retention of non-epiIESs. Silencing of the PiggyMac gene (PGM) was previously used to identify all known Paramecium IESs and has an average IES retention of ~67%. Compared to PGM silencing, most IESs are weakly or not retained after DCL2/3 cosilencing, with a mean of 3.0% retention for all IESs (compared to 0.4% for the control). In contrast to short IESs, both Sardine transposons and long IESs, including Anchois transposon-derived IESs, are strongly retained after DCL2/3 cosilencing. Both DCL2/3 cosilencing and PGM silencing substantially reduce iesRNA levels. PGM silencing also appears to reduce scnRNA levels. Knockdown of DCL5 effectively eliminates iesRNA production but not scnRNAs. Like the individual silencings of DCL2 and DLC3, DCL5 silencing did not result in lethality of the progeny. Like the DCL2/3 cosilencing, DCL5 silencing leads to relatively low levels of IES retention. The most striking difference between the IESs affected by DCL5 silencing compared to DCL2/3 silencing is that the DCL5 silencing does not result in pronounced retention of long IESs (including Anchois IESs) or Sardine transposons beyond that of IESs in general, whereas DCL2/3 cosilencing certainly does.

Twenty-five-nucleotide sRNAs are substantially depleted by both DCL2 (DCL2-KD) and DCL3 silencing (DCL3-KD), suggesting that both of these Dicer-like proteins contribute to scnRNA production. Independent DCL2 and DCL3 silencing had no lethal effect on the progeny of the silenced cells, whereas DCL2/3 cosilencing was 100% lethal. Only the most sensitive epiIES that we examined were retained in these independent silencings, whereas combined DCL2/3 silencing led to retention of most epiIESs (5/7) but no retention of non-epiIESs. Silencing of the PiggyMac gene (PGM) was previously used to identify all known Paramecium IESs and has an average IES retention of ~67%. Compared to PGM silencing, most IESs are weakly or not retained after DCL2/3 cosilencing, with a mean of 3.0% retention for all IESs (compared to 0.4% for the control). In contrast to short IESs, both Sardine transposons and long IESs, including Anchois transposon-derived IESs, are strongly retained after DCL2/3 cosilencing. Both DCL2/3 cosilencing and PGM silencing substantially reduce iesRNA levels. PGM silencing also appears to reduce scnRNA levels. Knockdown of DCL5 effectively eliminates iesRNA production but not scnRNAs. Like the individual silencings of DCL2 and DLC3, DCL5 silencing did not result in lethality of the progeny. Like the DCL2/3 cosilencing, DCL5 silencing leads to relatively low levels of IES retention. The most striking difference between the IESs affected by DCL5 silencing compared to DCL2/3 silencing is that the DCL5 silencing does not result in pronounced retention of long IESs (including Anchois IESs) or Sardine transposons beyond that of IESs in general, whereas DCL2/3 cosilencing certainly does.

RNA interference (RNAi)-mediated silencing of mtA in E cells resulted in the default O phenotype during sexual reactivity.The MIC gene is interrupted by four IESs that are excised in both MAC types; however, a 195-base-pair (bp) segment containing the transcription start site and the first 26 bp of the coding sequence was found to be excised as an IES in mating type O MACs, but retained in E MACs. This segment contains the mtA promoter, as indicated by microinjection of different constructs into the MACs of O cells: its presence upstream of the coding sequence was sufficient for transformed clones to express mating type E instead of mating type O.In contrast to mtAO lines, mtBO and mtCO O-expressing lines did not produce mtA mRNA on sexual reactivity.

RNA interference (RNAi)-mediated silencing of mtA in E cells resulted in the default O phenotype during sexual reactivity.The MIC gene is interrupted by four IESs that are excised in both MAC types; however, a 195-base-pair (bp) segment containing the transcription start site and the first 26 bp of the coding sequence was found to be excised as an IES in mating type O MACs, but retained in E MACs. This segment contains the mtA promoter, as indicated by microinjection of different constructs into the MACs of O cells: its presence upstream of the coding sequence was sufficient for transformed clones to express mating type E instead of mating type O.In contrast to mtAO lines, mtBO and mtCO O-expressing lines did not produce mtA mRNA on sexual reactivity.

The effects of silencing of DCL2/3, and DCL5 on IES excision. Weak to modest positive correlations can be seen among the IRSs following the DCL2/3, DCL5 and PGM silencings, suggesting that there may be some indirect associations between scnRNAs, iesRNAs and the PiggyMac excisase complex. For the DCL2/3- and DCL5- knockdowns, the correlation between these IESs was negligible. For the DCL2/3- and DCL5- knockdowns, the correlation between these IESs was negligible. This correlation between PGM-KD IRSs of pairs of adjacent IESs increases when the distance between the adjacent IESs decreases. Gene silencing reveals new properties of eliminated DNA in Paramecium. The trends in base frequency changes over both longer and shorter scales (hundreds and tens of bp, respectively) are similar for IESs with DCL2/3-KD and DCL5-KD IRSs of zero. For the DCL2/3-KD, with the exception of the most abundant and short IESs (26–36 bp) the first sub-terminal position's T frequency increases and then plateaus with IRS (mirrored by the C frequency), while the second position's A frequency decreases with IRS (mirrored by a T-frequency increase). Similar base frequency changes are visible in the DCL5-KD. The effects of silencing of DCL2/3 and DCL5 on aberrant DNA excision. Both cryptic IES excision and the use of alternative boundaries are reduced in PGM-KD cells, in agreement with the hypothesis that TA-indels are produced by excision errors. In contrast, increased TA-indel rates in the DCL2/3 and DCL5-KD samples compared to the control suggest that interfering with the sRNA-dependent IES targeting machinery results in increased erroneous excision. Sequence logos for the low-frequency TA-indels in all of the samples show a weak preference for a 5′-TATNR-3′ consensus at the ends of the excised segments, as shown for the control and DCL2/3-KD TA-indels. Relationships between IRSs and sRNA densities. In the DCL5-KD, it can be seen that scnRNA densities are more or less constant relative to the IRSs of all the knockdowns we examined. Furthermore, we observe the same overall 25 nt sRNA density in control samples for IESs showing no retention after DCL2/3 cosilencing. The strongest relationship between iesRNA densities and IRSs is for the DCL5-KD IRSs, i.e. IESs most sensitive to iesRNA depletion normally have the highest densities of iesRNAs.

The effects of silencing of DCL2/3, and DCL5 on IES excision. Weak to modest positive correlations can be seen among the IRSs following the DCL2/3, DCL5 and PGM silencings, suggesting that there may be some indirect associations between scnRNAs, iesRNAs and the PiggyMac excisase complex. For the DCL2/3- and DCL5- knockdowns, the correlation between these IESs was negligible. For the DCL2/3- and DCL5- knockdowns, the correlation between these IESs was negligible. This correlation between PGM-KD IRSs of pairs of adjacent IESs increases when the distance between the adjacent IESs decreases. Gene silencing reveals new properties of eliminated DNA in Paramecium. The trends in base frequency changes over both longer and shorter scales (hundreds and tens of bp, respectively) are similar for IESs with DCL2/3-KD and DCL5-KD IRSs of zero. For the DCL2/3-KD, with the exception of the most abundant and short IESs (26–36 bp) the first sub-terminal position's T frequency increases and then plateaus with IRS (mirrored by the C frequency), while the second position's A frequency decreases with IRS (mirrored by a T-frequency increase). Similar base frequency changes are visible in the DCL5-KD. The effects of silencing of DCL2/3 and DCL5 on aberrant DNA excision. Both cryptic IES excision and the use of alternative boundaries are reduced in PGM-KD cells, in agreement with the hypothesis that TA-indels are produced by excision errors. In contrast, increased TA-indel rates in the DCL2/3 and DCL5-KD samples compared to the control suggest that interfering with the sRNA-dependent IES targeting machinery results in increased erroneous excision. Sequence logos for the low-frequency TA-indels in all of the samples show a weak preference for a 5′-TATNR-3′ consensus at the ends of the excised segments, as shown for the control and DCL2/3-KD TA-indels. Relationships between IRSs and sRNA densities. In the DCL5-KD, it can be seen that scnRNA densities are more or less constant relative to the IRSs of all the knockdowns we examined. Furthermore, we observe the same overall 25 nt sRNA density in control samples for IESs showing no retention after DCL2/3 cosilencing. The strongest relationship between iesRNA densities and IRSs is for the DCL5-KD IRSs, i.e. IESs most sensitive to iesRNA depletion normally have the highest densities of iesRNAs.

Knockdown of PTCAF-1 impairs cell survival and DNA elimination. The PTCAF-1 KD impairs the elimination of mcIES from the developing MAC but does not affect the excision of non-mcIES. PTCAF-1 knockdown affects RNA scanning by preventing the elimination of MAC-specific scnRNAs. PTCAF-1 knockdown reduces the levels of H3K9me3 and H3K27me3 during development.

Knockdown of PTCAF-1 impairs cell survival and DNA elimination. The PTCAF-1 KD impairs the elimination of mcIES from the developing MAC but does not affect the excision of non-mcIES. PTCAF-1 knockdown affects RNA scanning by preventing the elimination of MAC-specific scnRNAs. PTCAF-1 knockdown reduces the levels of H3K9me3 and H3K27me3 during development.

Pdsg1 and Pdsg2 are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development.

Pdsg1 and Pdsg2 are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development.

Pdsg1 and Pdsg2 are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development.

Pdsg1 and Pdsg2 are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development.

More scnRNAs were co-precipitated with Twi1p in a GIW1 KO cell lysate than in the wild-type cell lysate without an ATP supply, suggesting that Giw1p indeed has an inhibitory effect on scnRNA loading into Twi1p in cell lysates. Although the addition of ATP enhanced loading in the wild-type cell lysate, the presence of ATP did not further enhance the loading of scnRNAs in the GIW1 KO cell lysate. These results indicate that the primary role of the ATP-dependent scnRNA loading-enhancing activity in the cell lysate is to counteract the loading inhibitory effect of Giw1p. The removal of Coi12p from the GIW1 KO cell lysate did not inhibit the loading of 27-nt RNA duplexes into Twi1p. In the wild-type cell lysate, 27- and 31-nt RNAs were mainly co-precipitated with Twi1p, whereas 23- and 35-nt RNAs were largely excluded from the Twi1p-bound RNA fraction. This result indicates that a sorting mechanism preferentially loads scnRNA-sized RNA duplexes into Twi1p in the wild-type cell lysate. In contrast, all of the RNAs were co-precipitated with Twi1p in the GIW1 KO cell lysate.

A large fraction of sRNAs maps to genes targeted by dsRNA feeding. This yielded numerous sRNAs matching the genes targeted by dsRNA in the 3 cultures of the wild type (WT) strain 51 in which one or two non-essential genes(ND169, ICL7a) were silenced, but not in a control culture fed with the standard Klebsiella pneumoniae. The numbers of target-gene sRNAs varied between 15 000 and 94 000 rpm, which represents a large fraction of MAC-mapping sRNAs in each case: 36% for ND169 and 20% for ICL7a in sample WT-1. To broaden the analysis, we used sRNA sequencing data sets from other dsRNA-feeding experiments: the single silencing of RDR3 or PTIWI14, both specifically involved in transgene- but not in dsRNA-induced RNAi (29,30), PTIWI08 (a WGD1 paralog of PTWI14), or ICL7a. Similarly, large numbers of sRNAs specifically mapped to the cognate target gene in each case.

A large fraction of sRNAs maps to genes targeted by dsRNA feeding. This yielded numerous sRNAs matching the genes targeted by dsRNA in the 3 cultures of the wild type (WT) strain 51 in which one or two non-essential genes(ND169, ICL7a) were silenced, but not in a control culture fed with the standard Klebsiella pneumoniae. The numbers of target-gene sRNAs varied between 15 000 and 94 000 rpm, which represents a large fraction of MAC-mapping sRNAs in each case: 36% for ND169 and 20% for ICL7a in sample WT-1. To broaden the analysis, we used sRNA sequencing data sets from other dsRNA-feeding experiments: the single silencing of RDR3 or PTIWI14, both specifically involved in transgene- but not in dsRNA-induced RNAi (29,30), PTIWI08 (a WGD1 paralog of PTWI14), or ICL7a. Similarly, large numbers of sRNAs specifically mapped to the cognate target gene in each case.

A large fraction of sRNAs maps to genes targeted by dsRNA feeding. This yielded numerous sRNAs matching the genes targeted by dsRNA in the 3 cultures of the wild type (WT) strain 51 in which one or two non-essential genes(ND169, ICL7a) were silenced, but not in a control culture fed with the standard Klebsiella pneumoniae. The numbers of target-gene sRNAs varied between 15 000 and 94 000 rpm, which represents a large fraction of MAC-mapping sRNAs in each case: 36% for ND169 and 20% for ICL7a in sample WT-1. To broaden the analysis, we used sRNA sequencing data sets from other dsRNA-feeding experiments: the single silencing of RDR3 or PTIWI14, both specifically involved in transgene- but not in dsRNA-induced RNAi (29,30), PTIWI08 (a WGD1 paralog of PTWI14), or ICL7a. Similarly, large numbers of sRNAs specifically mapped to the cognate target gene in each case.

A large fraction of sRNAs maps to genes targeted by dsRNA feeding. This yielded numerous sRNAs matching the genes targeted by dsRNA in the 3 cultures of the wild type (WT) strain 51 in which one or two non-essential genes(ND169, ICL7a) were silenced, but not in a control culture fed with the standard Klebsiella pneumoniae. The numbers of target-gene sRNAs varied between 15 000 and 94 000 rpm, which represents a large fraction of MAC-mapping sRNAs in each case: 36% for ND169 and 20% for ICL7a in sample WT-1. To broaden the analysis, we used sRNA sequencing data sets from other dsRNA-feeding experiments: the single silencing of RDR3 or PTIWI14, both specifically involved in transgene- but not in dsRNA-induced RNAi (29,30), PTIWI08 (a WGD1 paralog of PTWI14), or ICL7a. Similarly, large numbers of sRNAs specifically mapped to the cognate target gene in each case.

A large fraction of sRNAs maps to genes targeted by dsRNA feeding. This yielded numerous sRNAs matching the genes targeted by dsRNA in the 3 cultures of the wild type (WT) strain 51 in which one or two non-essential genes(ND169, ICL7a) were silenced, but not in a control culture fed with the standard Klebsiella pneumoniae. The numbers of target-gene sRNAs varied between 15 000 and 94 000 rpm, which represents a large fraction of MAC-mapping sRNAs in each case: 36% for ND169 and 20% for ICL7a in sample WT-1. To broaden the analysis, we used sRNA sequencing data sets from other dsRNA-feeding experiments: the single silencing of RDR3 or PTIWI14, both specifically involved in transgene- but not in dsRNA-induced RNAi (29,30), PTIWI08 (a WGD1 paralog of PTWI14), or ICL7a. Similarly, large numbers of sRNAs specifically mapped to the cognate target gene in each case.

In TWI1/TWI11 MIC-KO cells, in which Late-scnRNAs are unstable, RIs of some of Type-B IESs were ~1 (not eliminated), whereas those of most Type-A IESs were ~0.1 (eliminated). This result was in sharp contrast to TWI1 MAC-KO cells, in which both Early- and Late-scnRNAs were lost, and the RIs of most IESs were ~1 regardless of type. These findings suggest that Early-scnRNAs are sufficient to induce DNA elimination for a majority of IESs, whereas Late-scnRNAs are important for DNA elimination of some, mainly Type-B, IESs. Elimination of type-C IESs, which lack their own scnRNA production, was also affected in TWI1 MAC-KO cells and, to a lesser extent, in TWI1/TWI11 MIC-KO cells, indicating that scnRNAs also play a role in the elimination of Type-C IESs. We analyzed the accumulation of scnRNAs in RDR1 MIC-KO cells and found that Late-scnRNAs were accumulated normally in the absence of zygotic expression of Rdr1p. We found that EZL1, which encodes H3K9/K27 methyltransferase, and PDD1 were necessary for the production of Late-scnRNAs, although they were not required for the accumulation of Early-scnRNAs.

In TWI1/TWI11 MIC-KO cells, in which Late-scnRNAs are unstable, RIs of some of Type-B IESs were ~1 (not eliminated), whereas those of most Type-A IESs were ~0.1 (eliminated). This result was in sharp contrast to TWI1 MAC-KO cells, in which both Early- and Late-scnRNAs were lost, and the RIs of most IESs were ~1 regardless of type. These findings suggest that Early-scnRNAs are sufficient to induce DNA elimination for a majority of IESs, whereas Late-scnRNAs are important for DNA elimination of some, mainly Type-B, IESs. Elimination of type-C IESs, which lack their own scnRNA production, was also affected in TWI1 MAC-KO cells and, to a lesser extent, in TWI1/TWI11 MIC-KO cells, indicating that scnRNAs also play a role in the elimination of Type-C IESs. We analyzed the accumulation of scnRNAs in RDR1 MIC-KO cells and found that Late-scnRNAs were accumulated normally in the absence of zygotic expression of Rdr1p. We found that EZL1, which encodes H3K9/K27 methyltransferase, and PDD1 were necessary for the production of Late-scnRNAs, although they were not required for the accumulation of Early-scnRNAs.

In TWI1/TWI11 MIC-KO cells, in which Late-scnRNAs are unstable, RIs of some of Type-B IESs were ~1 (not eliminated), whereas those of most Type-A IESs were ~0.1 (eliminated). This result was in sharp contrast to TWI1 MAC-KO cells, in which both Early- and Late-scnRNAs were lost, and the RIs of most IESs were ~1 regardless of type. These findings suggest that Early-scnRNAs are sufficient to induce DNA elimination for a majority of IESs, whereas Late-scnRNAs are important for DNA elimination of some, mainly Type-B, IESs. Elimination of type-C IESs, which lack their own scnRNA production, was also affected in TWI1 MAC-KO cells and, to a lesser extent, in TWI1/TWI11 MIC-KO cells, indicating that scnRNAs also play a role in the elimination of Type-C IESs. We analyzed the accumulation of scnRNAs in RDR1 MIC-KO cells and found that Late-scnRNAs were accumulated normally in the absence of zygotic expression of Rdr1p. We found that EZL1, which encodes H3K9/K27 methyltransferase, and PDD1 were necessary for the production of Late-scnRNAs, although they were not required for the accumulation of Early-scnRNAs.

In TWI1/TWI11 MIC-KO cells, in which Late-scnRNAs are unstable, RIs of some of Type-B IESs were ~1 (not eliminated), whereas those of most Type-A IESs were ~0.1 (eliminated). This result was in sharp contrast to TWI1 MAC-KO cells, in which both Early- and Late-scnRNAs were lost, and the RIs of most IESs were ~1 regardless of type. These findings suggest that Early-scnRNAs are sufficient to induce DNA elimination for a majority of IESs, whereas Late-scnRNAs are important for DNA elimination of some, mainly Type-B, IESs. Elimination of type-C IESs, which lack their own scnRNA production, was also affected in TWI1 MAC-KO cells and, to a lesser extent, in TWI1/TWI11 MIC-KO cells, indicating that scnRNAs also play a role in the elimination of Type-C IESs. We analyzed the accumulation of scnRNAs in RDR1 MIC-KO cells and found that Late-scnRNAs were accumulated normally in the absence of zygotic expression of Rdr1p. We found that EZL1, which encodes H3K9/K27 methyltransferase, and PDD1 were necessary for the production of Late-scnRNAs, although they were not required for the accumulation of Early-scnRNAs.

In TWI1/TWI11 MIC-KO cells, in which Late-scnRNAs are unstable, RIs of some of Type-B IESs were ~1 (not eliminated), whereas those of most Type-A IESs were ~0.1 (eliminated). This result was in sharp contrast to TWI1 MAC-KO cells, in which both Early- and Late-scnRNAs were lost, and the RIs of most IESs were ~1 regardless of type. These findings suggest that Early-scnRNAs are sufficient to induce DNA elimination for a majority of IESs, whereas Late-scnRNAs are important for DNA elimination of some, mainly Type-B, IESs. Elimination of type-C IESs, which lack their own scnRNA production, was also affected in TWI1 MAC-KO cells and, to a lesser extent, in TWI1/TWI11 MIC-KO cells, indicating that scnRNAs also play a role in the elimination of Type-C IESs. We analyzed the accumulation of scnRNAs in RDR1 MIC-KO cells and found that Late-scnRNAs were accumulated normally in the absence of zygotic expression of Rdr1p. We found that EZL1, which encodes H3K9/K27 methyltransferase, and PDD1 were necessary for the production of Late-scnRNAs, although they were not required for the accumulation of Early-scnRNAs.

In TWI1/TWI11 MIC-KO cells, in which Late-scnRNAs are unstable, RIs of some of Type-B IESs were ~1 (not eliminated), whereas those of most Type-A IESs were ~0.1 (eliminated). This result was in sharp contrast to TWI1 MAC-KO cells, in which both Early- and Late-scnRNAs were lost, and the RIs of most IESs were ~1 regardless of type. These findings suggest that Early-scnRNAs are sufficient to induce DNA elimination for a majority of IESs, whereas Late-scnRNAs are important for DNA elimination of some, mainly Type-B, IESs. Elimination of type-C IESs, which lack their own scnRNA production, was also affected in TWI1 MAC-KO cells and, to a lesser extent, in TWI1/TWI11 MIC-KO cells, indicating that scnRNAs also play a role in the elimination of Type-C IESs. We analyzed the accumulation of scnRNAs in RDR1 MIC-KO cells and found that Late-scnRNAs were accumulated normally in the absence of zygotic expression of Rdr1p. We found that EZL1, which encodes H3K9/K27 methyltransferase, and PDD1 were necessary for the production of Late-scnRNAs, although they were not required for the accumulation of Early-scnRNAs.

In TWI1/TWI11 MIC-KO cells, in which Late-scnRNAs are unstable, RIs of some of Type-B IESs were ~1 (not eliminated), whereas those of most Type-A IESs were ~0.1 (eliminated). This result was in sharp contrast to TWI1 MAC-KO cells, in which both Early- and Late-scnRNAs were lost, and the RIs of most IESs were ~1 regardless of type. These findings suggest that Early-scnRNAs are sufficient to induce DNA elimination for a majority of IESs, whereas Late-scnRNAs are important for DNA elimination of some, mainly Type-B, IESs. Elimination of type-C IESs, which lack their own scnRNA production, was also affected in TWI1 MAC-KO cells and, to a lesser extent, in TWI1/TWI11 MIC-KO cells, indicating that scnRNAs also play a role in the elimination of Type-C IESs. We analyzed the accumulation of scnRNAs in RDR1 MIC-KO cells and found that Late-scnRNAs were accumulated normally in the absence of zygotic expression of Rdr1p. We found that EZL1, which encodes H3K9/K27 methyltransferase, and PDD1 were necessary for the production of Late-scnRNAs, although they were not required for the accumulation of Early-scnRNAs.

In TWI1/TWI11 MIC-KO cells, in which Late-scnRNAs are unstable, RIs of some of Type-B IESs were ~1 (not eliminated), whereas those of most Type-A IESs were ~0.1 (eliminated). This result was in sharp contrast to TWI1 MAC-KO cells, in which both Early- and Late-scnRNAs were lost, and the RIs of most IESs were ~1 regardless of type. These findings suggest that Early-scnRNAs are sufficient to induce DNA elimination for a majority of IESs, whereas Late-scnRNAs are important for DNA elimination of some, mainly Type-B, IESs. Elimination of type-C IESs, which lack their own scnRNA production, was also affected in TWI1 MAC-KO cells and, to a lesser extent, in TWI1/TWI11 MIC-KO cells, indicating that scnRNAs also play a role in the elimination of Type-C IESs. We analyzed the accumulation of scnRNAs in RDR1 MIC-KO cells and found that Late-scnRNAs were accumulated normally in the absence of zygotic expression of Rdr1p. We found that EZL1, which encodes H3K9/K27 methyltransferase, and PDD1 were necessary for the production of Late-scnRNAs, although they were not required for the accumulation of Early-scnRNAs.

The heterochromatin body component Jub1p facilitates the dephosphorylation of Pdd1p. Both JUB1 KO and phosphor-mimic mutations of Pdd1p severely compromised heterochromatin body formation and DNA elimination without affecting local heterochromatin assembly. Most, if not all, IESs are retained in the new MAC in the absence of Jub1p. A similar IES elimination defect was detected in TWI1 KO cells. Jub1p was less enriched on IESs in PDD1 KO cells than in WT cells. This was not due to an overall reduction or aberrant cellular localization of Jub1p, because Jub1p accumulated normally and localized to the new MAC in PDD1 KO cells. Pdd1p was phosphorylated in EZL1 and TWI1 KO cells.

The heterochromatin body component Jub1p facilitates the dephosphorylation of Pdd1p. Both JUB1 KO and phosphor-mimic mutations of Pdd1p severely compromised heterochromatin body formation and DNA elimination without affecting local heterochromatin assembly. Most, if not all, IESs are retained in the new MAC in the absence of Jub1p. A similar IES elimination defect was detected in TWI1 KO cells. Jub1p was less enriched on IESs in PDD1 KO cells than in WT cells. This was not due to an overall reduction or aberrant cellular localization of Jub1p, because Jub1p accumulated normally and localized to the new MAC in PDD1 KO cells. Pdd1p was phosphorylated in EZL1 and TWI1 KO cells.

Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5–3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. Plenty of DEGs between CYC2 KO and wild type strains were functionally enriched in DNA mismatch repair and DNA replication field. ΔCYC2 displayed conspicuously defect of meiotic micronuclei elongation and meiotic DSBs formation in compatible with previously published phenotype of SPO11 knockout strains (ΔSPO11) during the prophase of meiosis I.

Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5–3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. Plenty of DEGs between CYC2 KO and wild type strains were functionally enriched in DNA mismatch repair and DNA replication field. ΔCYC2 displayed conspicuously defect of meiotic micronuclei elongation and meiotic DSBs formation in compatible with previously published phenotype of SPO11 knockout strains (ΔSPO11) during the prophase of meiosis I.

Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5–3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. Plenty of DEGs between CYC2 KO and wild type strains were functionally enriched in DNA mismatch repair and DNA replication field. ΔCYC2 displayed conspicuously defect of meiotic micronuclei elongation and meiotic DSBs formation in compatible with previously published phenotype of SPO11 knockout strains (ΔSPO11) during the prophase of meiosis I.

Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5–3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. Plenty of DEGs between CYC2 KO and wild type strains were functionally enriched in DNA mismatch repair and DNA replication field. ΔCYC2 displayed conspicuously defect of meiotic micronuclei elongation and meiotic DSBs formation in compatible with previously published phenotype of SPO11 knockout strains (ΔSPO11) during the prophase of meiosis I.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Microarray analysis of RDR3 knockdown cultures revealed that ~20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family. Interestingly, silencing of RDR3 does not solely activate gene expression, but many genes are down-regulated. GO term enrichment of these DEGs indicating processes of chromatin assembly being down-regulated, and processes associated with transcriptional activity up-regulated, thus suggesting an association of RDR3 and heterochromatin formation.Knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs.RDR2 & RDR3 and PTIWI13 & PTIWI08/14 are necessary for primary siRNA accumulation. Knockdown of RNAi components reduces primary 23 nt small RNAs.knockdown of each of the genes within the two sets PTIWI13/8-14 and RDR2/3 resulted in the reduction of K28 siRNAs.RDR2 knockdown resulted in deficiency of heterochromatin formation at the endoND169 locus.

Deletion of CYC17 led to meiotic arrest at the diakinesis-like metaphase I stage. Expression of genes involved in DNA metabolism and chromosome organization (chromatin remodeling and basic chromosomal structure) was repressed in cyc17 knockout matings.

Deletion of CYC17 led to meiotic arrest at the diakinesis-like metaphase I stage. Expression of genes involved in DNA metabolism and chromosome organization (chromatin remodeling and basic chromosomal structure) was repressed in cyc17 knockout matings.

Deletion of CYC17 led to meiotic arrest at the diakinesis-like metaphase I stage. Expression of genes involved in DNA metabolism and chromosome organization (chromatin remodeling and basic chromosomal structure) was repressed in cyc17 knockout matings.

Deletion of CYC17 led to meiotic arrest at the diakinesis-like metaphase I stage. Expression of genes involved in DNA metabolism and chromosome organization (chromatin remodeling and basic chromosomal structure) was repressed in cyc17 knockout matings.

Meiosis is arrested at the pair formation stage in cdk3Δ. In WT cells, MICs started to elongate at meiosis initiation; in contrast, MIC elongation did not occur in cdk3Δ cells. Cdk3 acts upstream of DSB formation in T. thermophila. DSB formation and repair are inhibited in Cdk3 deficient cells. Genes related to DSB repair were down-regulated in cdk3Δ cells.Cdk3 may initiate meiosis together with Cyc2. In the absence of Cdk3, the abundance of the specific phosphopeptide was strongly decreased. In both cdk3Δ and cyc2Δ cells, most meiosis-related genes were downregulated.

Meiosis is arrested at the pair formation stage in cdk3Δ. In WT cells, MICs started to elongate at meiosis initiation; in contrast, MIC elongation did not occur in cdk3Δ cells. Cdk3 acts upstream of DSB formation in T. thermophila. DSB formation and repair are inhibited in Cdk3 deficient cells. Genes related to DSB repair were down-regulated in cdk3Δ cells.Cdk3 may initiate meiosis together with Cyc2. In the absence of Cdk3, the abundance of the specific phosphopeptide was strongly decreased. In both cdk3Δ and cyc2Δ cells, most meiosis-related genes were downregulated.

Meiosis is arrested at the pair formation stage in cdk3Δ. In WT cells, MICs started to elongate at meiosis initiation; in contrast, MIC elongation did not occur in cdk3Δ cells. Cdk3 acts upstream of DSB formation in T. thermophila. DSB formation and repair are inhibited in Cdk3 deficient cells. Genes related to DSB repair were down-regulated in cdk3Δ cells.Cdk3 may initiate meiosis together with Cyc2. In the absence of Cdk3, the abundance of the specific phosphopeptide was strongly decreased. In both cdk3Δ and cyc2Δ cells, most meiosis-related genes were downregulated.

The loss of Jub4p disturbs both Pdd1p phosphorylation and heterochromatin body formation. Heterochromatin is formed properly on IESs in the absence of Jub4p and thus Jub4p acts downstream of heterochromatin establishment for heterochromatin body formation.Unphosphorylatable mutations of Pdd1p inhibit heterochromatin body formation.

The loss of Jub4p disturbs both Pdd1p phosphorylation and heterochromatin body formation. Heterochromatin is formed properly on IESs in the absence of Jub4p and thus Jub4p acts downstream of heterochromatin establishment for heterochromatin body formation.Unphosphorylatable mutations of Pdd1p inhibit heterochromatin body formation.

The loss of Jub4p disturbs both Pdd1p phosphorylation and heterochromatin body formation. Heterochromatin is formed properly on IESs in the absence of Jub4p and thus Jub4p acts downstream of heterochromatin establishment for heterochromatin body formation.Unphosphorylatable mutations of Pdd1p inhibit heterochromatin body formation.

The loss of Jub4p disturbs both Pdd1p phosphorylation and heterochromatin body formation. Heterochromatin is formed properly on IESs in the absence of Jub4p and thus Jub4p acts downstream of heterochromatin establishment for heterochromatin body formation.Unphosphorylatable mutations of Pdd1p inhibit heterochromatin body formation.

The loss of Jub4p disturbs both Pdd1p phosphorylation and heterochromatin body formation. Heterochromatin is formed properly on IESs in the absence of Jub4p and thus Jub4p acts downstream of heterochromatin establishment for heterochromatin body formation.Unphosphorylatable mutations of Pdd1p inhibit heterochromatin body formation.

The loss of Jub4p disturbs both Pdd1p phosphorylation and heterochromatin body formation. Heterochromatin is formed properly on IESs in the absence of Jub4p and thus Jub4p acts downstream of heterochromatin establishment for heterochromatin body formation.Unphosphorylatable mutations of Pdd1p inhibit heterochromatin body formation.

Loss of this gene resulted in meiotic arrest prior to anaphase I. The cytological experiments revealed that the meiotic homologous pairing was not affected in the absence of E2FL1, but the paired homologous chromosomes did not separate and assumed a peculiar tandem arrangement. This is the first time that an E2F family member has been shown to regulate meiotic events. Moreover, BrdU incorporation showed that DSB processing during meiosis was abnormal upon the deletion of E2FL1. Transcriptome sequencing analysis revealed that E2FL1 knockout decreased the expression of genes involved in DNA replication and DNA repair in T. thermophila, suggesting that the function of E2F is highly conserved in eukaryotes. In addition, E2FL1 deletion inhibited the expression of related homologous chromosome segregation genes in T. thermophila. The result may explain the meiotic arrest phenotype at anaphase I.

Loss of this gene resulted in meiotic arrest prior to anaphase I. The cytological experiments revealed that the meiotic homologous pairing was not affected in the absence of E2FL1, but the paired homologous chromosomes did not separate and assumed a peculiar tandem arrangement. This is the first time that an E2F family member has been shown to regulate meiotic events. Moreover, BrdU incorporation showed that DSB processing during meiosis was abnormal upon the deletion of E2FL1. Transcriptome sequencing analysis revealed that E2FL1 knockout decreased the expression of genes involved in DNA replication and DNA repair in T. thermophila, suggesting that the function of E2F is highly conserved in eukaryotes. In addition, E2FL1 deletion inhibited the expression of related homologous chromosome segregation genes in T. thermophila. The result may explain the meiotic arrest phenotype at anaphase I.

Loss of this gene resulted in meiotic arrest prior to anaphase I. The cytological experiments revealed that the meiotic homologous pairing was not affected in the absence of E2FL1, but the paired homologous chromosomes did not separate and assumed a peculiar tandem arrangement. This is the first time that an E2F family member has been shown to regulate meiotic events. Moreover, BrdU incorporation showed that DSB processing during meiosis was abnormal upon the deletion of E2FL1. Transcriptome sequencing analysis revealed that E2FL1 knockout decreased the expression of genes involved in DNA replication and DNA repair in T. thermophila, suggesting that the function of E2F is highly conserved in eukaryotes. In addition, E2FL1 deletion inhibited the expression of related homologous chromosome segregation genes in T. thermophila. The result may explain the meiotic arrest phenotype at anaphase I.

Loss of this gene resulted in meiotic arrest prior to anaphase I. The cytological experiments revealed that the meiotic homologous pairing was not affected in the absence of E2FL1, but the paired homologous chromosomes did not separate and assumed a peculiar tandem arrangement. This is the first time that an E2F family member has been shown to regulate meiotic events. Moreover, BrdU incorporation showed that DSB processing during meiosis was abnormal upon the deletion of E2FL1. Transcriptome sequencing analysis revealed that E2FL1 knockout decreased the expression of genes involved in DNA replication and DNA repair in T. thermophila, suggesting that the function of E2F is highly conserved in eukaryotes. In addition, E2FL1 deletion inhibited the expression of related homologous chromosome segregation genes in T. thermophila. The result may explain the meiotic arrest phenotype at anaphase I.

Loss of this gene resulted in meiotic arrest prior to anaphase I. The cytological experiments revealed that the meiotic homologous pairing was not affected in the absence of E2FL1, but the paired homologous chromosomes did not separate and assumed a peculiar tandem arrangement. This is the first time that an E2F family member has been shown to regulate meiotic events. Moreover, BrdU incorporation showed that DSB processing during meiosis was abnormal upon the deletion of E2FL1. Transcriptome sequencing analysis revealed that E2FL1 knockout decreased the expression of genes involved in DNA replication and DNA repair in T. thermophila, suggesting that the function of E2F is highly conserved in eukaryotes. In addition, E2FL1 deletion inhibited the expression of related homologous chromosome segregation genes in T. thermophila. The result may explain the meiotic arrest phenotype at anaphase I.

Loss of this gene resulted in meiotic arrest prior to anaphase I. The cytological experiments revealed that the meiotic homologous pairing was not affected in the absence of E2FL1, but the paired homologous chromosomes did not separate and assumed a peculiar tandem arrangement. This is the first time that an E2F family member has been shown to regulate meiotic events. Moreover, BrdU incorporation showed that DSB processing during meiosis was abnormal upon the deletion of E2FL1. Transcriptome sequencing analysis revealed that E2FL1 knockout decreased the expression of genes involved in DNA replication and DNA repair in T. thermophila, suggesting that the function of E2F is highly conserved in eukaryotes. In addition, E2FL1 deletion inhibited the expression of related homologous chromosome segregation genes in T. thermophila. The result may explain the meiotic arrest phenotype at anaphase I.

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. Although the loss of Coi6p or Coi7p resulted in abnormal DNA elimination, it also severely blocked DNA elimination. The latter is probably not simply caused by the disturbance of heterochromatin borders because although we observed a similar degree of heterochromatin and Late-scnRNA spreading in ΔCOI6 and ΔJMJ1 cells, DNA elimination was only mildly inhibited in ΔJMJ1 cells. The phenotype of ΔLIA5 cells diverged from that of ΔCOI6 and COI7fs1/fs2 cells with a more severe DNA elimination block, no detectable ectopic DNA elimination, and Late-scnRNA upregulation from both MDSs and IESs

The silencing of Ligase IV has been shown to prevent the repair of genomic DNA following IES excision during Paramecium development, leading to a massively fragmented genome and cell death.Ligase IV silencing almost completely abrogated iesRNA production.

The silencing of Ligase IV has been shown to prevent the repair of genomic DNA following IES excision during Paramecium development, leading to a massively fragmented genome and cell death.Ligase IV silencing almost completely abrogated iesRNA production.

Complete UPF1a knockout is not lethal, leading only to a modest extension in generation time. uORF-containing genes are significantly enriched in the pool of genes upregulated in ΔUPF1a. PTC-containing isoform levels were significantly higher in ΔUPF1a than in WT cells. UPF1a and UPF2 gene locus disruption causes the greatest retention of nonsense transcripts, while UPF3 knockout has a reduced effect. There is modest fold change in expression of transcripts with long 3΄-UTRs in ΔUPF1a. Levels of the vast majority of NMD targets tested were significantly perturbed in SMG6L knockout cells. Many PTC-containing transcripts are dramatically retained in ΔSMG6L cells. ΔSMG6L mutant cells showed that around 50% of PTC-containing transcripts are specifically retained in ΔUPF1a cells. Mag1 protein does not contribute to the Tetrahymena NMD pathway. Very few NMD targets were upregulated in MAG1 knockout cells.

Complete UPF1a knockout is not lethal, leading only to a modest extension in generation time. uORF-containing genes are significantly enriched in the pool of genes upregulated in ΔUPF1a. PTC-containing isoform levels were significantly higher in ΔUPF1a than in WT cells. UPF1a and UPF2 gene locus disruption causes the greatest retention of nonsense transcripts, while UPF3 knockout has a reduced effect. There is modest fold change in expression of transcripts with long 3΄-UTRs in ΔUPF1a. Levels of the vast majority of NMD targets tested were significantly perturbed in SMG6L knockout cells. Many PTC-containing transcripts are dramatically retained in ΔSMG6L cells. ΔSMG6L mutant cells showed that around 50% of PTC-containing transcripts are specifically retained in ΔUPF1a cells. Mag1 protein does not contribute to the Tetrahymena NMD pathway. Very few NMD targets were upregulated in MAG1 knockout cells.

Complete UPF1a knockout is not lethal, leading only to a modest extension in generation time. uORF-containing genes are significantly enriched in the pool of genes upregulated in ΔUPF1a. PTC-containing isoform levels were significantly higher in ΔUPF1a than in WT cells. UPF1a and UPF2 gene locus disruption causes the greatest retention of nonsense transcripts, while UPF3 knockout has a reduced effect. There is modest fold change in expression of transcripts with long 3΄-UTRs in ΔUPF1a. Levels of the vast majority of NMD targets tested were significantly perturbed in SMG6L knockout cells. Many PTC-containing transcripts are dramatically retained in ΔSMG6L cells. ΔSMG6L mutant cells showed that around 50% of PTC-containing transcripts are specifically retained in ΔUPF1a cells. Mag1 protein does not contribute to the Tetrahymena NMD pathway. Very few NMD targets were upregulated in MAG1 knockout cells.

PTIWI01/09-KD Affects Dcl2/3-Dependent IESs, and PTIWI10/11-KD Affects Dcl5-Dependent IESs. PTIWI01/09-KD and PTIWI10/11-KD Lead to scnRNA and iesRNA Depletion, Respectively.Only PTIWI01/09-KD, but not PTIWI10/11-KD, affects the excision of the two known P. tetraurelia transposon classes Sardine and Thon, consistent with dependence of transposon elimination on scnRNAs but not on iesRNAs.

PTIWI01/09-KD Affects Dcl2/3-Dependent IESs, and PTIWI10/11-KD Affects Dcl5-Dependent IESs. PTIWI01/09-KD and PTIWI10/11-KD Lead to scnRNA and iesRNA Depletion, Respectively.Only PTIWI01/09-KD, but not PTIWI10/11-KD, affects the excision of the two known P. tetraurelia transposon classes Sardine and Thon, consistent with dependence of transposon elimination on scnRNAs but not on iesRNAs.

PTIWI01/09-KD Affects Dcl2/3-Dependent IESs, and PTIWI10/11-KD Affects Dcl5-Dependent IESs. PTIWI01/09-KD and PTIWI10/11-KD Lead to scnRNA and iesRNA Depletion, Respectively.Only PTIWI01/09-KD, but not PTIWI10/11-KD, affects the excision of the two known P. tetraurelia transposon classes Sardine and Thon, consistent with dependence of transposon elimination on scnRNAs but not on iesRNAs.

PTIWI01/09-KD Affects Dcl2/3-Dependent IESs, and PTIWI10/11-KD Affects Dcl5-Dependent IESs. PTIWI01/09-KD and PTIWI10/11-KD Lead to scnRNA and iesRNA Depletion, Respectively.Only PTIWI01/09-KD, but not PTIWI10/11-KD, affects the excision of the two known P. tetraurelia transposon classes Sardine and Thon, consistent with dependence of transposon elimination on scnRNAs but not on iesRNAs.

PTIWI01/09-KD Affects Dcl2/3-Dependent IESs, and PTIWI10/11-KD Affects Dcl5-Dependent IESs. PTIWI01/09-KD and PTIWI10/11-KD Lead to scnRNA and iesRNA Depletion, Respectively.Only PTIWI01/09-KD, but not PTIWI10/11-KD, affects the excision of the two known P. tetraurelia transposon classes Sardine and Thon, consistent with dependence of transposon elimination on scnRNAs but not on iesRNAs.

PTIWI01/09-KD Affects Dcl2/3-Dependent IESs, and PTIWI10/11-KD Affects Dcl5-Dependent IESs. PTIWI01/09-KD and PTIWI10/11-KD Lead to scnRNA and iesRNA Depletion, Respectively.Only PTIWI01/09-KD, but not PTIWI10/11-KD, affects the excision of the two known P. tetraurelia transposon classes Sardine and Thon, consistent with dependence of transposon elimination on scnRNAs but not on iesRNAs.

A subset of IES, efficiently removed from all WT samples, was consistently retained in ΔNMC-3 samples. Systematic search of the over 10 000 IES in the Tetrahymena MIC genome revealed only 11 IES affected in this way, hereafter referred to as IES-1 to 11. Removal of all 11 IES is dependent on NMC-3, likely because of TPB6 which contained in NMC-3. ΔDCL1 cells are defective in DNA elimination. Like regular IES, TPB6-dependent IES were not processed in conjugation progeny of mutants deficient in RNA interference and Polycomb repression, including ΔDCL1, ΔEZL1 and ΔPDD1. High levels of bi-directional scnRNA were found in the regular IES nested in IES-11, but not in the remainder of IES-11. This regular IES was eliminated, with variable boundaries, in ΔNMC-3 cells, but not ΔDCL1 cells. Formation of the developing MAC in the absence of TPB6 (ΔNMC-3 progeny) leads to interruption of exons in all 10 genes by the retained IES​. ΔNMC-3 progeny exhibited a severe growth phenotype and RNA-Seq revealed an altered global transcription profile. Genes normally controlled by starvation had aberrant expression in ΔNMC-3 progeny grown in rich medium: many starvation-induced genes were significantly up-regulated, while many starvation-repressed genes were significantly down-regulated. These included ngoA, a well-studied starvation-induced gene in Tetrahymena. Conspicuously, many ΔNMC-3 progeny cells were bloated with an extraordinarily large contractile vacuole, which failed to undergo the normal diastolic-systolic cycle for periodic expulsion of fluid. The percentage of abnormal cells increased in hypotonic media and decreased in hypertonic media.

A subset of IES, efficiently removed from all WT samples, was consistently retained in ΔNMC-3 samples. Systematic search of the over 10 000 IES in the Tetrahymena MIC genome revealed only 11 IES affected in this way, hereafter referred to as IES-1 to 11. Removal of all 11 IES is dependent on NMC-3, likely because of TPB6 which contained in NMC-3. ΔDCL1 cells are defective in DNA elimination. Like regular IES, TPB6-dependent IES were not processed in conjugation progeny of mutants deficient in RNA interference and Polycomb repression, including ΔDCL1, ΔEZL1 and ΔPDD1. High levels of bi-directional scnRNA were found in the regular IES nested in IES-11, but not in the remainder of IES-11. This regular IES was eliminated, with variable boundaries, in ΔNMC-3 cells, but not ΔDCL1 cells. Formation of the developing MAC in the absence of TPB6 (ΔNMC-3 progeny) leads to interruption of exons in all 10 genes by the retained IES​. ΔNMC-3 progeny exhibited a severe growth phenotype and RNA-Seq revealed an altered global transcription profile. Genes normally controlled by starvation had aberrant expression in ΔNMC-3 progeny grown in rich medium: many starvation-induced genes were significantly up-regulated, while many starvation-repressed genes were significantly down-regulated. These included ngoA, a well-studied starvation-induced gene in Tetrahymena. Conspicuously, many ΔNMC-3 progeny cells were bloated with an extraordinarily large contractile vacuole, which failed to undergo the normal diastolic-systolic cycle for periodic expulsion of fluid. The percentage of abnormal cells increased in hypotonic media and decreased in hypertonic media.

A subset of IES, efficiently removed from all WT samples, was consistently retained in ΔNMC-3 samples. Systematic search of the over 10 000 IES in the Tetrahymena MIC genome revealed only 11 IES affected in this way, hereafter referred to as IES-1 to 11. Removal of all 11 IES is dependent on NMC-3, likely because of TPB6 which contained in NMC-3. ΔDCL1 cells are defective in DNA elimination. Like regular IES, TPB6-dependent IES were not processed in conjugation progeny of mutants deficient in RNA interference and Polycomb repression, including ΔDCL1, ΔEZL1 and ΔPDD1. High levels of bi-directional scnRNA were found in the regular IES nested in IES-11, but not in the remainder of IES-11. This regular IES was eliminated, with variable boundaries, in ΔNMC-3 cells, but not ΔDCL1 cells. Formation of the developing MAC in the absence of TPB6 (ΔNMC-3 progeny) leads to interruption of exons in all 10 genes by the retained IES​. ΔNMC-3 progeny exhibited a severe growth phenotype and RNA-Seq revealed an altered global transcription profile. Genes normally controlled by starvation had aberrant expression in ΔNMC-3 progeny grown in rich medium: many starvation-induced genes were significantly up-regulated, while many starvation-repressed genes were significantly down-regulated. These included ngoA, a well-studied starvation-induced gene in Tetrahymena. Conspicuously, many ΔNMC-3 progeny cells were bloated with an extraordinarily large contractile vacuole, which failed to undergo the normal diastolic-systolic cycle for periodic expulsion of fluid. The percentage of abnormal cells increased in hypotonic media and decreased in hypertonic media.

A subset of IES, efficiently removed from all WT samples, was consistently retained in ΔNMC-3 samples. Systematic search of the over 10 000 IES in the Tetrahymena MIC genome revealed only 11 IES affected in this way, hereafter referred to as IES-1 to 11. Removal of all 11 IES is dependent on NMC-3, likely because of TPB6 which contained in NMC-3. ΔDCL1 cells are defective in DNA elimination. Like regular IES, TPB6-dependent IES were not processed in conjugation progeny of mutants deficient in RNA interference and Polycomb repression, including ΔDCL1, ΔEZL1 and ΔPDD1. High levels of bi-directional scnRNA were found in the regular IES nested in IES-11, but not in the remainder of IES-11. This regular IES was eliminated, with variable boundaries, in ΔNMC-3 cells, but not ΔDCL1 cells. Formation of the developing MAC in the absence of TPB6 (ΔNMC-3 progeny) leads to interruption of exons in all 10 genes by the retained IES​. ΔNMC-3 progeny exhibited a severe growth phenotype and RNA-Seq revealed an altered global transcription profile. Genes normally controlled by starvation had aberrant expression in ΔNMC-3 progeny grown in rich medium: many starvation-induced genes were significantly up-regulated, while many starvation-repressed genes were significantly down-regulated. These included ngoA, a well-studied starvation-induced gene in Tetrahymena. Conspicuously, many ΔNMC-3 progeny cells were bloated with an extraordinarily large contractile vacuole, which failed to undergo the normal diastolic-systolic cycle for periodic expulsion of fluid. The percentage of abnormal cells increased in hypotonic media and decreased in hypertonic media.

A subset of IES, efficiently removed from all WT samples, was consistently retained in ΔNMC-3 samples. Systematic search of the over 10 000 IES in the Tetrahymena MIC genome revealed only 11 IES affected in this way, hereafter referred to as IES-1 to 11. Removal of all 11 IES is dependent on NMC-3, likely because of TPB6 which contained in NMC-3. ΔDCL1 cells are defective in DNA elimination. Like regular IES, TPB6-dependent IES were not processed in conjugation progeny of mutants deficient in RNA interference and Polycomb repression, including ΔDCL1, ΔEZL1 and ΔPDD1. High levels of bi-directional scnRNA were found in the regular IES nested in IES-11, but not in the remainder of IES-11. This regular IES was eliminated, with variable boundaries, in ΔNMC-3 cells, but not ΔDCL1 cells. Formation of the developing MAC in the absence of TPB6 (ΔNMC-3 progeny) leads to interruption of exons in all 10 genes by the retained IES​. ΔNMC-3 progeny exhibited a severe growth phenotype and RNA-Seq revealed an altered global transcription profile. Genes normally controlled by starvation had aberrant expression in ΔNMC-3 progeny grown in rich medium: many starvation-induced genes were significantly up-regulated, while many starvation-repressed genes were significantly down-regulated. These included ngoA, a well-studied starvation-induced gene in Tetrahymena. Conspicuously, many ΔNMC-3 progeny cells were bloated with an extraordinarily large contractile vacuole, which failed to undergo the normal diastolic-systolic cycle for periodic expulsion of fluid. The percentage of abnormal cells increased in hypotonic media and decreased in hypertonic media.

Mutations that stall the processing or production of 21-nt sRNAs do not lead to increased transposon activity or increased target mRNA levels, but instead lead to reduced DNA copy number, potential S-phase arrest, and DNA damage. Furthermore, the 21-nt sRNAs map to the entire somatic genome and associate with chromatin. The dcl-1 cells showed reduced levels of 21-nt small RNAs and increased accumulation of 30- to 50-nt RNA. rdrp mutant lines also displayed reduced 21-nt RNA levels relative to wild-type control cells, demonstrating that sRNA production in asexually growing Oxytricha cells is dependent on both Dcl-1 and RdRP. But the sRNA reduction is partial. Comparison of 17- to 25-nt RNAs from wild-type versus mutant cells revealed reduced proportions of 21-nt RNAs mapping to 10,513 fully sequenced MAC chromosomes in dcl-141 cells. However, the sRNA mapping and sequence characteristics remain unaltered in dcl-1 or rdrp mutant cells; only their relative abundance is affected, compared to wild type. Both dcl-1 and rdrp mutants show reduced 17- to 25-nt sRNAs versus wild-type control cells, while dcl-1 cells display an overabundance of 30- to 50-nt RNAs, which are undetectable in wild-type or rdrp mutants. Rather than deriving from mRNA precursors, these sRNAs, like Oxytricha piRNAs, could be processed from long, noncoding RNAs, possibly from the same RNA molecules that establish chromosome copy number post-conjugation. In contrast to MAC-mapping sRNAs, dcl-1 cells show no reduction in the levels of MIC-mapping sRNAs. However, rdrp cells showed a small yet significant reduction in MIC-satellite and MIC gene mapping small RNAs. And they found genetically distinct classes of small RNAs in asexually growing Oxytricha cells; Dcl-1-dependent MAC-mapping 21-nt sRNAs and RdRP-dependent MIC-mapping 21- to 22-nt sRNAs. qPCR analysis of absolute DNA quantification for four representative MAC contigs shows statistically significant reduction of DNA copy number in dcl-1 and rdrp mutant strains. Mutant strains of dcl-1 and rdrp display slower growth and increased death rate with age. sRNA mutants display compromised genome integrity and defects in cell-cycle progression.

Mutations that stall the processing or production of 21-nt sRNAs do not lead to increased transposon activity or increased target mRNA levels, but instead lead to reduced DNA copy number, potential S-phase arrest, and DNA damage. Furthermore, the 21-nt sRNAs map to the entire somatic genome and associate with chromatin. The dcl-1 cells showed reduced levels of 21-nt small RNAs and increased accumulation of 30- to 50-nt RNA. rdrp mutant lines also displayed reduced 21-nt RNA levels relative to wild-type control cells, demonstrating that sRNA production in asexually growing Oxytricha cells is dependent on both Dcl-1 and RdRP. But the sRNA reduction is partial. Comparison of 17- to 25-nt RNAs from wild-type versus mutant cells revealed reduced proportions of 21-nt RNAs mapping to 10,513 fully sequenced MAC chromosomes in dcl-141 cells. However, the sRNA mapping and sequence characteristics remain unaltered in dcl-1 or rdrp mutant cells; only their relative abundance is affected, compared to wild type. Both dcl-1 and rdrp mutants show reduced 17- to 25-nt sRNAs versus wild-type control cells, while dcl-1 cells display an overabundance of 30- to 50-nt RNAs, which are undetectable in wild-type or rdrp mutants. Rather than deriving from mRNA precursors, these sRNAs, like Oxytricha piRNAs, could be processed from long, noncoding RNAs, possibly from the same RNA molecules that establish chromosome copy number post-conjugation. In contrast to MAC-mapping sRNAs, dcl-1 cells show no reduction in the levels of MIC-mapping sRNAs. However, rdrp cells showed a small yet significant reduction in MIC-satellite and MIC gene mapping small RNAs. And they found genetically distinct classes of small RNAs in asexually growing Oxytricha cells; Dcl-1-dependent MAC-mapping 21-nt sRNAs and RdRP-dependent MIC-mapping 21- to 22-nt sRNAs. qPCR analysis of absolute DNA quantification for four representative MAC contigs shows statistically significant reduction of DNA copy number in dcl-1 and rdrp mutant strains. Mutant strains of dcl-1 and rdrp display slower growth and increased death rate with age. sRNA mutants display compromised genome integrity and defects in cell-cycle progression.

Mutations that stall the processing or production of 21-nt sRNAs do not lead to increased transposon activity or increased target mRNA levels, but instead lead to reduced DNA copy number, potential S-phase arrest, and DNA damage. Furthermore, the 21-nt sRNAs map to the entire somatic genome and associate with chromatin. The dcl-1 cells showed reduced levels of 21-nt small RNAs and increased accumulation of 30- to 50-nt RNA. rdrp mutant lines also displayed reduced 21-nt RNA levels relative to wild-type control cells, demonstrating that sRNA production in asexually growing Oxytricha cells is dependent on both Dcl-1 and RdRP. But the sRNA reduction is partial. Comparison of 17- to 25-nt RNAs from wild-type versus mutant cells revealed reduced proportions of 21-nt RNAs mapping to 10,513 fully sequenced MAC chromosomes in dcl-141 cells. However, the sRNA mapping and sequence characteristics remain unaltered in dcl-1 or rdrp mutant cells; only their relative abundance is affected, compared to wild type. Both dcl-1 and rdrp mutants show reduced 17- to 25-nt sRNAs versus wild-type control cells, while dcl-1 cells display an overabundance of 30- to 50-nt RNAs, which are undetectable in wild-type or rdrp mutants. Rather than deriving from mRNA precursors, these sRNAs, like Oxytricha piRNAs, could be processed from long, noncoding RNAs, possibly from the same RNA molecules that establish chromosome copy number post-conjugation. In contrast to MAC-mapping sRNAs, dcl-1 cells show no reduction in the levels of MIC-mapping sRNAs. However, rdrp cells showed a small yet significant reduction in MIC-satellite and MIC gene mapping small RNAs. And they found genetically distinct classes of small RNAs in asexually growing Oxytricha cells; Dcl-1-dependent MAC-mapping 21-nt sRNAs and RdRP-dependent MIC-mapping 21- to 22-nt sRNAs. qPCR analysis of absolute DNA quantification for four representative MAC contigs shows statistically significant reduction of DNA copy number in dcl-1 and rdrp mutant strains. Mutant strains of dcl-1 and rdrp display slower growth and increased death rate with age. sRNA mutants display compromised genome integrity and defects in cell-cycle progression.

Mutations that stall the processing or production of 21-nt sRNAs do not lead to increased transposon activity or increased target mRNA levels, but instead lead to reduced DNA copy number, potential S-phase arrest, and DNA damage. Furthermore, the 21-nt sRNAs map to the entire somatic genome and associate with chromatin. The dcl-1 cells showed reduced levels of 21-nt small RNAs and increased accumulation of 30- to 50-nt RNA. rdrp mutant lines also displayed reduced 21-nt RNA levels relative to wild-type control cells, demonstrating that sRNA production in asexually growing Oxytricha cells is dependent on both Dcl-1 and RdRP. But the sRNA reduction is partial. Comparison of 17- to 25-nt RNAs from wild-type versus mutant cells revealed reduced proportions of 21-nt RNAs mapping to 10,513 fully sequenced MAC chromosomes in dcl-141 cells. However, the sRNA mapping and sequence characteristics remain unaltered in dcl-1 or rdrp mutant cells; only their relative abundance is affected, compared to wild type. Both dcl-1 and rdrp mutants show reduced 17- to 25-nt sRNAs versus wild-type control cells, while dcl-1 cells display an overabundance of 30- to 50-nt RNAs, which are undetectable in wild-type or rdrp mutants. Rather than deriving from mRNA precursors, these sRNAs, like Oxytricha piRNAs, could be processed from long, noncoding RNAs, possibly from the same RNA molecules that establish chromosome copy number post-conjugation. In contrast to MAC-mapping sRNAs, dcl-1 cells show no reduction in the levels of MIC-mapping sRNAs. However, rdrp cells showed a small yet significant reduction in MIC-satellite and MIC gene mapping small RNAs. And they found genetically distinct classes of small RNAs in asexually growing Oxytricha cells; Dcl-1-dependent MAC-mapping 21-nt sRNAs and RdRP-dependent MIC-mapping 21- to 22-nt sRNAs. qPCR analysis of absolute DNA quantification for four representative MAC contigs shows statistically significant reduction of DNA copy number in dcl-1 and rdrp mutant strains. Mutant strains of dcl-1 and rdrp display slower growth and increased death rate with age. sRNA mutants display compromised genome integrity and defects in cell-cycle progression.

Mutations that stall the processing or production of 21-nt sRNAs do not lead to increased transposon activity or increased target mRNA levels, but instead lead to reduced DNA copy number, potential S-phase arrest, and DNA damage. Furthermore, the 21-nt sRNAs map to the entire somatic genome and associate with chromatin. The dcl-1 cells showed reduced levels of 21-nt small RNAs and increased accumulation of 30- to 50-nt RNA. rdrp mutant lines also displayed reduced 21-nt RNA levels relative to wild-type control cells, demonstrating that sRNA production in asexually growing Oxytricha cells is dependent on both Dcl-1 and RdRP. But the sRNA reduction is partial. Comparison of 17- to 25-nt RNAs from wild-type versus mutant cells revealed reduced proportions of 21-nt RNAs mapping to 10,513 fully sequenced MAC chromosomes in dcl-141 cells. However, the sRNA mapping and sequence characteristics remain unaltered in dcl-1 or rdrp mutant cells; only their relative abundance is affected, compared to wild type. Both dcl-1 and rdrp mutants show reduced 17- to 25-nt sRNAs versus wild-type control cells, while dcl-1 cells display an overabundance of 30- to 50-nt RNAs, which are undetectable in wild-type or rdrp mutants. Rather than deriving from mRNA precursors, these sRNAs, like Oxytricha piRNAs, could be processed from long, noncoding RNAs, possibly from the same RNA molecules that establish chromosome copy number post-conjugation. In contrast to MAC-mapping sRNAs, dcl-1 cells show no reduction in the levels of MIC-mapping sRNAs. However, rdrp cells showed a small yet significant reduction in MIC-satellite and MIC gene mapping small RNAs. And they found genetically distinct classes of small RNAs in asexually growing Oxytricha cells; Dcl-1-dependent MAC-mapping 21-nt sRNAs and RdRP-dependent MIC-mapping 21- to 22-nt sRNAs. qPCR analysis of absolute DNA quantification for four representative MAC contigs shows statistically significant reduction of DNA copy number in dcl-1 and rdrp mutant strains. Mutant strains of dcl-1 and rdrp display slower growth and increased death rate with age. sRNA mutants display compromised genome integrity and defects in cell-cycle progression.

Meiotic progression was abnormal at 6 h post mixing in dpl2Δ cells: cells arrested at a stage prior to anaphase I with five dense chromatin masses in an orderly arrangement. The chromatin masses began to condense and by 7 h post mixing some chromatin had condensed into a single MIC. More genes were differentially expresses at later than at early time points, indicating that Dpl2 had an important role in the later stages of meiosis. Gene ontology (GO) enrichment analysis of downregulated DEGs showed that GO terms related to DNA metabolism (which included DNA replication and DNA repair) were significantly enriched at all time points post mixing. The phenotype of dpl2Δ cells was similar to that of e2fl1Δ cells. Transcriptome sequencing analysis also showed that GO enrichment profiles for downregulated DEGs were similar in dpl2Δ and e2fl1Δ cells at each time point post mixing. Dpl2 and E2fl1 interacted to form a complex during meiosis in Tetrahymena. CYC2 and TCDK3 expression was increased in both e2fl1Δ and dpl2Δ cells. Conversely, E2FL1 and DPL2 were downregulated in both cyc2Δ and tcdk3Δ cells. CYC17 was downregulated in both e2fl1Δ and dpl2Δ cells, and both E2FL1 and DPL2 were also downregulated in cyc17Δ cell. Therefore, Dpl2 complex formation with E2fl1 regulated meiosis in T. thermophila.

Meiotic progression was abnormal at 6 h post mixing in dpl2Δ cells: cells arrested at a stage prior to anaphase I with five dense chromatin masses in an orderly arrangement. The chromatin masses began to condense and by 7 h post mixing some chromatin had condensed into a single MIC. More genes were differentially expresses at later than at early time points, indicating that Dpl2 had an important role in the later stages of meiosis. Gene ontology (GO) enrichment analysis of downregulated DEGs showed that GO terms related to DNA metabolism (which included DNA replication and DNA repair) were significantly enriched at all time points post mixing. The phenotype of dpl2Δ cells was similar to that of e2fl1Δ cells. Transcriptome sequencing analysis also showed that GO enrichment profiles for downregulated DEGs were similar in dpl2Δ and e2fl1Δ cells at each time point post mixing. Dpl2 and E2fl1 interacted to form a complex during meiosis in Tetrahymena. CYC2 and TCDK3 expression was increased in both e2fl1Δ and dpl2Δ cells. Conversely, E2FL1 and DPL2 were downregulated in both cyc2Δ and tcdk3Δ cells. CYC17 was downregulated in both e2fl1Δ and dpl2Δ cells, and both E2FL1 and DPL2 were also downregulated in cyc17Δ cell. Therefore, Dpl2 complex formation with E2fl1 regulated meiosis in T. thermophila.

Meiotic progression was abnormal at 6 h post mixing in dpl2Δ cells: cells arrested at a stage prior to anaphase I with five dense chromatin masses in an orderly arrangement. The chromatin masses began to condense and by 7 h post mixing some chromatin had condensed into a single MIC. More genes were differentially expresses at later than at early time points, indicating that Dpl2 had an important role in the later stages of meiosis. Gene ontology (GO) enrichment analysis of downregulated DEGs showed that GO terms related to DNA metabolism (which included DNA replication and DNA repair) were significantly enriched at all time points post mixing. The phenotype of dpl2Δ cells was similar to that of e2fl1Δ cells. Transcriptome sequencing analysis also showed that GO enrichment profiles for downregulated DEGs were similar in dpl2Δ and e2fl1Δ cells at each time point post mixing. Dpl2 and E2fl1 interacted to form a complex during meiosis in Tetrahymena. CYC2 and TCDK3 expression was increased in both e2fl1Δ and dpl2Δ cells. Conversely, E2FL1 and DPL2 were downregulated in both cyc2Δ and tcdk3Δ cells. CYC17 was downregulated in both e2fl1Δ and dpl2Δ cells, and both E2FL1 and DPL2 were also downregulated in cyc17Δ cell. Therefore, Dpl2 complex formation with E2fl1 regulated meiosis in T. thermophila.

Meiotic progression was abnormal at 6 h post mixing in dpl2Δ cells: cells arrested at a stage prior to anaphase I with five dense chromatin masses in an orderly arrangement. The chromatin masses began to condense and by 7 h post mixing some chromatin had condensed into a single MIC. More genes were differentially expresses at later than at early time points, indicating that Dpl2 had an important role in the later stages of meiosis. Gene ontology (GO) enrichment analysis of downregulated DEGs showed that GO terms related to DNA metabolism (which included DNA replication and DNA repair) were significantly enriched at all time points post mixing. The phenotype of dpl2Δ cells was similar to that of e2fl1Δ cells. Transcriptome sequencing analysis also showed that GO enrichment profiles for downregulated DEGs were similar in dpl2Δ and e2fl1Δ cells at each time point post mixing. Dpl2 and E2fl1 interacted to form a complex during meiosis in Tetrahymena. CYC2 and TCDK3 expression was increased in both e2fl1Δ and dpl2Δ cells. Conversely, E2FL1 and DPL2 were downregulated in both cyc2Δ and tcdk3Δ cells. CYC17 was downregulated in both e2fl1Δ and dpl2Δ cells, and both E2FL1 and DPL2 were also downregulated in cyc17Δ cell. Therefore, Dpl2 complex formation with E2fl1 regulated meiosis in T. thermophila.

Meiotic progression was abnormal at 6 h post mixing in dpl2Δ cells: cells arrested at a stage prior to anaphase I with five dense chromatin masses in an orderly arrangement. The chromatin masses began to condense and by 7 h post mixing some chromatin had condensed into a single MIC. More genes were differentially expresses at later than at early time points, indicating that Dpl2 had an important role in the later stages of meiosis. Gene ontology (GO) enrichment analysis of downregulated DEGs showed that GO terms related to DNA metabolism (which included DNA replication and DNA repair) were significantly enriched at all time points post mixing. The phenotype of dpl2Δ cells was similar to that of e2fl1Δ cells. Transcriptome sequencing analysis also showed that GO enrichment profiles for downregulated DEGs were similar in dpl2Δ and e2fl1Δ cells at each time point post mixing. Dpl2 and E2fl1 interacted to form a complex during meiosis in Tetrahymena. CYC2 and TCDK3 expression was increased in both e2fl1Δ and dpl2Δ cells. Conversely, E2FL1 and DPL2 were downregulated in both cyc2Δ and tcdk3Δ cells. CYC17 was downregulated in both e2fl1Δ and dpl2Δ cells, and both E2FL1 and DPL2 were also downregulated in cyc17Δ cell. Therefore, Dpl2 complex formation with E2fl1 regulated meiosis in T. thermophila.

We identified significant alteration of global transcriptome in KO cells, with hundreds of genes being significantly up- or downregulated. TAMT-1 knockout suppresses 6mA levels in vivo. By disrupting TAMT-1, we observed a dramatic decline of 6mA levels in living cells.

We identified significant alteration of global transcriptome in KO cells, with hundreds of genes being significantly up- or downregulated. TAMT-1 knockout suppresses 6mA levels in vivo. By disrupting TAMT-1, we observed a dramatic decline of 6mA levels in living cells.

We identified significant alteration of global transcriptome in KO cells, with hundreds of genes being significantly up- or downregulated. TAMT-1 knockout suppresses 6mA levels in vivo. By disrupting TAMT-1, we observed a dramatic decline of 6mA levels in living cells.

We identified significant alteration of global transcriptome in KO cells, with hundreds of genes being significantly up- or downregulated. TAMT-1 knockout suppresses 6mA levels in vivo. By disrupting TAMT-1, we observed a dramatic decline of 6mA levels in living cells.

In the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)—many related to transposable elements (TEs)—become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. Widespread production of IES-specific polyadenylated RNA in mutants deficient in RNAi-dependent Polycomb repression. Many of the IES-specific transcripts highly induced in the mutants display mRNA hallmarks, including strand specificity, abundant and efficiently processed splice sites, poly-A tailing, and protein-coding capacity. Broad transcriptional activation of TE-related sequences in mutants deficient in RNAi-dependent Polycomb repression. RNAi-dependent Polycomb repression is required for controlling the excision and, by inference, mobilization of the Tc1 element. scnRNA production from IES-specific loci is abolished in ΔDCL1 cells. In ΔEZL1 and ΔPDD1 cells, even though global scnRNA levels were not significantly affected, IES-specific loci with strong late-scnRNA bias were preferentially depleted of scnRNA at late conjugation.

In the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)—many related to transposable elements (TEs)—become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. Widespread production of IES-specific polyadenylated RNA in mutants deficient in RNAi-dependent Polycomb repression. Many of the IES-specific transcripts highly induced in the mutants display mRNA hallmarks, including strand specificity, abundant and efficiently processed splice sites, poly-A tailing, and protein-coding capacity. Broad transcriptional activation of TE-related sequences in mutants deficient in RNAi-dependent Polycomb repression. RNAi-dependent Polycomb repression is required for controlling the excision and, by inference, mobilization of the Tc1 element. scnRNA production from IES-specific loci is abolished in ΔDCL1 cells. In ΔEZL1 and ΔPDD1 cells, even though global scnRNA levels were not significantly affected, IES-specific loci with strong late-scnRNA bias were preferentially depleted of scnRNA at late conjugation.

In the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)—many related to transposable elements (TEs)—become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. Widespread production of IES-specific polyadenylated RNA in mutants deficient in RNAi-dependent Polycomb repression. Many of the IES-specific transcripts highly induced in the mutants display mRNA hallmarks, including strand specificity, abundant and efficiently processed splice sites, poly-A tailing, and protein-coding capacity. Broad transcriptional activation of TE-related sequences in mutants deficient in RNAi-dependent Polycomb repression. RNAi-dependent Polycomb repression is required for controlling the excision and, by inference, mobilization of the Tc1 element. scnRNA production from IES-specific loci is abolished in ΔDCL1 cells. In ΔEZL1 and ΔPDD1 cells, even though global scnRNA levels were not significantly affected, IES-specific loci with strong late-scnRNA bias were preferentially depleted of scnRNA at late conjugation.

In the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)—many related to transposable elements (TEs)—become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. Widespread production of IES-specific polyadenylated RNA in mutants deficient in RNAi-dependent Polycomb repression. Many of the IES-specific transcripts highly induced in the mutants display mRNA hallmarks, including strand specificity, abundant and efficiently processed splice sites, poly-A tailing, and protein-coding capacity. Broad transcriptional activation of TE-related sequences in mutants deficient in RNAi-dependent Polycomb repression. RNAi-dependent Polycomb repression is required for controlling the excision and, by inference, mobilization of the Tc1 element. scnRNA production from IES-specific loci is abolished in ΔDCL1 cells. In ΔEZL1 and ΔPDD1 cells, even though global scnRNA levels were not significantly affected, IES-specific loci with strong late-scnRNA bias were preferentially depleted of scnRNA at late conjugation.

In the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)—many related to transposable elements (TEs)—become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. Widespread production of IES-specific polyadenylated RNA in mutants deficient in RNAi-dependent Polycomb repression. Many of the IES-specific transcripts highly induced in the mutants display mRNA hallmarks, including strand specificity, abundant and efficiently processed splice sites, poly-A tailing, and protein-coding capacity. Broad transcriptional activation of TE-related sequences in mutants deficient in RNAi-dependent Polycomb repression. RNAi-dependent Polycomb repression is required for controlling the excision and, by inference, mobilization of the Tc1 element. scnRNA production from IES-specific loci is abolished in ΔDCL1 cells. In ΔEZL1 and ΔPDD1 cells, even though global scnRNA levels were not significantly affected, IES-specific loci with strong late-scnRNA bias were preferentially depleted of scnRNA at late conjugation.

In the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)—many related to transposable elements (TEs)—become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. Widespread production of IES-specific polyadenylated RNA in mutants deficient in RNAi-dependent Polycomb repression. Many of the IES-specific transcripts highly induced in the mutants display mRNA hallmarks, including strand specificity, abundant and efficiently processed splice sites, poly-A tailing, and protein-coding capacity. Broad transcriptional activation of TE-related sequences in mutants deficient in RNAi-dependent Polycomb repression. RNAi-dependent Polycomb repression is required for controlling the excision and, by inference, mobilization of the Tc1 element. scnRNA production from IES-specific loci is abolished in ΔDCL1 cells. In ΔEZL1 and ΔPDD1 cells, even though global scnRNA levels were not significantly affected, IES-specific loci with strong late-scnRNA bias were preferentially depleted of scnRNA at late conjugation.

In the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)—many related to transposable elements (TEs)—become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. Widespread production of IES-specific polyadenylated RNA in mutants deficient in RNAi-dependent Polycomb repression. Many of the IES-specific transcripts highly induced in the mutants display mRNA hallmarks, including strand specificity, abundant and efficiently processed splice sites, poly-A tailing, and protein-coding capacity. Broad transcriptional activation of TE-related sequences in mutants deficient in RNAi-dependent Polycomb repression. RNAi-dependent Polycomb repression is required for controlling the excision and, by inference, mobilization of the Tc1 element. scnRNA production from IES-specific loci is abolished in ΔDCL1 cells. In ΔEZL1 and ΔPDD1 cells, even though global scnRNA levels were not significantly affected, IES-specific loci with strong late-scnRNA bias were preferentially depleted of scnRNA at late conjugation.

In the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)—many related to transposable elements (TEs)—become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. Widespread production of IES-specific polyadenylated RNA in mutants deficient in RNAi-dependent Polycomb repression. Many of the IES-specific transcripts highly induced in the mutants display mRNA hallmarks, including strand specificity, abundant and efficiently processed splice sites, poly-A tailing, and protein-coding capacity. Broad transcriptional activation of TE-related sequences in mutants deficient in RNAi-dependent Polycomb repression. RNAi-dependent Polycomb repression is required for controlling the excision and, by inference, mobilization of the Tc1 element. scnRNA production from IES-specific loci is abolished in ΔDCL1 cells. In ΔEZL1 and ΔPDD1 cells, even though global scnRNA levels were not significantly affected, IES-specific loci with strong late-scnRNA bias were preferentially depleted of scnRNA at late conjugation.

In the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)—many related to transposable elements (TEs)—become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. Widespread production of IES-specific polyadenylated RNA in mutants deficient in RNAi-dependent Polycomb repression. Many of the IES-specific transcripts highly induced in the mutants display mRNA hallmarks, including strand specificity, abundant and efficiently processed splice sites, poly-A tailing, and protein-coding capacity. Broad transcriptional activation of TE-related sequences in mutants deficient in RNAi-dependent Polycomb repression. RNAi-dependent Polycomb repression is required for controlling the excision and, by inference, mobilization of the Tc1 element. scnRNA production from IES-specific loci is abolished in ΔDCL1 cells. In ΔEZL1 and ΔPDD1 cells, even though global scnRNA levels were not significantly affected, IES-specific loci with strong late-scnRNA bias were preferentially depleted of scnRNA at late conjugation.

In the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)—many related to transposable elements (TEs)—become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. Widespread production of IES-specific polyadenylated RNA in mutants deficient in RNAi-dependent Polycomb repression. Many of the IES-specific transcripts highly induced in the mutants display mRNA hallmarks, including strand specificity, abundant and efficiently processed splice sites, poly-A tailing, and protein-coding capacity. Broad transcriptional activation of TE-related sequences in mutants deficient in RNAi-dependent Polycomb repression. RNAi-dependent Polycomb repression is required for controlling the excision and, by inference, mobilization of the Tc1 element. scnRNA production from IES-specific loci is abolished in ΔDCL1 cells. In ΔEZL1 and ΔPDD1 cells, even though global scnRNA levels were not significantly affected, IES-specific loci with strong late-scnRNA bias were preferentially depleted of scnRNA at late conjugation.

In the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)—many related to transposable elements (TEs)—become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. Widespread production of IES-specific polyadenylated RNA in mutants deficient in RNAi-dependent Polycomb repression. Many of the IES-specific transcripts highly induced in the mutants display mRNA hallmarks, including strand specificity, abundant and efficiently processed splice sites, poly-A tailing, and protein-coding capacity. Broad transcriptional activation of TE-related sequences in mutants deficient in RNAi-dependent Polycomb repression. RNAi-dependent Polycomb repression is required for controlling the excision and, by inference, mobilization of the Tc1 element. scnRNA production from IES-specific loci is abolished in ΔDCL1 cells. In ΔEZL1 and ΔPDD1 cells, even though global scnRNA levels were not significantly affected, IES-specific loci with strong late-scnRNA bias were preferentially depleted of scnRNA at late conjugation.

In the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)—many related to transposable elements (TEs)—become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. Widespread production of IES-specific polyadenylated RNA in mutants deficient in RNAi-dependent Polycomb repression. Many of the IES-specific transcripts highly induced in the mutants display mRNA hallmarks, including strand specificity, abundant and efficiently processed splice sites, poly-A tailing, and protein-coding capacity. Broad transcriptional activation of TE-related sequences in mutants deficient in RNAi-dependent Polycomb repression. RNAi-dependent Polycomb repression is required for controlling the excision and, by inference, mobilization of the Tc1 element. scnRNA production from IES-specific loci is abolished in ΔDCL1 cells. In ΔEZL1 and ΔPDD1 cells, even though global scnRNA levels were not significantly affected, IES-specific loci with strong late-scnRNA bias were preferentially depleted of scnRNA at late conjugation.

As a conjugation specific cyclin gene, CYC2 knockout mutants failed to form an elongated crescent structure and aborted meiosis progress in T. thermophila. γ-H2A.X staining revealed fewer micronuclear DNA double-strand breaks (DSBs) in cyc2Δ cells than in wild-type cells. Furthermore, cyc2Δ cells still failed to form a crescent structure even though DSBs were induced by exogenous agents, indicating that a lack of DSBs was not completely responsible for failure to enter the crescent stage. Tubulin staining showed that impaired perinuclear microtubule structure may contribute to the blockage in micronuclear elongation. At the same time, expression of microtubule-associated kinesin genes, KIN11 and KIN141, was significantly downregulated in cyc2Δ cells. Moreover, micronuclear specific accumulation of heterochromatin marker trimethylated H3K23 abnormally increased in the cyc2Δ mutants. Together, these results show that cyclin Cyc2p is required for micronuclear bouquet formation via controlling microtubule-directed nuclear elongation in Tetrahymena.

Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression.

Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression.

Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression.

Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression.

Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression.

Knockdown of the EZL1 gene results in loss of H3K27me3 and H3K9me3 during development, blockage of programmed DNA elimination and lethality of the sexual progeny. EZL1 knockdown results in massive transcriptional upregulation of transposable elements, with only a minor effect on protein-coding gene expression. PGM knockdown cells are blocked at a late developmental stage, after H3K9me3 and H3K27me3 are normally deposited and accumulated. ChIP-seq reads were mapped on the 61 TE consensus sequences. Consistent with qPCR data, enrichment of H3K27me3 and H3K9me3 marks with respect to the input was observed on the TE consensus sequences, while there was no enrichment of H3K4me3. Furthermore, there is a statistically significant correlation between the enrichment of H3K27me3 and the enrichment of H3K9me3. Interestingly, all TE consensus sequences showed enriched occupancy of H3K27me3 and H3K9me3, regardless of whether the elements were transcriptionally upregulated upon EZL1 knockdown or not.

Knockdown of the EZL1 gene results in loss of H3K27me3 and H3K9me3 during development, blockage of programmed DNA elimination and lethality of the sexual progeny. EZL1 knockdown results in massive transcriptional upregulation of transposable elements, with only a minor effect on protein-coding gene expression. PGM knockdown cells are blocked at a late developmental stage, after H3K9me3 and H3K27me3 are normally deposited and accumulated. ChIP-seq reads were mapped on the 61 TE consensus sequences. Consistent with qPCR data, enrichment of H3K27me3 and H3K9me3 marks with respect to the input was observed on the TE consensus sequences, while there was no enrichment of H3K4me3. Furthermore, there is a statistically significant correlation between the enrichment of H3K27me3 and the enrichment of H3K9me3. Interestingly, all TE consensus sequences showed enriched occupancy of H3K27me3 and H3K9me3, regardless of whether the elements were transcriptionally upregulated upon EZL1 knockdown or not.

Knockdown of the EZL1 gene results in loss of H3K27me3 and H3K9me3 during development, blockage of programmed DNA elimination and lethality of the sexual progeny. EZL1 knockdown results in massive transcriptional upregulation of transposable elements, with only a minor effect on protein-coding gene expression. PGM knockdown cells are blocked at a late developmental stage, after H3K9me3 and H3K27me3 are normally deposited and accumulated. ChIP-seq reads were mapped on the 61 TE consensus sequences. Consistent with qPCR data, enrichment of H3K27me3 and H3K9me3 marks with respect to the input was observed on the TE consensus sequences, while there was no enrichment of H3K4me3. Furthermore, there is a statistically significant correlation between the enrichment of H3K27me3 and the enrichment of H3K9me3. Interestingly, all TE consensus sequences showed enriched occupancy of H3K27me3 and H3K9me3, regardless of whether the elements were transcriptionally upregulated upon EZL1 knockdown or not.

Two RNA-dependent RNA Polymerase mutants, RDR1 and RDR2, show a drastic loss of siRNAs especially in phased SRCs accompanied with increased mRNA levels. Many phased SRCs depend on both, RDR1 and RDR2. RDR1 and RDR2 produce phased siRNAs from exogenous and endogenous substrates.A statistically significant reduction of siRNA reads in many SRCs is observed in both RDR mutants. The majority of differentially downregulated SRCs depend on both RDRs. Several SRCs are upregulated. This could be due to general secondary or indirect effects, because we also observed that RDR mutants induce massive transcriptomic alterations.GSRCs that overlap with downregulated SRCs (D1D2, D1, D2) are often fully covered by siRNAs and contain the largest percentage of SRCs that are phased in WT. The D1 and D2 SRCs showed a stronger antisense bias compared to other categories. D1D2 category SRCs are downregulated in both mutants, suggesting that small RNA production in these SRCs differs as well. In both RDR mutants, the sRNA fold change of phased SRCs is statistically significantly lower than unphased. This is not the case for the non phased SRCs.

Two RNA-dependent RNA Polymerase mutants, RDR1 and RDR2, show a drastic loss of siRNAs especially in phased SRCs accompanied with increased mRNA levels. Many phased SRCs depend on both, RDR1 and RDR2. RDR1 and RDR2 produce phased siRNAs from exogenous and endogenous substrates.A statistically significant reduction of siRNA reads in many SRCs is observed in both RDR mutants. The majority of differentially downregulated SRCs depend on both RDRs. Several SRCs are upregulated. This could be due to general secondary or indirect effects, because we also observed that RDR mutants induce massive transcriptomic alterations.GSRCs that overlap with downregulated SRCs (D1D2, D1, D2) are often fully covered by siRNAs and contain the largest percentage of SRCs that are phased in WT. The D1 and D2 SRCs showed a stronger antisense bias compared to other categories. D1D2 category SRCs are downregulated in both mutants, suggesting that small RNA production in these SRCs differs as well. In both RDR mutants, the sRNA fold change of phased SRCs is statistically significantly lower than unphased. This is not the case for the non phased SRCs.

Two RNA-dependent RNA Polymerase mutants, RDR1 and RDR2, show a drastic loss of siRNAs especially in phased SRCs accompanied with increased mRNA levels. Many phased SRCs depend on both, RDR1 and RDR2. RDR1 and RDR2 produce phased siRNAs from exogenous and endogenous substrates.A statistically significant reduction of siRNA reads in many SRCs is observed in both RDR mutants. The majority of differentially downregulated SRCs depend on both RDRs. Several SRCs are upregulated. This could be due to general secondary or indirect effects, because we also observed that RDR mutants induce massive transcriptomic alterations.GSRCs that overlap with downregulated SRCs (D1D2, D1, D2) are often fully covered by siRNAs and contain the largest percentage of SRCs that are phased in WT. The D1 and D2 SRCs showed a stronger antisense bias compared to other categories. D1D2 category SRCs are downregulated in both mutants, suggesting that small RNA production in these SRCs differs as well. In both RDR mutants, the sRNA fold change of phased SRCs is statistically significantly lower than unphased. This is not the case for the non phased SRCs.

Two RNA-dependent RNA Polymerase mutants, RDR1 and RDR2, show a drastic loss of siRNAs especially in phased SRCs accompanied with increased mRNA levels. Many phased SRCs depend on both, RDR1 and RDR2. RDR1 and RDR2 produce phased siRNAs from exogenous and endogenous substrates.A statistically significant reduction of siRNA reads in many SRCs is observed in both RDR mutants. The majority of differentially downregulated SRCs depend on both RDRs. Several SRCs are upregulated. This could be due to general secondary or indirect effects, because we also observed that RDR mutants induce massive transcriptomic alterations.GSRCs that overlap with downregulated SRCs (D1D2, D1, D2) are often fully covered by siRNAs and contain the largest percentage of SRCs that are phased in WT. The D1 and D2 SRCs showed a stronger antisense bias compared to other categories. D1D2 category SRCs are downregulated in both mutants, suggesting that small RNA production in these SRCs differs as well. In both RDR mutants, the sRNA fold change of phased SRCs is statistically significantly lower than unphased. This is not the case for the non phased SRCs.

AMT1 loss-of-function leads to severe defects in growth and development. Single Molecule, Real-Time (SMRT) sequencing reveals that AMT1 is required for the bulk of 6mA and all symmetric methylation at the ApT dinucleotides. The detection of hemi-methylated ApT sites suggests a semi-conservative mechanism for maintaining symmetric methylation. AMT1 affects expression of many genes; in particular, RAB46, encoding a Rab family GTPase involved in contractile vacuole function, is likely a direct target. The distribution of 6mA resembles H3K4 methylation and H2A.Z, two conserved epigenetic marks associated with RNA polymerase II transcription. Furthermore, strong 6mA and nucleosome positioning in wild-type cells is attenuated in ΔAMT1 cells.

AMT1 loss-of-function leads to severe defects in growth and development. Single Molecule, Real-Time (SMRT) sequencing reveals that AMT1 is required for the bulk of 6mA and all symmetric methylation at the ApT dinucleotides. The detection of hemi-methylated ApT sites suggests a semi-conservative mechanism for maintaining symmetric methylation. AMT1 affects expression of many genes; in particular, RAB46, encoding a Rab family GTPase involved in contractile vacuole function, is likely a direct target. The distribution of 6mA resembles H3K4 methylation and H2A.Z, two conserved epigenetic marks associated with RNA polymerase II transcription. Furthermore, strong 6mA and nucleosome positioning in wild-type cells is attenuated in ΔAMT1 cells.

Either knockout CDK19 or CYC9 prevents cells from completing co-stimulation. The absence of the CDK19 or CYC9 gene products leads to a change in expression of many genes that are presumably important for control of the sexual cycle as well.

Either knockout CDK19 or CYC9 prevents cells from completing co-stimulation. The absence of the CDK19 or CYC9 gene products leads to a change in expression of many genes that are presumably important for control of the sexual cycle as well.

Either knockout CDK19 or CYC9 prevents cells from completing co-stimulation. The absence of the CDK19 or CYC9 gene products leads to a change in expression of many genes that are presumably important for control of the sexual cycle as well.

Either knockout CDK19 or CYC9 prevents cells from completing co-stimulation. The absence of the CDK19 or CYC9 gene products leads to a change in expression of many genes that are presumably important for control of the sexual cycle as well.

We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. DCR1 feeding shows a lower amount of siRNAs compared with ND169 feeding. A statistically significant reduction (Wilcoxon test P-value < 0.05) of siRNA abundance is apparent in each feeding culture compared with the WTs without dsRNA diet. In both serotypes, the endo-siRNA accumulation pattern indicates ND169 feeding to be closer to DCR1 silencing than to WTs suggesting that ND169 silencing does not solely affect the ND169 mRNA. In serotypes 51A and 51B, the DCR1 feeding samples have 371 and 367 differentially expressed endo-siRNAs, respectively. Of them approximately 70% of the endo-siRNAs (257 in 51A and 254 in 51B) are differentially expressed in ND169 feeding as well, which suggest a common response to exo-dsRNA. Feeding of dsRNA causes de-regulation of gene expression. Similar to the analysis of siRNAs, we observe that there are large changes in the mRNA transcriptome after ND169 and DCR1 feeding. However, the ND169 feeding replicates (except one replicate in serotype 51A) are relatively closer to the WT replicates than what we observed in endo-siRNA accumulation, according to our clustering analysis. Albeit observing a rather equal amount of endo-siRNA loss in both feeding samples, the loss in mRNA is different among them, with stronger reduction of mRNA expression in DCR1 feeding. In both serotypes, DCR1 feeding has the highest and unique set of differentially expressed genes. However, approximately 30–40% of the differentially expressed genes in DCR1 are commonly found in the ND169 control feeding as well.

We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. DCR1 feeding shows a lower amount of siRNAs compared with ND169 feeding. A statistically significant reduction (Wilcoxon test P-value < 0.05) of siRNA abundance is apparent in each feeding culture compared with the WTs without dsRNA diet. In both serotypes, the endo-siRNA accumulation pattern indicates ND169 feeding to be closer to DCR1 silencing than to WTs suggesting that ND169 silencing does not solely affect the ND169 mRNA. In serotypes 51A and 51B, the DCR1 feeding samples have 371 and 367 differentially expressed endo-siRNAs, respectively. Of them approximately 70% of the endo-siRNAs (257 in 51A and 254 in 51B) are differentially expressed in ND169 feeding as well, which suggest a common response to exo-dsRNA. Feeding of dsRNA causes de-regulation of gene expression. Similar to the analysis of siRNAs, we observe that there are large changes in the mRNA transcriptome after ND169 and DCR1 feeding. However, the ND169 feeding replicates (except one replicate in serotype 51A) are relatively closer to the WT replicates than what we observed in endo-siRNA accumulation, according to our clustering analysis. Albeit observing a rather equal amount of endo-siRNA loss in both feeding samples, the loss in mRNA is different among them, with stronger reduction of mRNA expression in DCR1 feeding. In both serotypes, DCR1 feeding has the highest and unique set of differentially expressed genes. However, approximately 30–40% of the differentially expressed genes in DCR1 are commonly found in the ND169 control feeding as well.

We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. DCR1 feeding shows a lower amount of siRNAs compared with ND169 feeding. A statistically significant reduction (Wilcoxon test P-value < 0.05) of siRNA abundance is apparent in each feeding culture compared with the WTs without dsRNA diet. In both serotypes, the endo-siRNA accumulation pattern indicates ND169 feeding to be closer to DCR1 silencing than to WTs suggesting that ND169 silencing does not solely affect the ND169 mRNA. In serotypes 51A and 51B, the DCR1 feeding samples have 371 and 367 differentially expressed endo-siRNAs, respectively. Of them approximately 70% of the endo-siRNAs (257 in 51A and 254 in 51B) are differentially expressed in ND169 feeding as well, which suggest a common response to exo-dsRNA. Feeding of dsRNA causes de-regulation of gene expression. Similar to the analysis of siRNAs, we observe that there are large changes in the mRNA transcriptome after ND169 and DCR1 feeding. However, the ND169 feeding replicates (except one replicate in serotype 51A) are relatively closer to the WT replicates than what we observed in endo-siRNA accumulation, according to our clustering analysis. Albeit observing a rather equal amount of endo-siRNA loss in both feeding samples, the loss in mRNA is different among them, with stronger reduction of mRNA expression in DCR1 feeding. In both serotypes, DCR1 feeding has the highest and unique set of differentially expressed genes. However, approximately 30–40% of the differentially expressed genes in DCR1 are commonly found in the ND169 control feeding as well.

We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. DCR1 feeding shows a lower amount of siRNAs compared with ND169 feeding. A statistically significant reduction (Wilcoxon test P-value < 0.05) of siRNA abundance is apparent in each feeding culture compared with the WTs without dsRNA diet. In both serotypes, the endo-siRNA accumulation pattern indicates ND169 feeding to be closer to DCR1 silencing than to WTs suggesting that ND169 silencing does not solely affect the ND169 mRNA. In serotypes 51A and 51B, the DCR1 feeding samples have 371 and 367 differentially expressed endo-siRNAs, respectively. Of them approximately 70% of the endo-siRNAs (257 in 51A and 254 in 51B) are differentially expressed in ND169 feeding as well, which suggest a common response to exo-dsRNA. Feeding of dsRNA causes de-regulation of gene expression. Similar to the analysis of siRNAs, we observe that there are large changes in the mRNA transcriptome after ND169 and DCR1 feeding. However, the ND169 feeding replicates (except one replicate in serotype 51A) are relatively closer to the WT replicates than what we observed in endo-siRNA accumulation, according to our clustering analysis. Albeit observing a rather equal amount of endo-siRNA loss in both feeding samples, the loss in mRNA is different among them, with stronger reduction of mRNA expression in DCR1 feeding. In both serotypes, DCR1 feeding has the highest and unique set of differentially expressed genes. However, approximately 30–40% of the differentially expressed genes in DCR1 are commonly found in the ND169 control feeding as well.

We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. DCR1 feeding shows a lower amount of siRNAs compared with ND169 feeding. A statistically significant reduction (Wilcoxon test P-value < 0.05) of siRNA abundance is apparent in each feeding culture compared with the WTs without dsRNA diet. In both serotypes, the endo-siRNA accumulation pattern indicates ND169 feeding to be closer to DCR1 silencing than to WTs suggesting that ND169 silencing does not solely affect the ND169 mRNA. In serotypes 51A and 51B, the DCR1 feeding samples have 371 and 367 differentially expressed endo-siRNAs, respectively. Of them approximately 70% of the endo-siRNAs (257 in 51A and 254 in 51B) are differentially expressed in ND169 feeding as well, which suggest a common response to exo-dsRNA. Feeding of dsRNA causes de-regulation of gene expression. Similar to the analysis of siRNAs, we observe that there are large changes in the mRNA transcriptome after ND169 and DCR1 feeding. However, the ND169 feeding replicates (except one replicate in serotype 51A) are relatively closer to the WT replicates than what we observed in endo-siRNA accumulation, according to our clustering analysis. Albeit observing a rather equal amount of endo-siRNA loss in both feeding samples, the loss in mRNA is different among them, with stronger reduction of mRNA expression in DCR1 feeding. In both serotypes, DCR1 feeding has the highest and unique set of differentially expressed genes. However, approximately 30–40% of the differentially expressed genes in DCR1 are commonly found in the ND169 control feeding as well.

We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. DCR1 feeding shows a lower amount of siRNAs compared with ND169 feeding. A statistically significant reduction (Wilcoxon test P-value < 0.05) of siRNA abundance is apparent in each feeding culture compared with the WTs without dsRNA diet. In both serotypes, the endo-siRNA accumulation pattern indicates ND169 feeding to be closer to DCR1 silencing than to WTs suggesting that ND169 silencing does not solely affect the ND169 mRNA. In serotypes 51A and 51B, the DCR1 feeding samples have 371 and 367 differentially expressed endo-siRNAs, respectively. Of them approximately 70% of the endo-siRNAs (257 in 51A and 254 in 51B) are differentially expressed in ND169 feeding as well, which suggest a common response to exo-dsRNA. Feeding of dsRNA causes de-regulation of gene expression. Similar to the analysis of siRNAs, we observe that there are large changes in the mRNA transcriptome after ND169 and DCR1 feeding. However, the ND169 feeding replicates (except one replicate in serotype 51A) are relatively closer to the WT replicates than what we observed in endo-siRNA accumulation, according to our clustering analysis. Albeit observing a rather equal amount of endo-siRNA loss in both feeding samples, the loss in mRNA is different among them, with stronger reduction of mRNA expression in DCR1 feeding. In both serotypes, DCR1 feeding has the highest and unique set of differentially expressed genes. However, approximately 30–40% of the differentially expressed genes in DCR1 are commonly found in the ND169 control feeding as well.

We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. DCR1 feeding shows a lower amount of siRNAs compared with ND169 feeding. A statistically significant reduction (Wilcoxon test P-value < 0.05) of siRNA abundance is apparent in each feeding culture compared with the WTs without dsRNA diet. In both serotypes, the endo-siRNA accumulation pattern indicates ND169 feeding to be closer to DCR1 silencing than to WTs suggesting that ND169 silencing does not solely affect the ND169 mRNA. In serotypes 51A and 51B, the DCR1 feeding samples have 371 and 367 differentially expressed endo-siRNAs, respectively. Of them approximately 70% of the endo-siRNAs (257 in 51A and 254 in 51B) are differentially expressed in ND169 feeding as well, which suggest a common response to exo-dsRNA. Feeding of dsRNA causes de-regulation of gene expression. Similar to the analysis of siRNAs, we observe that there are large changes in the mRNA transcriptome after ND169 and DCR1 feeding. However, the ND169 feeding replicates (except one replicate in serotype 51A) are relatively closer to the WT replicates than what we observed in endo-siRNA accumulation, according to our clustering analysis. Albeit observing a rather equal amount of endo-siRNA loss in both feeding samples, the loss in mRNA is different among them, with stronger reduction of mRNA expression in DCR1 feeding. In both serotypes, DCR1 feeding has the highest and unique set of differentially expressed genes. However, approximately 30–40% of the differentially expressed genes in DCR1 are commonly found in the ND169 control feeding as well.

We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. DCR1 feeding shows a lower amount of siRNAs compared with ND169 feeding. A statistically significant reduction (Wilcoxon test P-value < 0.05) of siRNA abundance is apparent in each feeding culture compared with the WTs without dsRNA diet. In both serotypes, the endo-siRNA accumulation pattern indicates ND169 feeding to be closer to DCR1 silencing than to WTs suggesting that ND169 silencing does not solely affect the ND169 mRNA. In serotypes 51A and 51B, the DCR1 feeding samples have 371 and 367 differentially expressed endo-siRNAs, respectively. Of them approximately 70% of the endo-siRNAs (257 in 51A and 254 in 51B) are differentially expressed in ND169 feeding as well, which suggest a common response to exo-dsRNA. Feeding of dsRNA causes de-regulation of gene expression. Similar to the analysis of siRNAs, we observe that there are large changes in the mRNA transcriptome after ND169 and DCR1 feeding. However, the ND169 feeding replicates (except one replicate in serotype 51A) are relatively closer to the WT replicates than what we observed in endo-siRNA accumulation, according to our clustering analysis. Albeit observing a rather equal amount of endo-siRNA loss in both feeding samples, the loss in mRNA is different among them, with stronger reduction of mRNA expression in DCR1 feeding. In both serotypes, DCR1 feeding has the highest and unique set of differentially expressed genes. However, approximately 30–40% of the differentially expressed genes in DCR1 are commonly found in the ND169 control feeding as well.

We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. DCR1 feeding shows a lower amount of siRNAs compared with ND169 feeding. A statistically significant reduction (Wilcoxon test P-value < 0.05) of siRNA abundance is apparent in each feeding culture compared with the WTs without dsRNA diet. In both serotypes, the endo-siRNA accumulation pattern indicates ND169 feeding to be closer to DCR1 silencing than to WTs suggesting that ND169 silencing does not solely affect the ND169 mRNA. In serotypes 51A and 51B, the DCR1 feeding samples have 371 and 367 differentially expressed endo-siRNAs, respectively. Of them approximately 70% of the endo-siRNAs (257 in 51A and 254 in 51B) are differentially expressed in ND169 feeding as well, which suggest a common response to exo-dsRNA. Feeding of dsRNA causes de-regulation of gene expression. Similar to the analysis of siRNAs, we observe that there are large changes in the mRNA transcriptome after ND169 and DCR1 feeding. However, the ND169 feeding replicates (except one replicate in serotype 51A) are relatively closer to the WT replicates than what we observed in endo-siRNA accumulation, according to our clustering analysis. Albeit observing a rather equal amount of endo-siRNA loss in both feeding samples, the loss in mRNA is different among them, with stronger reduction of mRNA expression in DCR1 feeding. In both serotypes, DCR1 feeding has the highest and unique set of differentially expressed genes. However, approximately 30–40% of the differentially expressed genes in DCR1 are commonly found in the ND169 control feeding as well.

We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. DCR1 feeding shows a lower amount of siRNAs compared with ND169 feeding. A statistically significant reduction (Wilcoxon test P-value < 0.05) of siRNA abundance is apparent in each feeding culture compared with the WTs without dsRNA diet. In both serotypes, the endo-siRNA accumulation pattern indicates ND169 feeding to be closer to DCR1 silencing than to WTs suggesting that ND169 silencing does not solely affect the ND169 mRNA. In serotypes 51A and 51B, the DCR1 feeding samples have 371 and 367 differentially expressed endo-siRNAs, respectively. Of them approximately 70% of the endo-siRNAs (257 in 51A and 254 in 51B) are differentially expressed in ND169 feeding as well, which suggest a common response to exo-dsRNA. Feeding of dsRNA causes de-regulation of gene expression. Similar to the analysis of siRNAs, we observe that there are large changes in the mRNA transcriptome after ND169 and DCR1 feeding. However, the ND169 feeding replicates (except one replicate in serotype 51A) are relatively closer to the WT replicates than what we observed in endo-siRNA accumulation, according to our clustering analysis. Albeit observing a rather equal amount of endo-siRNA loss in both feeding samples, the loss in mRNA is different among them, with stronger reduction of mRNA expression in DCR1 feeding. In both serotypes, DCR1 feeding has the highest and unique set of differentially expressed genes. However, approximately 30–40% of the differentially expressed genes in DCR1 are commonly found in the ND169 control feeding as well.

We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. DCR1 feeding shows a lower amount of siRNAs compared with ND169 feeding. A statistically significant reduction (Wilcoxon test P-value < 0.05) of siRNA abundance is apparent in each feeding culture compared with the WTs without dsRNA diet. In both serotypes, the endo-siRNA accumulation pattern indicates ND169 feeding to be closer to DCR1 silencing than to WTs suggesting that ND169 silencing does not solely affect the ND169 mRNA. In serotypes 51A and 51B, the DCR1 feeding samples have 371 and 367 differentially expressed endo-siRNAs, respectively. Of them approximately 70% of the endo-siRNAs (257 in 51A and 254 in 51B) are differentially expressed in ND169 feeding as well, which suggest a common response to exo-dsRNA. Feeding of dsRNA causes de-regulation of gene expression. Similar to the analysis of siRNAs, we observe that there are large changes in the mRNA transcriptome after ND169 and DCR1 feeding. However, the ND169 feeding replicates (except one replicate in serotype 51A) are relatively closer to the WT replicates than what we observed in endo-siRNA accumulation, according to our clustering analysis. Albeit observing a rather equal amount of endo-siRNA loss in both feeding samples, the loss in mRNA is different among them, with stronger reduction of mRNA expression in DCR1 feeding. In both serotypes, DCR1 feeding has the highest and unique set of differentially expressed genes. However, approximately 30–40% of the differentially expressed genes in DCR1 are commonly found in the ND169 control feeding as well.

We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. DCR1 feeding shows a lower amount of siRNAs compared with ND169 feeding. A statistically significant reduction (Wilcoxon test P-value < 0.05) of siRNA abundance is apparent in each feeding culture compared with the WTs without dsRNA diet. In both serotypes, the endo-siRNA accumulation pattern indicates ND169 feeding to be closer to DCR1 silencing than to WTs suggesting that ND169 silencing does not solely affect the ND169 mRNA. In serotypes 51A and 51B, the DCR1 feeding samples have 371 and 367 differentially expressed endo-siRNAs, respectively. Of them approximately 70% of the endo-siRNAs (257 in 51A and 254 in 51B) are differentially expressed in ND169 feeding as well, which suggest a common response to exo-dsRNA. Feeding of dsRNA causes de-regulation of gene expression. Similar to the analysis of siRNAs, we observe that there are large changes in the mRNA transcriptome after ND169 and DCR1 feeding. However, the ND169 feeding replicates (except one replicate in serotype 51A) are relatively closer to the WT replicates than what we observed in endo-siRNA accumulation, according to our clustering analysis. Albeit observing a rather equal amount of endo-siRNA loss in both feeding samples, the loss in mRNA is different among them, with stronger reduction of mRNA expression in DCR1 feeding. In both serotypes, DCR1 feeding has the highest and unique set of differentially expressed genes. However, approximately 30–40% of the differentially expressed genes in DCR1 are commonly found in the ND169 control feeding as well.

Resequencing the genome upon SPT16-1 knockdown (KD) revealed that Spt16-1 is required for the elimination of all germline specific sequences. SPT16-1-KD small RNA sequencing indicated that Spt16-1 is essential for iesRNA accumulation but not for the biogenesis or selection of scanRNAs. SPT16-1 KD did not affect the deposition of H3K9me3 and H3K27me3 histone marks in the new developing MAC, suggesting that Spt16-1 acts downstream of scanRNA-directed heterochromatin formation. We further showed that Spt16-1 is required for the correct nuclear localization of Pgm in the new developing MAC but not for its expression or stability. Autogamy commenced normally in SPT16-1 KD cells, which eventually formed two new developing MACs. However, post-autogamous cells were unable to resume vegetative growth when returned to normal medium, as observed upon PGM silencing. Post-autogamous cells displayed two large macronuclei and died before the first cellular division, similar to PGM silenced cells. Programmed genome rearrangements did not proceed normally after Spt16-1 depletion. Excision of all IESs is altered and 98.6% (44,334) IESs are statistically significantly retained in the developing MAC after SPT16-1 KD, similar to the 99% (44,491) observed after PGM KD. Spt16-1, like Pgm, is essential for all DNA elimination events. In contrast to control KD, SPT16-1 KD substantially reduces iesRNA production, but does not affect the biogenesis or selection of scanRNAs. Depletion of Spt16-1 did not affect the deposition of H3K9me3 and H3K27me3 in the developing MACs. Confocal microscopy indicated that Pgm accumulated in the developing new MAC at early stages of development (T5) in control cells. In contrast, we could not detect Pgm accumulation in the developing new MAC in SPT16-1 KD cells.

Resequencing the genome upon SPT16-1 knockdown (KD) revealed that Spt16-1 is required for the elimination of all germline specific sequences. SPT16-1-KD small RNA sequencing indicated that Spt16-1 is essential for iesRNA accumulation but not for the biogenesis or selection of scanRNAs. SPT16-1 KD did not affect the deposition of H3K9me3 and H3K27me3 histone marks in the new developing MAC, suggesting that Spt16-1 acts downstream of scanRNA-directed heterochromatin formation. We further showed that Spt16-1 is required for the correct nuclear localization of Pgm in the new developing MAC but not for its expression or stability. Autogamy commenced normally in SPT16-1 KD cells, which eventually formed two new developing MACs. However, post-autogamous cells were unable to resume vegetative growth when returned to normal medium, as observed upon PGM silencing. Post-autogamous cells displayed two large macronuclei and died before the first cellular division, similar to PGM silenced cells. Programmed genome rearrangements did not proceed normally after Spt16-1 depletion. Excision of all IESs is altered and 98.6% (44,334) IESs are statistically significantly retained in the developing MAC after SPT16-1 KD, similar to the 99% (44,491) observed after PGM KD. Spt16-1, like Pgm, is essential for all DNA elimination events. In contrast to control KD, SPT16-1 KD substantially reduces iesRNA production, but does not affect the biogenesis or selection of scanRNAs. Depletion of Spt16-1 did not affect the deposition of H3K9me3 and H3K27me3 in the developing MACs. Confocal microscopy indicated that Pgm accumulated in the developing new MAC at early stages of development (T5) in control cells. In contrast, we could not detect Pgm accumulation in the developing new MAC in SPT16-1 KD cells.

Resequencing the genome upon SPT16-1 knockdown (KD) revealed that Spt16-1 is required for the elimination of all germline specific sequences. SPT16-1-KD small RNA sequencing indicated that Spt16-1 is essential for iesRNA accumulation but not for the biogenesis or selection of scanRNAs. SPT16-1 KD did not affect the deposition of H3K9me3 and H3K27me3 histone marks in the new developing MAC, suggesting that Spt16-1 acts downstream of scanRNA-directed heterochromatin formation. We further showed that Spt16-1 is required for the correct nuclear localization of Pgm in the new developing MAC but not for its expression or stability. Autogamy commenced normally in SPT16-1 KD cells, which eventually formed two new developing MACs. However, post-autogamous cells were unable to resume vegetative growth when returned to normal medium, as observed upon PGM silencing. Post-autogamous cells displayed two large macronuclei and died before the first cellular division, similar to PGM silenced cells. Programmed genome rearrangements did not proceed normally after Spt16-1 depletion. Excision of all IESs is altered and 98.6% (44,334) IESs are statistically significantly retained in the developing MAC after SPT16-1 KD, similar to the 99% (44,491) observed after PGM KD. Spt16-1, like Pgm, is essential for all DNA elimination events. In contrast to control KD, SPT16-1 KD substantially reduces iesRNA production, but does not affect the biogenesis or selection of scanRNAs. Depletion of Spt16-1 did not affect the deposition of H3K9me3 and H3K27me3 in the developing MACs. Confocal microscopy indicated that Pgm accumulated in the developing new MAC at early stages of development (T5) in control cells. In contrast, we could not detect Pgm accumulation in the developing new MAC in SPT16-1 KD cells.

Slower proliferation and increased expression of genes involved in DNA metabolism and chromosome organization and maintenance in sRNA biogenesis mutants RSP1Δ, RDN2Δ, and RDF2Δ. In addition, RSP1Δ and RDN2Δ cells frequently exhibited enlarged chromatin extrusion bodies, which are nonnuclear, DNA-containing structures that may be akin to mammalian micronuclei. Expression of homologous recombination factor Rad51 was specifically elevated in RSP1Δ and RDN2Δ strains, with Rad51 and double-stranded DNA break marker γ-H2A.X localized to discrete macronuclear foci. In addition, an increase in Rad51 and γ-H2A.X foci was also found in knockouts of TWI8, a macronucleus-localized PIWI protein. 

Slower proliferation and increased expression of genes involved in DNA metabolism and chromosome organization and maintenance in sRNA biogenesis mutants RSP1Δ, RDN2Δ, and RDF2Δ. In addition, RSP1Δ and RDN2Δ cells frequently exhibited enlarged chromatin extrusion bodies, which are nonnuclear, DNA-containing structures that may be akin to mammalian micronuclei. Expression of homologous recombination factor Rad51 was specifically elevated in RSP1Δ and RDN2Δ strains, with Rad51 and double-stranded DNA break marker γ-H2A.X localized to discrete macronuclear foci. In addition, an increase in Rad51 and γ-H2A.X foci was also found in knockouts of TWI8, a macronucleus-localized PIWI protein. 

Slower proliferation and increased expression of genes involved in DNA metabolism and chromosome organization and maintenance in sRNA biogenesis mutants RSP1Δ, RDN2Δ, and RDF2Δ. In addition, RSP1Δ and RDN2Δ cells frequently exhibited enlarged chromatin extrusion bodies, which are nonnuclear, DNA-containing structures that may be akin to mammalian micronuclei. Expression of homologous recombination factor Rad51 was specifically elevated in RSP1Δ and RDN2Δ strains, with Rad51 and double-stranded DNA break marker γ-H2A.X localized to discrete macronuclear foci. In addition, an increase in Rad51 and γ-H2A.X foci was also found in knockouts of TWI8, a macronucleus-localized PIWI protein. 

Slower proliferation and increased expression of genes involved in DNA metabolism and chromosome organization and maintenance in sRNA biogenesis mutants RSP1Δ, RDN2Δ, and RDF2Δ. In addition, RSP1Δ and RDN2Δ cells frequently exhibited enlarged chromatin extrusion bodies, which are nonnuclear, DNA-containing structures that may be akin to mammalian micronuclei. Expression of homologous recombination factor Rad51 was specifically elevated in RSP1Δ and RDN2Δ strains, with Rad51 and double-stranded DNA break marker γ-H2A.X localized to discrete macronuclear foci. In addition, an increase in Rad51 and γ-H2A.X foci was also found in knockouts of TWI8, a macronucleus-localized PIWI protein. 

Knockdown of SPO11, MSH4-1 and MSH5 is lethal and that though scnRNAs are not notably affected by the knockdowns of these genes, the effect on iesRNAs is pronounced. IESs and transposons (irrespective of whether they are maternally controlled or not) are generally affected more strongly by the knockdowns of these genes than those involved in scanRNA and iesRNA production.

Knockdown of SPO11, MSH4-1 and MSH5 is lethal and that though scnRNAs are not notably affected by the knockdowns of these genes, the effect on iesRNAs is pronounced. IESs and transposons (irrespective of whether they are maternally controlled or not) are generally affected more strongly by the knockdowns of these genes than those involved in scanRNA and iesRNA production.

Knockdown of SPO11, MSH4-1 and MSH5 is lethal and that though scnRNAs are not notably affected by the knockdowns of these genes, the effect on iesRNAs is pronounced. IESs and transposons (irrespective of whether they are maternally controlled or not) are generally affected more strongly by the knockdowns of these genes than those involved in scanRNA and iesRNA production.

Knockdown of SPO11, MSH4-1 and MSH5 is lethal and that though scnRNAs are not notably affected by the knockdowns of these genes, the effect on iesRNAs is pronounced. IESs and transposons (irrespective of whether they are maternally controlled or not) are generally affected more strongly by the knockdowns of these genes than those involved in scanRNA and iesRNA production.

Knockdown of SPO11, MSH4-1 and MSH5 is lethal and that though scnRNAs are not notably affected by the knockdowns of these genes, the effect on iesRNAs is pronounced. IESs and transposons (irrespective of whether they are maternally controlled or not) are generally affected more strongly by the knockdowns of these genes than those involved in scanRNA and iesRNA production.

Knockdown of SPO11, MSH4-1 and MSH5 is lethal and that though scnRNAs are not notably affected by the knockdowns of these genes, the effect on iesRNAs is pronounced. IESs and transposons (irrespective of whether they are maternally controlled or not) are generally affected more strongly by the knockdowns of these genes than those involved in scanRNA and iesRNA production.

Simultaneous silencing of the three SPT4 genes with high expression levels results in high lethality and only 8% of the progeny is able to produce a functional macronucleus. Depletion of Spt4 affects the sRNA biosynthesis pathway. The silencing of three SPT4 genes but not the silencing of two SPT4m autogamy-specific genes leads to a substantial reduction in scnRNA production, most probably due to a block in the synthesis of dsRNA scnRNA precursors. Spt4 contribution to the elimination of germline-specific DNA sequences. SPT4m-RNAi presents a phenotype close to the wild-type, while SPT4-RNAi leads to retention of MIC-specific sequences. The effect observed for SPT4-RNAi was comparable with results obtained for the silencing of other components of the scnRNA pathway—Dcl2/3 (DICER like proteins), and we observed significant retention of all TEs. expression of TEs is up-regulated during MAC development upon SPT5m-RNAi and the effect was comparable with de-repression observed for EZL1. On the other hand, SPT4-RNAi resulted in a very small change in TEs expression. Analysis of IES excision revealed that SPT4m-RNAi practically does not influence excision of IESs—only ~200 IESs were retained—while SPT4-RNAi causes significant retention of ~6800 IESs—nearly 15% of all IESs. All IESs affected by Spt4 are included in a larger set of those retained by SPT5m RNAi and we observe that IESs sensitive to SPT4 show higher RS in SPT5m RNAi. PIWI entrance to the MAC is disrupted upon depletion of Spt4 and Spt5. Absence of the Spt4/Spt5 proteins changes the fate of the Ptiwi09 protein – it can enter the MIC but is not transferred to the old and then to the new MAC.

Simultaneous silencing of the three SPT4 genes with high expression levels results in high lethality and only 8% of the progeny is able to produce a functional macronucleus. Depletion of Spt4 affects the sRNA biosynthesis pathway. The silencing of three SPT4 genes but not the silencing of two SPT4m autogamy-specific genes leads to a substantial reduction in scnRNA production, most probably due to a block in the synthesis of dsRNA scnRNA precursors. Spt4 contribution to the elimination of germline-specific DNA sequences. SPT4m-RNAi presents a phenotype close to the wild-type, while SPT4-RNAi leads to retention of MIC-specific sequences. The effect observed for SPT4-RNAi was comparable with results obtained for the silencing of other components of the scnRNA pathway—Dcl2/3 (DICER like proteins), and we observed significant retention of all TEs. expression of TEs is up-regulated during MAC development upon SPT5m-RNAi and the effect was comparable with de-repression observed for EZL1. On the other hand, SPT4-RNAi resulted in a very small change in TEs expression. Analysis of IES excision revealed that SPT4m-RNAi practically does not influence excision of IESs—only ~200 IESs were retained—while SPT4-RNAi causes significant retention of ~6800 IESs—nearly 15% of all IESs. All IESs affected by Spt4 are included in a larger set of those retained by SPT5m RNAi and we observe that IESs sensitive to SPT4 show higher RS in SPT5m RNAi. PIWI entrance to the MAC is disrupted upon depletion of Spt4 and Spt5. Absence of the Spt4/Spt5 proteins changes the fate of the Ptiwi09 protein – it can enter the MIC but is not transferred to the old and then to the new MAC.

Simultaneous silencing of the three SPT4 genes with high expression levels results in high lethality and only 8% of the progeny is able to produce a functional macronucleus. Depletion of Spt4 affects the sRNA biosynthesis pathway. The silencing of three SPT4 genes but not the silencing of two SPT4m autogamy-specific genes leads to a substantial reduction in scnRNA production, most probably due to a block in the synthesis of dsRNA scnRNA precursors. Spt4 contribution to the elimination of germline-specific DNA sequences. SPT4m-RNAi presents a phenotype close to the wild-type, while SPT4-RNAi leads to retention of MIC-specific sequences. The effect observed for SPT4-RNAi was comparable with results obtained for the silencing of other components of the scnRNA pathway—Dcl2/3 (DICER like proteins), and we observed significant retention of all TEs. expression of TEs is up-regulated during MAC development upon SPT5m-RNAi and the effect was comparable with de-repression observed for EZL1. On the other hand, SPT4-RNAi resulted in a very small change in TEs expression. Analysis of IES excision revealed that SPT4m-RNAi practically does not influence excision of IESs—only ~200 IESs were retained—while SPT4-RNAi causes significant retention of ~6800 IESs—nearly 15% of all IESs. All IESs affected by Spt4 are included in a larger set of those retained by SPT5m RNAi and we observe that IESs sensitive to SPT4 show higher RS in SPT5m RNAi. PIWI entrance to the MAC is disrupted upon depletion of Spt4 and Spt5. Absence of the Spt4/Spt5 proteins changes the fate of the Ptiwi09 protein – it can enter the MIC but is not transferred to the old and then to the new MAC.

Simultaneous silencing of the three SPT4 genes with high expression levels results in high lethality and only 8% of the progeny is able to produce a functional macronucleus. Depletion of Spt4 affects the sRNA biosynthesis pathway. The silencing of three SPT4 genes but not the silencing of two SPT4m autogamy-specific genes leads to a substantial reduction in scnRNA production, most probably due to a block in the synthesis of dsRNA scnRNA precursors. Spt4 contribution to the elimination of germline-specific DNA sequences. SPT4m-RNAi presents a phenotype close to the wild-type, while SPT4-RNAi leads to retention of MIC-specific sequences. The effect observed for SPT4-RNAi was comparable with results obtained for the silencing of other components of the scnRNA pathway—Dcl2/3 (DICER like proteins), and we observed significant retention of all TEs. expression of TEs is up-regulated during MAC development upon SPT5m-RNAi and the effect was comparable with de-repression observed for EZL1. On the other hand, SPT4-RNAi resulted in a very small change in TEs expression. Analysis of IES excision revealed that SPT4m-RNAi practically does not influence excision of IESs—only ~200 IESs were retained—while SPT4-RNAi causes significant retention of ~6800 IESs—nearly 15% of all IESs. All IESs affected by Spt4 are included in a larger set of those retained by SPT5m RNAi and we observe that IESs sensitive to SPT4 show higher RS in SPT5m RNAi. PIWI entrance to the MAC is disrupted upon depletion of Spt4 and Spt5. Absence of the Spt4/Spt5 proteins changes the fate of the Ptiwi09 protein – it can enter the MIC but is not transferred to the old and then to the new MAC.

Simultaneous silencing of the three SPT4 genes with high expression levels results in high lethality and only 8% of the progeny is able to produce a functional macronucleus. Depletion of Spt4 affects the sRNA biosynthesis pathway. The silencing of three SPT4 genes but not the silencing of two SPT4m autogamy-specific genes leads to a substantial reduction in scnRNA production, most probably due to a block in the synthesis of dsRNA scnRNA precursors. Spt4 contribution to the elimination of germline-specific DNA sequences. SPT4m-RNAi presents a phenotype close to the wild-type, while SPT4-RNAi leads to retention of MIC-specific sequences. The effect observed for SPT4-RNAi was comparable with results obtained for the silencing of other components of the scnRNA pathway—Dcl2/3 (DICER like proteins), and we observed significant retention of all TEs. expression of TEs is up-regulated during MAC development upon SPT5m-RNAi and the effect was comparable with de-repression observed for EZL1. On the other hand, SPT4-RNAi resulted in a very small change in TEs expression. Analysis of IES excision revealed that SPT4m-RNAi practically does not influence excision of IESs—only ~200 IESs were retained—while SPT4-RNAi causes significant retention of ~6800 IESs—nearly 15% of all IESs. All IESs affected by Spt4 are included in a larger set of those retained by SPT5m RNAi and we observe that IESs sensitive to SPT4 show higher RS in SPT5m RNAi. PIWI entrance to the MAC is disrupted upon depletion of Spt4 and Spt5. Absence of the Spt4/Spt5 proteins changes the fate of the Ptiwi09 protein – it can enter the MIC but is not transferred to the old and then to the new MAC.

Simultaneous silencing of the three SPT4 genes with high expression levels results in high lethality and only 8% of the progeny is able to produce a functional macronucleus. Depletion of Spt4 affects the sRNA biosynthesis pathway. The silencing of three SPT4 genes but not the silencing of two SPT4m autogamy-specific genes leads to a substantial reduction in scnRNA production, most probably due to a block in the synthesis of dsRNA scnRNA precursors. Spt4 contribution to the elimination of germline-specific DNA sequences. SPT4m-RNAi presents a phenotype close to the wild-type, while SPT4-RNAi leads to retention of MIC-specific sequences. The effect observed for SPT4-RNAi was comparable with results obtained for the silencing of other components of the scnRNA pathway—Dcl2/3 (DICER like proteins), and we observed significant retention of all TEs. expression of TEs is up-regulated during MAC development upon SPT5m-RNAi and the effect was comparable with de-repression observed for EZL1. On the other hand, SPT4-RNAi resulted in a very small change in TEs expression. Analysis of IES excision revealed that SPT4m-RNAi practically does not influence excision of IESs—only ~200 IESs were retained—while SPT4-RNAi causes significant retention of ~6800 IESs—nearly 15% of all IESs. All IESs affected by Spt4 are included in a larger set of those retained by SPT5m RNAi and we observe that IESs sensitive to SPT4 show higher RS in SPT5m RNAi. PIWI entrance to the MAC is disrupted upon depletion of Spt4 and Spt5. Absence of the Spt4/Spt5 proteins changes the fate of the Ptiwi09 protein – it can enter the MIC but is not transferred to the old and then to the new MAC.

Simultaneous silencing of the three SPT4 genes with high expression levels results in high lethality and only 8% of the progeny is able to produce a functional macronucleus. Depletion of Spt4 affects the sRNA biosynthesis pathway. The silencing of three SPT4 genes but not the silencing of two SPT4m autogamy-specific genes leads to a substantial reduction in scnRNA production, most probably due to a block in the synthesis of dsRNA scnRNA precursors. Spt4 contribution to the elimination of germline-specific DNA sequences. SPT4m-RNAi presents a phenotype close to the wild-type, while SPT4-RNAi leads to retention of MIC-specific sequences. The effect observed for SPT4-RNAi was comparable with results obtained for the silencing of other components of the scnRNA pathway—Dcl2/3 (DICER like proteins), and we observed significant retention of all TEs. expression of TEs is up-regulated during MAC development upon SPT5m-RNAi and the effect was comparable with de-repression observed for EZL1. On the other hand, SPT4-RNAi resulted in a very small change in TEs expression. Analysis of IES excision revealed that SPT4m-RNAi practically does not influence excision of IESs—only ~200 IESs were retained—while SPT4-RNAi causes significant retention of ~6800 IESs—nearly 15% of all IESs. All IESs affected by Spt4 are included in a larger set of those retained by SPT5m RNAi and we observe that IESs sensitive to SPT4 show higher RS in SPT5m RNAi. PIWI entrance to the MAC is disrupted upon depletion of Spt4 and Spt5. Absence of the Spt4/Spt5 proteins changes the fate of the Ptiwi09 protein – it can enter the MIC but is not transferred to the old and then to the new MAC.

Simultaneous silencing of the three SPT4 genes with high expression levels results in high lethality and only 8% of the progeny is able to produce a functional macronucleus. Depletion of Spt4 affects the sRNA biosynthesis pathway. The silencing of three SPT4 genes but not the silencing of two SPT4m autogamy-specific genes leads to a substantial reduction in scnRNA production, most probably due to a block in the synthesis of dsRNA scnRNA precursors. Spt4 contribution to the elimination of germline-specific DNA sequences. SPT4m-RNAi presents a phenotype close to the wild-type, while SPT4-RNAi leads to retention of MIC-specific sequences. The effect observed for SPT4-RNAi was comparable with results obtained for the silencing of other components of the scnRNA pathway—Dcl2/3 (DICER like proteins), and we observed significant retention of all TEs. expression of TEs is up-regulated during MAC development upon SPT5m-RNAi and the effect was comparable with de-repression observed for EZL1. On the other hand, SPT4-RNAi resulted in a very small change in TEs expression. Analysis of IES excision revealed that SPT4m-RNAi practically does not influence excision of IESs—only ~200 IESs were retained—while SPT4-RNAi causes significant retention of ~6800 IESs—nearly 15% of all IESs. All IESs affected by Spt4 are included in a larger set of those retained by SPT5m RNAi and we observe that IESs sensitive to SPT4 show higher RS in SPT5m RNAi. PIWI entrance to the MAC is disrupted upon depletion of Spt4 and Spt5. Absence of the Spt4/Spt5 proteins changes the fate of the Ptiwi09 protein – it can enter the MIC but is not transferred to the old and then to the new MAC.

Simultaneous silencing of the three SPT4 genes with high expression levels results in high lethality and only 8% of the progeny is able to produce a functional macronucleus. Depletion of Spt4 affects the sRNA biosynthesis pathway. The silencing of three SPT4 genes but not the silencing of two SPT4m autogamy-specific genes leads to a substantial reduction in scnRNA production, most probably due to a block in the synthesis of dsRNA scnRNA precursors. Spt4 contribution to the elimination of germline-specific DNA sequences. SPT4m-RNAi presents a phenotype close to the wild-type, while SPT4-RNAi leads to retention of MIC-specific sequences. The effect observed for SPT4-RNAi was comparable with results obtained for the silencing of other components of the scnRNA pathway—Dcl2/3 (DICER like proteins), and we observed significant retention of all TEs. expression of TEs is up-regulated during MAC development upon SPT5m-RNAi and the effect was comparable with de-repression observed for EZL1. On the other hand, SPT4-RNAi resulted in a very small change in TEs expression. Analysis of IES excision revealed that SPT4m-RNAi practically does not influence excision of IESs—only ~200 IESs were retained—while SPT4-RNAi causes significant retention of ~6800 IESs—nearly 15% of all IESs. All IESs affected by Spt4 are included in a larger set of those retained by SPT5m RNAi and we observe that IESs sensitive to SPT4 show higher RS in SPT5m RNAi. PIWI entrance to the MAC is disrupted upon depletion of Spt4 and Spt5. Absence of the Spt4/Spt5 proteins changes the fate of the Ptiwi09 protein – it can enter the MIC but is not transferred to the old and then to the new MAC.

Simultaneous silencing of the three SPT4 genes with high expression levels results in high lethality and only 8% of the progeny is able to produce a functional macronucleus. Depletion of Spt4 affects the sRNA biosynthesis pathway. The silencing of three SPT4 genes but not the silencing of two SPT4m autogamy-specific genes leads to a substantial reduction in scnRNA production, most probably due to a block in the synthesis of dsRNA scnRNA precursors. Spt4 contribution to the elimination of germline-specific DNA sequences. SPT4m-RNAi presents a phenotype close to the wild-type, while SPT4-RNAi leads to retention of MIC-specific sequences. The effect observed for SPT4-RNAi was comparable with results obtained for the silencing of other components of the scnRNA pathway—Dcl2/3 (DICER like proteins), and we observed significant retention of all TEs. expression of TEs is up-regulated during MAC development upon SPT5m-RNAi and the effect was comparable with de-repression observed for EZL1. On the other hand, SPT4-RNAi resulted in a very small change in TEs expression. Analysis of IES excision revealed that SPT4m-RNAi practically does not influence excision of IESs—only ~200 IESs were retained—while SPT4-RNAi causes significant retention of ~6800 IESs—nearly 15% of all IESs. All IESs affected by Spt4 are included in a larger set of those retained by SPT5m RNAi and we observe that IESs sensitive to SPT4 show higher RS in SPT5m RNAi. PIWI entrance to the MAC is disrupted upon depletion of Spt4 and Spt5. Absence of the Spt4/Spt5 proteins changes the fate of the Ptiwi09 protein – it can enter the MIC but is not transferred to the old and then to the new MAC.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Ezl1 and its interacting partners control H3K9me3/H3K27me3 accumulation and localization. KD of EZL1, CAF1, or PTIWI01/09 led to lethality of the sexual progeny, whereas KD of a control, non-essential gene did not impair survival of the post-autogamous progeny. Meanwhile, KD of EED and SUZ12.like, RF2, RF4, and EAP1 also led to lethality of the sexual progeny, whereas KD of RF1A and RF1B did not, either alone or in combination, suggesting they are not essential. We could not detect accumulation of H3K27me3 in the maternal MAC nor of H3K9me3 or H3K27me3 in the new developing MACs in EED, SUZ12.like, or RF2 KDs, as previously reported for EZL1 KD and CAF1 KD. Quantification of H3K9me3 and H3K27me3 fluorescence in the new developing MACs indicated that (1) H3K9me3 accumulation is significantly diminished upon Rf4 or Ptiwi01/09 depletion, whereas it is delayed upon Eap1 depletion; and (2) H3K27me3 accumulation is delayed upon Eap1 or Ptiwi01/09 depletion, and significantly increased upon Rf4 depletion. H3K27me3 accumulation in the fragmented maternal MAC was still observed upon depletion of Eap1 but not upon depletion of Rf4 or Ptiwi01/09. In Eap1-, Rf4-, and Ptiwi01/09-depleted cells, the H3K9me3 and H3K27me3 signals remain diffuse as development proceeds and no foci are detected. DNA elimination and TE repression require Ezl1, Caf1, Eed, Suz12.like, and Rf2. 70% of IESs are statistically significantly retained in the developing MAC after EED, SUZ12.like, or RF2 KD. Furthermore, 100% of TE copies classified as TIR, LINE, Solo-ORF, and SINE elements, which are mostly found in the MIC-limited sequences, are retained with at least 1 RPKM upon EZL1, CAF1, EED, SUZ12.like, or RF2 KD, compared with only 0.67% in the MAC control. TE copies are increasingly expressed during MAC development upon EZL1, EED, SUZ12.like, and RF2 RNAi. The core complex, Rf2, Eap1, and Rf4 are dispensable for scnRNA biogenesis. scnRNAs are produced from the MIC at very early stages of autogamy (T = 0 h). At this time point, 25-nt scnRNAs form the majority of sRNAs in the control sample, as also seen in EZL1, SUZ12.like, RF2, EAP1, and RF4 KD cells, and in contrast to the loss of scnRNAs upon depletion of the scnRNA biogenesis factors Ptiwi01/09. Eap1 and Rf4 are involved in DNA elimination and TE silencing. Depletion of Eap1 and Rf4 indicated that only 3.7% and 9.8% IESs (p value < 0.05) are retained, respectively, similar to PTIWI01/09 KD (5.6% IESs). TEs from all four of the major distinct TE families are retained in these conditions, and coverage was once again less than in EZL1 KD, with 50%, 79%, and 90% of TE copies retained with at least 1 RPKM in EAP1, RF4, and PTIWI01/09 KD, respectively. TE de-silencing in Eap1- and Rf4-depleted cells is weaker (with ~10% of all TE copies expressed >1 RPKM at T = 50 h) than in Ptiwi01/09-depleted cells. Depletion of the PRC2-Ezl1 cofactor Rf4 specifically disrupts the interaction between Ptiwi09 and Ezl1, suggesting that Rf4 bridges the PRC2-Ezl1 complex to Ptiwi09.

Silencing of Ezl1, PtCAF1, Suz12, Eed, or Rnf2 all abolish the methylation marks in both nuclei. Contrary to this, the depletion of Rnf1 does not affect the new MAC localization of either methylation mark, but the H3K27me3 staining in the maternal MAC was abolished. Depletion of any PRC2 subunit leads to a complete inability to resume vegetative growth. Each silencing leads to the retention of 60%–70% of all IESs, with both long and short IESs being affected. The depletion of the accessory subunit, Rnf1, has a very different effect on DNA elimination: much fewer IESs are retained, and the average retention score is very low. In total, 20.4% of IESs were retained, nearly all of which fall within the PtCAF1-KD affected subset. Furthermore, the IESs affected by Rnf1-KD are mainly sRNA dependent (Dcl2/3/5 dependent), although not all sRNA-dependent IESs require Rnf1. Loss of PRC2 impairs the RNA scanning process but does not affect scnRNA biogenesis. In addition, its depletion leads to a block in the iesRNA pathway. The depletion of PRC2 does not lead to a global upregulation of transcription. Interestingly, many genes were downregulated in the late time point. Moreover, we observed a significant upregulation of transcripts mapping to TEs, indicating that PRC2 is required for preventing TE expression. The nucleosome densities on IESs appear altered by PtCAF1-KD, but more notable is the greater association of higher nucleosome densities with stronger PtCAF1-KD effect on IES excision.

Silencing of Ezl1, PtCAF1, Suz12, Eed, or Rnf2 all abolish the methylation marks in both nuclei. Contrary to this, the depletion of Rnf1 does not affect the new MAC localization of either methylation mark, but the H3K27me3 staining in the maternal MAC was abolished. Depletion of any PRC2 subunit leads to a complete inability to resume vegetative growth. Each silencing leads to the retention of 60%–70% of all IESs, with both long and short IESs being affected. The depletion of the accessory subunit, Rnf1, has a very different effect on DNA elimination: much fewer IESs are retained, and the average retention score is very low. In total, 20.4% of IESs were retained, nearly all of which fall within the PtCAF1-KD affected subset. Furthermore, the IESs affected by Rnf1-KD are mainly sRNA dependent (Dcl2/3/5 dependent), although not all sRNA-dependent IESs require Rnf1. Loss of PRC2 impairs the RNA scanning process but does not affect scnRNA biogenesis. In addition, its depletion leads to a block in the iesRNA pathway. The depletion of PRC2 does not lead to a global upregulation of transcription. Interestingly, many genes were downregulated in the late time point. Moreover, we observed a significant upregulation of transcripts mapping to TEs, indicating that PRC2 is required for preventing TE expression. The nucleosome densities on IESs appear altered by PtCAF1-KD, but more notable is the greater association of higher nucleosome densities with stronger PtCAF1-KD effect on IES excision.

Silencing of Ezl1, PtCAF1, Suz12, Eed, or Rnf2 all abolish the methylation marks in both nuclei. Contrary to this, the depletion of Rnf1 does not affect the new MAC localization of either methylation mark, but the H3K27me3 staining in the maternal MAC was abolished. Depletion of any PRC2 subunit leads to a complete inability to resume vegetative growth. Each silencing leads to the retention of 60%–70% of all IESs, with both long and short IESs being affected. The depletion of the accessory subunit, Rnf1, has a very different effect on DNA elimination: much fewer IESs are retained, and the average retention score is very low. In total, 20.4% of IESs were retained, nearly all of which fall within the PtCAF1-KD affected subset. Furthermore, the IESs affected by Rnf1-KD are mainly sRNA dependent (Dcl2/3/5 dependent), although not all sRNA-dependent IESs require Rnf1. Loss of PRC2 impairs the RNA scanning process but does not affect scnRNA biogenesis. In addition, its depletion leads to a block in the iesRNA pathway. The depletion of PRC2 does not lead to a global upregulation of transcription. Interestingly, many genes were downregulated in the late time point. Moreover, we observed a significant upregulation of transcripts mapping to TEs, indicating that PRC2 is required for preventing TE expression. The nucleosome densities on IESs appear altered by PtCAF1-KD, but more notable is the greater association of higher nucleosome densities with stronger PtCAF1-KD effect on IES excision.

Silencing of Ezl1, PtCAF1, Suz12, Eed, or Rnf2 all abolish the methylation marks in both nuclei. Contrary to this, the depletion of Rnf1 does not affect the new MAC localization of either methylation mark, but the H3K27me3 staining in the maternal MAC was abolished. Depletion of any PRC2 subunit leads to a complete inability to resume vegetative growth. Each silencing leads to the retention of 60%–70% of all IESs, with both long and short IESs being affected. The depletion of the accessory subunit, Rnf1, has a very different effect on DNA elimination: much fewer IESs are retained, and the average retention score is very low. In total, 20.4% of IESs were retained, nearly all of which fall within the PtCAF1-KD affected subset. Furthermore, the IESs affected by Rnf1-KD are mainly sRNA dependent (Dcl2/3/5 dependent), although not all sRNA-dependent IESs require Rnf1. Loss of PRC2 impairs the RNA scanning process but does not affect scnRNA biogenesis. In addition, its depletion leads to a block in the iesRNA pathway. The depletion of PRC2 does not lead to a global upregulation of transcription. Interestingly, many genes were downregulated in the late time point. Moreover, we observed a significant upregulation of transcripts mapping to TEs, indicating that PRC2 is required for preventing TE expression. The nucleosome densities on IESs appear altered by PtCAF1-KD, but more notable is the greater association of higher nucleosome densities with stronger PtCAF1-KD effect on IES excision.

Silencing of Ezl1, PtCAF1, Suz12, Eed, or Rnf2 all abolish the methylation marks in both nuclei. Contrary to this, the depletion of Rnf1 does not affect the new MAC localization of either methylation mark, but the H3K27me3 staining in the maternal MAC was abolished. Depletion of any PRC2 subunit leads to a complete inability to resume vegetative growth. Each silencing leads to the retention of 60%–70% of all IESs, with both long and short IESs being affected. The depletion of the accessory subunit, Rnf1, has a very different effect on DNA elimination: much fewer IESs are retained, and the average retention score is very low. In total, 20.4% of IESs were retained, nearly all of which fall within the PtCAF1-KD affected subset. Furthermore, the IESs affected by Rnf1-KD are mainly sRNA dependent (Dcl2/3/5 dependent), although not all sRNA-dependent IESs require Rnf1. Loss of PRC2 impairs the RNA scanning process but does not affect scnRNA biogenesis. In addition, its depletion leads to a block in the iesRNA pathway. The depletion of PRC2 does not lead to a global upregulation of transcription. Interestingly, many genes were downregulated in the late time point. Moreover, we observed a significant upregulation of transcripts mapping to TEs, indicating that PRC2 is required for preventing TE expression. The nucleosome densities on IESs appear altered by PtCAF1-KD, but more notable is the greater association of higher nucleosome densities with stronger PtCAF1-KD effect on IES excision.

Silencing of Ezl1, PtCAF1, Suz12, Eed, or Rnf2 all abolish the methylation marks in both nuclei. Contrary to this, the depletion of Rnf1 does not affect the new MAC localization of either methylation mark, but the H3K27me3 staining in the maternal MAC was abolished. Depletion of any PRC2 subunit leads to a complete inability to resume vegetative growth. Each silencing leads to the retention of 60%–70% of all IESs, with both long and short IESs being affected. The depletion of the accessory subunit, Rnf1, has a very different effect on DNA elimination: much fewer IESs are retained, and the average retention score is very low. In total, 20.4% of IESs were retained, nearly all of which fall within the PtCAF1-KD affected subset. Furthermore, the IESs affected by Rnf1-KD are mainly sRNA dependent (Dcl2/3/5 dependent), although not all sRNA-dependent IESs require Rnf1. Loss of PRC2 impairs the RNA scanning process but does not affect scnRNA biogenesis. In addition, its depletion leads to a block in the iesRNA pathway. The depletion of PRC2 does not lead to a global upregulation of transcription. Interestingly, many genes were downregulated in the late time point. Moreover, we observed a significant upregulation of transcripts mapping to TEs, indicating that PRC2 is required for preventing TE expression. The nucleosome densities on IESs appear altered by PtCAF1-KD, but more notable is the greater association of higher nucleosome densities with stronger PtCAF1-KD effect on IES excision.

Silencing of Ezl1, PtCAF1, Suz12, Eed, or Rnf2 all abolish the methylation marks in both nuclei. Contrary to this, the depletion of Rnf1 does not affect the new MAC localization of either methylation mark, but the H3K27me3 staining in the maternal MAC was abolished. Depletion of any PRC2 subunit leads to a complete inability to resume vegetative growth. Each silencing leads to the retention of 60%–70% of all IESs, with both long and short IESs being affected. The depletion of the accessory subunit, Rnf1, has a very different effect on DNA elimination: much fewer IESs are retained, and the average retention score is very low. In total, 20.4% of IESs were retained, nearly all of which fall within the PtCAF1-KD affected subset. Furthermore, the IESs affected by Rnf1-KD are mainly sRNA dependent (Dcl2/3/5 dependent), although not all sRNA-dependent IESs require Rnf1. Loss of PRC2 impairs the RNA scanning process but does not affect scnRNA biogenesis. In addition, its depletion leads to a block in the iesRNA pathway. The depletion of PRC2 does not lead to a global upregulation of transcription. Interestingly, many genes were downregulated in the late time point. Moreover, we observed a significant upregulation of transcripts mapping to TEs, indicating that PRC2 is required for preventing TE expression. The nucleosome densities on IESs appear altered by PtCAF1-KD, but more notable is the greater association of higher nucleosome densities with stronger PtCAF1-KD effect on IES excision.

Silencing of Ezl1, PtCAF1, Suz12, Eed, or Rnf2 all abolish the methylation marks in both nuclei. Contrary to this, the depletion of Rnf1 does not affect the new MAC localization of either methylation mark, but the H3K27me3 staining in the maternal MAC was abolished. Depletion of any PRC2 subunit leads to a complete inability to resume vegetative growth. Each silencing leads to the retention of 60%–70% of all IESs, with both long and short IESs being affected. The depletion of the accessory subunit, Rnf1, has a very different effect on DNA elimination: much fewer IESs are retained, and the average retention score is very low. In total, 20.4% of IESs were retained, nearly all of which fall within the PtCAF1-KD affected subset. Furthermore, the IESs affected by Rnf1-KD are mainly sRNA dependent (Dcl2/3/5 dependent), although not all sRNA-dependent IESs require Rnf1. Loss of PRC2 impairs the RNA scanning process but does not affect scnRNA biogenesis. In addition, its depletion leads to a block in the iesRNA pathway. The depletion of PRC2 does not lead to a global upregulation of transcription. Interestingly, many genes were downregulated in the late time point. Moreover, we observed a significant upregulation of transcripts mapping to TEs, indicating that PRC2 is required for preventing TE expression. The nucleosome densities on IESs appear altered by PtCAF1-KD, but more notable is the greater association of higher nucleosome densities with stronger PtCAF1-KD effect on IES excision.

Silencing of Ezl1, PtCAF1, Suz12, Eed, or Rnf2 all abolish the methylation marks in both nuclei. Contrary to this, the depletion of Rnf1 does not affect the new MAC localization of either methylation mark, but the H3K27me3 staining in the maternal MAC was abolished. Depletion of any PRC2 subunit leads to a complete inability to resume vegetative growth. Each silencing leads to the retention of 60%–70% of all IESs, with both long and short IESs being affected. The depletion of the accessory subunit, Rnf1, has a very different effect on DNA elimination: much fewer IESs are retained, and the average retention score is very low. In total, 20.4% of IESs were retained, nearly all of which fall within the PtCAF1-KD affected subset. Furthermore, the IESs affected by Rnf1-KD are mainly sRNA dependent (Dcl2/3/5 dependent), although not all sRNA-dependent IESs require Rnf1. Loss of PRC2 impairs the RNA scanning process but does not affect scnRNA biogenesis. In addition, its depletion leads to a block in the iesRNA pathway. The depletion of PRC2 does not lead to a global upregulation of transcription. Interestingly, many genes were downregulated in the late time point. Moreover, we observed a significant upregulation of transcripts mapping to TEs, indicating that PRC2 is required for preventing TE expression. The nucleosome densities on IESs appear altered by PtCAF1-KD, but more notable is the greater association of higher nucleosome densities with stronger PtCAF1-KD effect on IES excision.

Knockdown of ISWI1 affects cell survival and DNA elimination. In a survival test of the post‐autogamous progeny after ISWI1‐KD over 3 days, 86% of the cells did not survive beyond the first day after cells were re‐fed and allowed to resume vegetative division. The remaining 14% of cells did not go through the usual rate of four vegetative divisions per day. For ISWI1–KD, most of the IESs we analyzed were retained. In ISWI1–KD, there was greater Sardine and Thon transposons retention, respectively, compared to the control ND7–KD. ISWI1 is required for the complete excision of most IESs. IES retention scores (IRSs) vary from 0.0 (complete IES excision) to 1.0 (complete failure of IES excision) upon knockdown. Approximately 35,000 (78%) IESs are sensitive to ISWI1–KD with a right‐skewed retention score distribution. As for most genes that influence IES excision, ISWI1–KD IES retention is length dependent. ISWI1‐KD enhances excision of IESs at alternative boundaries. In ISWI1–KD, alternative boundary excision occurs at ~65% of IESs. ISWI1–KD leads to erroneous DNA excision at the next closest available sites. Nucleosomal densities increase with IES dependence on ISWI1 and other genes involved in Paramecium IES excision. There appear to be differences in nucleosome density distributions between both ISWI1/PGM–KD and ND7/PGM–KD, and NOWA1/2/PGM–KD and EV/PGM–KD. However, these are much less pronounced than the difference in nucleosome density distributions between IESs that are more weakly and more strongly retained in knockdowns like ISWI1–KD.

Knockdown of ISWI1 affects cell survival and DNA elimination. In a survival test of the post‐autogamous progeny after ISWI1‐KD over 3 days, 86% of the cells did not survive beyond the first day after cells were re‐fed and allowed to resume vegetative division. The remaining 14% of cells did not go through the usual rate of four vegetative divisions per day. For ISWI1–KD, most of the IESs we analyzed were retained. In ISWI1–KD, there was greater Sardine and Thon transposons retention, respectively, compared to the control ND7–KD. ISWI1 is required for the complete excision of most IESs. IES retention scores (IRSs) vary from 0.0 (complete IES excision) to 1.0 (complete failure of IES excision) upon knockdown. Approximately 35,000 (78%) IESs are sensitive to ISWI1–KD with a right‐skewed retention score distribution. As for most genes that influence IES excision, ISWI1–KD IES retention is length dependent. ISWI1‐KD enhances excision of IESs at alternative boundaries. In ISWI1–KD, alternative boundary excision occurs at ~65% of IESs. ISWI1–KD leads to erroneous DNA excision at the next closest available sites. Nucleosomal densities increase with IES dependence on ISWI1 and other genes involved in Paramecium IES excision. There appear to be differences in nucleosome density distributions between both ISWI1/PGM–KD and ND7/PGM–KD, and NOWA1/2/PGM–KD and EV/PGM–KD. However, these are much less pronounced than the difference in nucleosome density distributions between IESs that are more weakly and more strongly retained in knockdowns like ISWI1–KD.

Mlp1 appears to promote the removal of 3’-trailers, as depletion of Mlp1 in vivo leads to a greater relative abundance of UUU-3’OH trailer extensions. Mlp1 depletion did not lead to changes in mature tRNA expression levels, suggesting that an Mlp1-independent tRNA maturation pathway also likely exists in T. thermophila.

Most mRNAs overlapping class I clusters are significantly upregulated in the absence of Ptiwi08, indicating a suppressive role of these sRNA. We observed an overall decrease in quantity of 23 nt sRNAs in the Dcr1-silenced sample (2.7-fold reduction), as expected if this size class is generated by Dcr1. Next, we examined the 23 nt sRNAs mapping to the class I clusters we identified in the Ptiwi08 RIP sample. We observed a 3.9-fold decrease in 23 nt sRNAs in class I SRCs in the Dcr1-silenced sample compared with the control. Calculating the 5′ to 5′ distance on opposite genomic strands revealed an increase in 21 bp overlaps (i.e., Dicer signal) from 2.3% in the control to 4.3% in the Dcr1 knockdown (KD). This increase mainly originates from cluster C1166 (cluster 22) and, to a minor extent, C909, C2056, and C2529. The remaining clusters do not display a difference in the Dicer signal strength compared with the control. Additionally, it appears that sRNAs mapping to C1166 do not display the same reduction as most of the Ptiwi08 clusters when Dcr1 is depleted. We observed an overall decrease in the quantity of 23 nt sRNAs mapping to most clusters in the Ptiwi08-KD culture yet a modest increase in Dicer signal from 2.267% in the EV control to 2.970% in the Ptiwi08 KD. Closer inspection of the clusters contributing to this signature revealed that the increase is almost entirely derived from cluster C1166 (and to a minor extent C909 and C338). we did not observe an increase in strand bias or Dicer signal strength of the sRNAs mapping to the clusters when Hen1 was depleted. On the contrary, the proportion of reads displaying a 21 bp overlap decreased from 2.022% to 0.953%, suggesting that the SRCs are in fact “more” single stranded in the absence of Hen1.

Most mRNAs overlapping class I clusters are significantly upregulated in the absence of Ptiwi08, indicating a suppressive role of these sRNA. We observed an overall decrease in quantity of 23 nt sRNAs in the Dcr1-silenced sample (2.7-fold reduction), as expected if this size class is generated by Dcr1. Next, we examined the 23 nt sRNAs mapping to the class I clusters we identified in the Ptiwi08 RIP sample. We observed a 3.9-fold decrease in 23 nt sRNAs in class I SRCs in the Dcr1-silenced sample compared with the control. Calculating the 5′ to 5′ distance on opposite genomic strands revealed an increase in 21 bp overlaps (i.e., Dicer signal) from 2.3% in the control to 4.3% in the Dcr1 knockdown (KD). This increase mainly originates from cluster C1166 (cluster 22) and, to a minor extent, C909, C2056, and C2529. The remaining clusters do not display a difference in the Dicer signal strength compared with the control. Additionally, it appears that sRNAs mapping to C1166 do not display the same reduction as most of the Ptiwi08 clusters when Dcr1 is depleted. We observed an overall decrease in the quantity of 23 nt sRNAs mapping to most clusters in the Ptiwi08-KD culture yet a modest increase in Dicer signal from 2.267% in the EV control to 2.970% in the Ptiwi08 KD. Closer inspection of the clusters contributing to this signature revealed that the increase is almost entirely derived from cluster C1166 (and to a minor extent C909 and C338). we did not observe an increase in strand bias or Dicer signal strength of the sRNAs mapping to the clusters when Hen1 was depleted. On the contrary, the proportion of reads displaying a 21 bp overlap decreased from 2.022% to 0.953%, suggesting that the SRCs are in fact “more” single stranded in the absence of Hen1.

Most mRNAs overlapping class I clusters are significantly upregulated in the absence of Ptiwi08, indicating a suppressive role of these sRNA. We observed an overall decrease in quantity of 23 nt sRNAs in the Dcr1-silenced sample (2.7-fold reduction), as expected if this size class is generated by Dcr1. Next, we examined the 23 nt sRNAs mapping to the class I clusters we identified in the Ptiwi08 RIP sample. We observed a 3.9-fold decrease in 23 nt sRNAs in class I SRCs in the Dcr1-silenced sample compared with the control. Calculating the 5′ to 5′ distance on opposite genomic strands revealed an increase in 21 bp overlaps (i.e., Dicer signal) from 2.3% in the control to 4.3% in the Dcr1 knockdown (KD). This increase mainly originates from cluster C1166 (cluster 22) and, to a minor extent, C909, C2056, and C2529. The remaining clusters do not display a difference in the Dicer signal strength compared with the control. Additionally, it appears that sRNAs mapping to C1166 do not display the same reduction as most of the Ptiwi08 clusters when Dcr1 is depleted. We observed an overall decrease in the quantity of 23 nt sRNAs mapping to most clusters in the Ptiwi08-KD culture yet a modest increase in Dicer signal from 2.267% in the EV control to 2.970% in the Ptiwi08 KD. Closer inspection of the clusters contributing to this signature revealed that the increase is almost entirely derived from cluster C1166 (and to a minor extent C909 and C338). we did not observe an increase in strand bias or Dicer signal strength of the sRNAs mapping to the clusters when Hen1 was depleted. On the contrary, the proportion of reads displaying a 21 bp overlap decreased from 2.022% to 0.953%, suggesting that the SRCs are in fact “more” single stranded in the absence of Hen1.

Most mRNAs overlapping class I clusters are significantly upregulated in the absence of Ptiwi08, indicating a suppressive role of these sRNA. We observed an overall decrease in quantity of 23 nt sRNAs in the Dcr1-silenced sample (2.7-fold reduction), as expected if this size class is generated by Dcr1. Next, we examined the 23 nt sRNAs mapping to the class I clusters we identified in the Ptiwi08 RIP sample. We observed a 3.9-fold decrease in 23 nt sRNAs in class I SRCs in the Dcr1-silenced sample compared with the control. Calculating the 5′ to 5′ distance on opposite genomic strands revealed an increase in 21 bp overlaps (i.e., Dicer signal) from 2.3% in the control to 4.3% in the Dcr1 knockdown (KD). This increase mainly originates from cluster C1166 (cluster 22) and, to a minor extent, C909, C2056, and C2529. The remaining clusters do not display a difference in the Dicer signal strength compared with the control. Additionally, it appears that sRNAs mapping to C1166 do not display the same reduction as most of the Ptiwi08 clusters when Dcr1 is depleted. We observed an overall decrease in the quantity of 23 nt sRNAs mapping to most clusters in the Ptiwi08-KD culture yet a modest increase in Dicer signal from 2.267% in the EV control to 2.970% in the Ptiwi08 KD. Closer inspection of the clusters contributing to this signature revealed that the increase is almost entirely derived from cluster C1166 (and to a minor extent C909 and C338). we did not observe an increase in strand bias or Dicer signal strength of the sRNAs mapping to the clusters when Hen1 was depleted. On the contrary, the proportion of reads displaying a 21 bp overlap decreased from 2.022% to 0.953%, suggesting that the SRCs are in fact “more” single stranded in the absence of Hen1.

Abolishing the formation of new MACs (CtIP KD) or perturbing the normal course of PGR in the new MACs (PGM, KU80c or XRCC4 KDs) induce significant variations of the P. tetraurelia transcriptome during autogamy. Upregulation of PGM mRNA in a KU80c KD results in accumulation of Pgm at late developmental stages. Deregulation of PGM expression (i) mostly takes place in the old MAC and (ii) is likely not at the protein level. RNAi-mediated silencing of either PGM or KU80c causes the retention of all IESs. DSBs introduced at IES ends in an XRCC4 KD are not repaired, resulting in the persistence of broken ends and a failure to assemble IES- junctions. 65% of IESs were retained at the T30 time-point of autogamy in an XRCC4 KD, in contrast to a control KD. As shown on western blots, we observed a strong increase in Pgm levels in an XRCC4 KD relative to control conditions. Many genes are significantly up- or downregulated at the LATE stage in a PGM, KU80c or XRCC4 KD. 628 genes are upregulated in all three KDs. mRNAs from genes involved in PGR accumulate (or persist) at LATE stages in PGM, KU80c or XRCC4 KDs as a response to defective IES excision in the new MAC. In a CtIP KD, the formation of the zygotic nucleus is inhibited and no new MAC is formed. In contrast to control conditions, we detected no expression of endogenous Pgm in a CtIP KD, whichever autogamy stage was examined. Following micro-injection of the reporter GFP transgene into the old MAC, we observed only a background GFP signal in a CtIP KD. Of note, 263 downregulated genes in a CtIP KD at EARLY or INTERMEDIATE stages are also upregulated in PGM, KU80c and XRCC4 KDs at LATE stages.

Abolishing the formation of new MACs (CtIP KD) or perturbing the normal course of PGR in the new MACs (PGM, KU80c or XRCC4 KDs) induce significant variations of the P. tetraurelia transcriptome during autogamy. Upregulation of PGM mRNA in a KU80c KD results in accumulation of Pgm at late developmental stages. Deregulation of PGM expression (i) mostly takes place in the old MAC and (ii) is likely not at the protein level. RNAi-mediated silencing of either PGM or KU80c causes the retention of all IESs. DSBs introduced at IES ends in an XRCC4 KD are not repaired, resulting in the persistence of broken ends and a failure to assemble IES- junctions. 65% of IESs were retained at the T30 time-point of autogamy in an XRCC4 KD, in contrast to a control KD. As shown on western blots, we observed a strong increase in Pgm levels in an XRCC4 KD relative to control conditions. Many genes are significantly up- or downregulated at the LATE stage in a PGM, KU80c or XRCC4 KD. 628 genes are upregulated in all three KDs. mRNAs from genes involved in PGR accumulate (or persist) at LATE stages in PGM, KU80c or XRCC4 KDs as a response to defective IES excision in the new MAC. In a CtIP KD, the formation of the zygotic nucleus is inhibited and no new MAC is formed. In contrast to control conditions, we detected no expression of endogenous Pgm in a CtIP KD, whichever autogamy stage was examined. Following micro-injection of the reporter GFP transgene into the old MAC, we observed only a background GFP signal in a CtIP KD. Of note, 263 downregulated genes in a CtIP KD at EARLY or INTERMEDIATE stages are also upregulated in PGM, KU80c and XRCC4 KDs at LATE stages.

Abolishing the formation of new MACs (CtIP KD) or perturbing the normal course of PGR in the new MACs (PGM, KU80c or XRCC4 KDs) induce significant variations of the P. tetraurelia transcriptome during autogamy. Upregulation of PGM mRNA in a KU80c KD results in accumulation of Pgm at late developmental stages. Deregulation of PGM expression (i) mostly takes place in the old MAC and (ii) is likely not at the protein level. RNAi-mediated silencing of either PGM or KU80c causes the retention of all IESs. DSBs introduced at IES ends in an XRCC4 KD are not repaired, resulting in the persistence of broken ends and a failure to assemble IES- junctions. 65% of IESs were retained at the T30 time-point of autogamy in an XRCC4 KD, in contrast to a control KD. As shown on western blots, we observed a strong increase in Pgm levels in an XRCC4 KD relative to control conditions. Many genes are significantly up- or downregulated at the LATE stage in a PGM, KU80c or XRCC4 KD. 628 genes are upregulated in all three KDs. mRNAs from genes involved in PGR accumulate (or persist) at LATE stages in PGM, KU80c or XRCC4 KDs as a response to defective IES excision in the new MAC. In a CtIP KD, the formation of the zygotic nucleus is inhibited and no new MAC is formed. In contrast to control conditions, we detected no expression of endogenous Pgm in a CtIP KD, whichever autogamy stage was examined. Following micro-injection of the reporter GFP transgene into the old MAC, we observed only a background GFP signal in a CtIP KD. Of note, 263 downregulated genes in a CtIP KD at EARLY or INTERMEDIATE stages are also upregulated in PGM, KU80c and XRCC4 KDs at LATE stages.

Abolishing the formation of new MACs (CtIP KD) or perturbing the normal course of PGR in the new MACs (PGM, KU80c or XRCC4 KDs) induce significant variations of the P. tetraurelia transcriptome during autogamy. Upregulation of PGM mRNA in a KU80c KD results in accumulation of Pgm at late developmental stages. Deregulation of PGM expression (i) mostly takes place in the old MAC and (ii) is likely not at the protein level. RNAi-mediated silencing of either PGM or KU80c causes the retention of all IESs. DSBs introduced at IES ends in an XRCC4 KD are not repaired, resulting in the persistence of broken ends and a failure to assemble IES- junctions. 65% of IESs were retained at the T30 time-point of autogamy in an XRCC4 KD, in contrast to a control KD. As shown on western blots, we observed a strong increase in Pgm levels in an XRCC4 KD relative to control conditions. Many genes are significantly up- or downregulated at the LATE stage in a PGM, KU80c or XRCC4 KD. 628 genes are upregulated in all three KDs. mRNAs from genes involved in PGR accumulate (or persist) at LATE stages in PGM, KU80c or XRCC4 KDs as a response to defective IES excision in the new MAC. In a CtIP KD, the formation of the zygotic nucleus is inhibited and no new MAC is formed. In contrast to control conditions, we detected no expression of endogenous Pgm in a CtIP KD, whichever autogamy stage was examined. Following micro-injection of the reporter GFP transgene into the old MAC, we observed only a background GFP signal in a CtIP KD. Of note, 263 downregulated genes in a CtIP KD at EARLY or INTERMEDIATE stages are also upregulated in PGM, KU80c and XRCC4 KDs at LATE stages.

We report strong correlations between the IESs affected by NOWA1/2 co-silencing (NOWA1/2-KD; KD=knockdown), DCL2/3/5 triple silencing (DCL2/3/5-KD) and TFIIS4 silencing (TFIIS4-KD), suggesting that Dcl2/3/5, Nowa1/2 and TFIIS4 are all components of an sRNA-guided DNA excision subsystem. Strong correlations also exist between PTCAF1-KD and EZL1-KD IES retention, whereas the correlations between the IES retention of these gene knockdowns and those of RNA-related genes are somewhat weaker. We also observe more IES retention due to the silencing of chromatin modifying genes (PTCAF1 and EZL1) than due to depletion of most IES-matching sRNAs by DCL2/3/5-KD, suggesting that chromatin modification due to PtCaf1 and Ezl1 also influences genome editing in an sRNA-independent manner. The correlations in OES retention following gene knockdown suggest most OESs are effectively the longest IESs.

We report strong correlations between the IESs affected by NOWA1/2 co-silencing (NOWA1/2-KD; KD=knockdown), DCL2/3/5 triple silencing (DCL2/3/5-KD) and TFIIS4 silencing (TFIIS4-KD), suggesting that Dcl2/3/5, Nowa1/2 and TFIIS4 are all components of an sRNA-guided DNA excision subsystem. Strong correlations also exist between PTCAF1-KD and EZL1-KD IES retention, whereas the correlations between the IES retention of these gene knockdowns and those of RNA-related genes are somewhat weaker. We also observe more IES retention due to the silencing of chromatin modifying genes (PTCAF1 and EZL1) than due to depletion of most IES-matching sRNAs by DCL2/3/5-KD, suggesting that chromatin modification due to PtCaf1 and Ezl1 also influences genome editing in an sRNA-independent manner. The correlations in OES retention following gene knockdown suggest most OESs are effectively the longest IESs.

We report strong correlations between the IESs affected by NOWA1/2 co-silencing (NOWA1/2-KD; KD=knockdown), DCL2/3/5 triple silencing (DCL2/3/5-KD) and TFIIS4 silencing (TFIIS4-KD), suggesting that Dcl2/3/5, Nowa1/2 and TFIIS4 are all components of an sRNA-guided DNA excision subsystem. Strong correlations also exist between PTCAF1-KD and EZL1-KD IES retention, whereas the correlations between the IES retention of these gene knockdowns and those of RNA-related genes are somewhat weaker. We also observe more IES retention due to the silencing of chromatin modifying genes (PTCAF1 and EZL1) than due to depletion of most IES-matching sRNAs by DCL2/3/5-KD, suggesting that chromatin modification due to PtCaf1 and Ezl1 also influences genome editing in an sRNA-independent manner. The correlations in OES retention following gene knockdown suggest most OESs are effectively the longest IESs.

We report strong correlations between the IESs affected by NOWA1/2 co-silencing (NOWA1/2-KD; KD=knockdown), DCL2/3/5 triple silencing (DCL2/3/5-KD) and TFIIS4 silencing (TFIIS4-KD), suggesting that Dcl2/3/5, Nowa1/2 and TFIIS4 are all components of an sRNA-guided DNA excision subsystem. Strong correlations also exist between PTCAF1-KD and EZL1-KD IES retention, whereas the correlations between the IES retention of these gene knockdowns and those of RNA-related genes are somewhat weaker. We also observe more IES retention due to the silencing of chromatin modifying genes (PTCAF1 and EZL1) than due to depletion of most IES-matching sRNAs by DCL2/3/5-KD, suggesting that chromatin modification due to PtCaf1 and Ezl1 also influences genome editing in an sRNA-independent manner. The correlations in OES retention following gene knockdown suggest most OESs are effectively the longest IESs.

We report strong correlations between the IESs affected by NOWA1/2 co-silencing (NOWA1/2-KD; KD=knockdown), DCL2/3/5 triple silencing (DCL2/3/5-KD) and TFIIS4 silencing (TFIIS4-KD), suggesting that Dcl2/3/5, Nowa1/2 and TFIIS4 are all components of an sRNA-guided DNA excision subsystem. Strong correlations also exist between PTCAF1-KD and EZL1-KD IES retention, whereas the correlations between the IES retention of these gene knockdowns and those of RNA-related genes are somewhat weaker. We also observe more IES retention due to the silencing of chromatin modifying genes (PTCAF1 and EZL1) than due to depletion of most IES-matching sRNAs by DCL2/3/5-KD, suggesting that chromatin modification due to PtCaf1 and Ezl1 also influences genome editing in an sRNA-independent manner. The correlations in OES retention following gene knockdown suggest most OESs are effectively the longest IESs.

We report strong correlations between the IESs affected by NOWA1/2 co-silencing (NOWA1/2-KD; KD=knockdown), DCL2/3/5 triple silencing (DCL2/3/5-KD) and TFIIS4 silencing (TFIIS4-KD), suggesting that Dcl2/3/5, Nowa1/2 and TFIIS4 are all components of an sRNA-guided DNA excision subsystem. Strong correlations also exist between PTCAF1-KD and EZL1-KD IES retention, whereas the correlations between the IES retention of these gene knockdowns and those of RNA-related genes are somewhat weaker. We also observe more IES retention due to the silencing of chromatin modifying genes (PTCAF1 and EZL1) than due to depletion of most IES-matching sRNAs by DCL2/3/5-KD, suggesting that chromatin modification due to PtCaf1 and Ezl1 also influences genome editing in an sRNA-independent manner. The correlations in OES retention following gene knockdown suggest most OESs are effectively the longest IESs.

Lack of SMYD1 in Tetrahymena causes mild DNA replication stress, manifested by the accumulation of DNA double strand breaks (DSBs) and single strand DNA (ssDNA), and activation of DNA damage response.