Limits...
Continuous requirement for the Clr4 complex but not RNAi for centromeric heterochromatin assembly in fission yeast harboring a disrupted RITS complex.

Shanker S, Job G, George OL, Creamer KM, Shaban A, Partridge JF - PLoS Genet. (2010)

Bottom Line: Positive feedback mechanisms that link the RNAi pathway and the Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in requirements for H3K9 methylation for full siRNA production and for siRNA production to achieve full histone methylation.Further supporting the existence of RNAi-independent mechanisms for establishment of centromeric heterochromatin, overexpression of clr4(+) in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at centromeres.Instead, our results indicate that RNAi cooperates with RNAi-independent factors in the assembly of heterochromatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, St Jude Children's Research Hospital, Memphis, Tennessee, United States of America.

ABSTRACT
Formation of centromeric heterochromatin in fission yeast requires the combined action of chromatin modifying enzymes and small RNAs derived from centromeric transcripts. Positive feedback mechanisms that link the RNAi pathway and the Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in requirements for H3K9 methylation for full siRNA production and for siRNA production to achieve full histone methylation. Nonetheless, it has been proposed that the Argonaute protein, Ago1, is the key initial trigger for heterochromatin assembly via its association with Dicer-independent "priRNAs." The RITS complex physically links Ago1 and the H3-K9me binding protein Chp1. Here we exploit an assay for heterochromatin assembly in which loss of silencing by deletion of RNAi or Clr-C components can be reversed by re-introduction of the deleted gene. We showed previously that a mutant version of the RITS complex (Tas3(WG)) that biochemically separates Ago1 from Chp1 and Tas3 proteins permits maintenance of heterochromatin, but prevents its formation when Clr4 is removed and re-introduced. Here we show that the block occurs with mutants in Clr-C, but not mutants in the RNAi pathway. Thus, Clr-C components, but not RNAi factors, play a more critical role in assembly when the integrity of RITS is disrupted. Consistent with previous reports, cells lacking Clr-C components completely lack H3K9me2 on centromeric DNA repeats, whereas RNAi pathway mutants accumulate low levels of H3K9me2. Further supporting the existence of RNAi-independent mechanisms for establishment of centromeric heterochromatin, overexpression of clr4(+) in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at centromeres. These findings and our observation that ago1Δ and dcr1Δ mutants display indistinguishable low levels of H3K9me2 (in contrast to a previous report) challenge the model that priRNAs trigger heterochromatin formation. Instead, our results indicate that RNAi cooperates with RNAi-independent factors in the assembly of heterochromatin.

Show MeSH

Related in: MedlinePlus

Clr-C components raf1+ and raf2+ facilitate establishment of repressive centromeric heterochromatin.A. Serial dilution assay of raf1+ reintegration strains bearing the cen::ura4+ reporter, and plated on specified media. Strains analyzed: PY2036, 3287, 3659, 3707, 3708, 3710, 3711. B. Serial dilution assay to monitor heterochromatin establishment in raf2Δ cells into which raf2+ has been reintegrated in tas3-TAP and tas3WG-TAP backgrounds. Strains analyzed: PY2036, 3675, 3676, 3781, 3783, 3791, 3792. C. Real time PCR analysis of dh centromeric transcripts relative to transcripts from adh1+. Data shown represent the mean ± SEM measurements from cDNA derived from two independent cultures, normalized to the wild type cen::ura4+ strain (PY2036). Strains analyzed: PY2036, 3287, 3659, 3707, 3710, 3711. See also Figure S1A. D. Northern blotting for dg and dh siRNAs and for the loading control SnoR69 on small RNA populations derived from indicated strains (as used in (C) and PY3708). E. Real time PCR analysis of cDNA to monitor centromeric transcript accumulation following reintegration of raf2+ into raf2Δ cells bearing the tas3-TAP and tas3WG-TAP alleles. Transcripts from dh were measured relative to the adh1+ control. Data were normalized to wild type cen::ura4+ strains, and represents mean ± SEM. Strains analyzed: PY2036, 3494, 3497, 3293, 3781, 3783, 3791, 3792. See also Figure S1B. F. Northern blot analysis of siRNAs derived from dg and dh regions of the centromere, with SnoR69 as a loading control. Strains were as listed in (E).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2965749&req=5

pgen-1001174-g002: Clr-C components raf1+ and raf2+ facilitate establishment of repressive centromeric heterochromatin.A. Serial dilution assay of raf1+ reintegration strains bearing the cen::ura4+ reporter, and plated on specified media. Strains analyzed: PY2036, 3287, 3659, 3707, 3708, 3710, 3711. B. Serial dilution assay to monitor heterochromatin establishment in raf2Δ cells into which raf2+ has been reintegrated in tas3-TAP and tas3WG-TAP backgrounds. Strains analyzed: PY2036, 3675, 3676, 3781, 3783, 3791, 3792. C. Real time PCR analysis of dh centromeric transcripts relative to transcripts from adh1+. Data shown represent the mean ± SEM measurements from cDNA derived from two independent cultures, normalized to the wild type cen::ura4+ strain (PY2036). Strains analyzed: PY2036, 3287, 3659, 3707, 3710, 3711. See also Figure S1A. D. Northern blotting for dg and dh siRNAs and for the loading control SnoR69 on small RNA populations derived from indicated strains (as used in (C) and PY3708). E. Real time PCR analysis of cDNA to monitor centromeric transcript accumulation following reintegration of raf2+ into raf2Δ cells bearing the tas3-TAP and tas3WG-TAP alleles. Transcripts from dh were measured relative to the adh1+ control. Data were normalized to wild type cen::ura4+ strains, and represents mean ± SEM. Strains analyzed: PY2036, 3494, 3497, 3293, 3781, 3783, 3791, 3792. See also Figure S1B. F. Northern blot analysis of siRNAs derived from dg and dh regions of the centromere, with SnoR69 as a loading control. Strains were as listed in (E).

Mentions: raf1Δ and raf2Δ were crossed into tas3-TAP and tas3WG -TAP backgrounds, and wild type genomic copies of raf1+ or raf2+ were reintegrated into the corresponding deletion mutants and assessed for heterochromatin assembly. As seen for transient depletion experiments with rik1, tas3-TAP cells efficiently re-assembled centromeric heterochromatin on reintroduction of raf1+ or raf2+, whereas silencing of the cen::ura4+ reporter was not apparent in tas3WG-TAP backgrounds (Figure 2A and 2B). Centromeric transcripts accumulate to high levels in raf1 and raf2 deleted cells, and although transcript levels drop following reintegration of raf1+ into raf1Δ tas3-TAP cells or of raf2+ into raf2Δ tas3-TAP cells, high levels of dg and dh transcripts are maintained in both raf1+ and raf2+ reconstituted tas3WG-TAP cells (Figure 2C and 2E, Figure S1A and S1B). Consistent with this failure to suppress high levels of centromeric transcription in tas3WG-TAP cells transiently depleted for raf1+ or raf2+, these cells fail to engage the RNAi pathway to promote destruction of centromeric transcripts into siRNAs (Figure 2D and 2F).


Continuous requirement for the Clr4 complex but not RNAi for centromeric heterochromatin assembly in fission yeast harboring a disrupted RITS complex.

Shanker S, Job G, George OL, Creamer KM, Shaban A, Partridge JF - PLoS Genet. (2010)

Clr-C components raf1+ and raf2+ facilitate establishment of repressive centromeric heterochromatin.A. Serial dilution assay of raf1+ reintegration strains bearing the cen::ura4+ reporter, and plated on specified media. Strains analyzed: PY2036, 3287, 3659, 3707, 3708, 3710, 3711. B. Serial dilution assay to monitor heterochromatin establishment in raf2Δ cells into which raf2+ has been reintegrated in tas3-TAP and tas3WG-TAP backgrounds. Strains analyzed: PY2036, 3675, 3676, 3781, 3783, 3791, 3792. C. Real time PCR analysis of dh centromeric transcripts relative to transcripts from adh1+. Data shown represent the mean ± SEM measurements from cDNA derived from two independent cultures, normalized to the wild type cen::ura4+ strain (PY2036). Strains analyzed: PY2036, 3287, 3659, 3707, 3710, 3711. See also Figure S1A. D. Northern blotting for dg and dh siRNAs and for the loading control SnoR69 on small RNA populations derived from indicated strains (as used in (C) and PY3708). E. Real time PCR analysis of cDNA to monitor centromeric transcript accumulation following reintegration of raf2+ into raf2Δ cells bearing the tas3-TAP and tas3WG-TAP alleles. Transcripts from dh were measured relative to the adh1+ control. Data were normalized to wild type cen::ura4+ strains, and represents mean ± SEM. Strains analyzed: PY2036, 3494, 3497, 3293, 3781, 3783, 3791, 3792. See also Figure S1B. F. Northern blot analysis of siRNAs derived from dg and dh regions of the centromere, with SnoR69 as a loading control. Strains were as listed in (E).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2965749&req=5

pgen-1001174-g002: Clr-C components raf1+ and raf2+ facilitate establishment of repressive centromeric heterochromatin.A. Serial dilution assay of raf1+ reintegration strains bearing the cen::ura4+ reporter, and plated on specified media. Strains analyzed: PY2036, 3287, 3659, 3707, 3708, 3710, 3711. B. Serial dilution assay to monitor heterochromatin establishment in raf2Δ cells into which raf2+ has been reintegrated in tas3-TAP and tas3WG-TAP backgrounds. Strains analyzed: PY2036, 3675, 3676, 3781, 3783, 3791, 3792. C. Real time PCR analysis of dh centromeric transcripts relative to transcripts from adh1+. Data shown represent the mean ± SEM measurements from cDNA derived from two independent cultures, normalized to the wild type cen::ura4+ strain (PY2036). Strains analyzed: PY2036, 3287, 3659, 3707, 3710, 3711. See also Figure S1A. D. Northern blotting for dg and dh siRNAs and for the loading control SnoR69 on small RNA populations derived from indicated strains (as used in (C) and PY3708). E. Real time PCR analysis of cDNA to monitor centromeric transcript accumulation following reintegration of raf2+ into raf2Δ cells bearing the tas3-TAP and tas3WG-TAP alleles. Transcripts from dh were measured relative to the adh1+ control. Data were normalized to wild type cen::ura4+ strains, and represents mean ± SEM. Strains analyzed: PY2036, 3494, 3497, 3293, 3781, 3783, 3791, 3792. See also Figure S1B. F. Northern blot analysis of siRNAs derived from dg and dh regions of the centromere, with SnoR69 as a loading control. Strains were as listed in (E).
Mentions: raf1Δ and raf2Δ were crossed into tas3-TAP and tas3WG -TAP backgrounds, and wild type genomic copies of raf1+ or raf2+ were reintegrated into the corresponding deletion mutants and assessed for heterochromatin assembly. As seen for transient depletion experiments with rik1, tas3-TAP cells efficiently re-assembled centromeric heterochromatin on reintroduction of raf1+ or raf2+, whereas silencing of the cen::ura4+ reporter was not apparent in tas3WG-TAP backgrounds (Figure 2A and 2B). Centromeric transcripts accumulate to high levels in raf1 and raf2 deleted cells, and although transcript levels drop following reintegration of raf1+ into raf1Δ tas3-TAP cells or of raf2+ into raf2Δ tas3-TAP cells, high levels of dg and dh transcripts are maintained in both raf1+ and raf2+ reconstituted tas3WG-TAP cells (Figure 2C and 2E, Figure S1A and S1B). Consistent with this failure to suppress high levels of centromeric transcription in tas3WG-TAP cells transiently depleted for raf1+ or raf2+, these cells fail to engage the RNAi pathway to promote destruction of centromeric transcripts into siRNAs (Figure 2D and 2F).

Bottom Line: Positive feedback mechanisms that link the RNAi pathway and the Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in requirements for H3K9 methylation for full siRNA production and for siRNA production to achieve full histone methylation.Further supporting the existence of RNAi-independent mechanisms for establishment of centromeric heterochromatin, overexpression of clr4(+) in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at centromeres.Instead, our results indicate that RNAi cooperates with RNAi-independent factors in the assembly of heterochromatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, St Jude Children's Research Hospital, Memphis, Tennessee, United States of America.

ABSTRACT
Formation of centromeric heterochromatin in fission yeast requires the combined action of chromatin modifying enzymes and small RNAs derived from centromeric transcripts. Positive feedback mechanisms that link the RNAi pathway and the Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in requirements for H3K9 methylation for full siRNA production and for siRNA production to achieve full histone methylation. Nonetheless, it has been proposed that the Argonaute protein, Ago1, is the key initial trigger for heterochromatin assembly via its association with Dicer-independent "priRNAs." The RITS complex physically links Ago1 and the H3-K9me binding protein Chp1. Here we exploit an assay for heterochromatin assembly in which loss of silencing by deletion of RNAi or Clr-C components can be reversed by re-introduction of the deleted gene. We showed previously that a mutant version of the RITS complex (Tas3(WG)) that biochemically separates Ago1 from Chp1 and Tas3 proteins permits maintenance of heterochromatin, but prevents its formation when Clr4 is removed and re-introduced. Here we show that the block occurs with mutants in Clr-C, but not mutants in the RNAi pathway. Thus, Clr-C components, but not RNAi factors, play a more critical role in assembly when the integrity of RITS is disrupted. Consistent with previous reports, cells lacking Clr-C components completely lack H3K9me2 on centromeric DNA repeats, whereas RNAi pathway mutants accumulate low levels of H3K9me2. Further supporting the existence of RNAi-independent mechanisms for establishment of centromeric heterochromatin, overexpression of clr4(+) in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at centromeres. These findings and our observation that ago1Δ and dcr1Δ mutants display indistinguishable low levels of H3K9me2 (in contrast to a previous report) challenge the model that priRNAs trigger heterochromatin formation. Instead, our results indicate that RNAi cooperates with RNAi-independent factors in the assembly of heterochromatin.

Show MeSH
Related in: MedlinePlus