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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.

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Table summarizing results from this study.Tas3WG cells lacking Clr-C components lack H3K9me, but retain heterochromatin independent siRNAs and priRNAs. These cells cannot support recruitment of Clr-C to centromeres following reintegration of the missing Clr-C component. In contrast, Tas3WG cells lacking RNAi components retain residual H3K9me, but lack siRNAs. On reintegration of the missing RNAi component, these cells convert to allow full heterochromatin assembly on centromeric repeats. These results suggest that Clr-C can function independently of the RNAi pathway in the assembly of centromeric heterochromatin. To further test this hypothesis, H3K9me was withdrawn from RNAi deficient cells to determine whether Clr-C can target centromeres de novo independently of the RNAi pathway. Overexpression of Clr4 in clr4Δago1Δ cells supports recruitment of Clr-C activity to centromeric repeats to allow initiation of heterochromatin. These data suggest that normally RNAi-independent and RNAi-dependent mechanisms cooperate for full heterochromatin assembly.
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pgen-1001174-g009: Table summarizing results from this study.Tas3WG cells lacking Clr-C components lack H3K9me, but retain heterochromatin independent siRNAs and priRNAs. These cells cannot support recruitment of Clr-C to centromeres following reintegration of the missing Clr-C component. In contrast, Tas3WG cells lacking RNAi components retain residual H3K9me, but lack siRNAs. On reintegration of the missing RNAi component, these cells convert to allow full heterochromatin assembly on centromeric repeats. These results suggest that Clr-C can function independently of the RNAi pathway in the assembly of centromeric heterochromatin. To further test this hypothesis, H3K9me was withdrawn from RNAi deficient cells to determine whether Clr-C can target centromeres de novo independently of the RNAi pathway. Overexpression of Clr4 in clr4Δago1Δ cells supports recruitment of Clr-C activity to centromeric repeats to allow initiation of heterochromatin. These data suggest that normally RNAi-independent and RNAi-dependent mechanisms cooperate for full heterochromatin assembly.

Mentions: Our demonstration that heterochromatin can assemble following transient depletion of RNAi components in tas3WG cells is suggestive that the RNAi pathway is acting downstream of Clr-C. However, given that low levels of centromeric H3K9me2 are maintained in RNAi-defective cells, it is difficult to assess whether RNAi is required for the initial step in heterochromatin initiation. To address this question, we removed residual H3K9me2 from RNAi defective cells by introduction of the clr4Δ allele. We then tested whether H3K9me2 could be deposited at centromeres following expression of Clr4 in these cells that lack both Clr4 and Ago1 or Clr4 and Dcr1 (Figure 8A). Following overexpression of clr4+ in ago1Δclr4Δ cells, H3K9me2 could be detected on centromeric repeats above the background observed in clr4 cells, and similar to levels found normally in ago1Δ cells. Similar results were obtained following overexpression of clr4+ in dcr1Δclr4Δ cells (Figure 8B). Thus, when overexpressed, Clr4 can target centromeric repeats to initiate H3K9me2 deposition in the absence of a functional RNAi pathway. We note, however, that reintroduction of clr4+ into its normal locus in these cells is not sufficient, in the absence of the RNAi pathway, for accumulation of detectable centromeric H3K9me2 (data not shown). Together, these experiments strongly indicate that Clr-C can initiate H3K9me deposition at centromeres via RNAi-independent mechanisms, but that cooperation between RNAi-dependent and RNAi-independent factors normally results in full heterochromatin assembly (summarized in Figure 9).


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)

Table summarizing results from this study.Tas3WG cells lacking Clr-C components lack H3K9me, but retain heterochromatin independent siRNAs and priRNAs. These cells cannot support recruitment of Clr-C to centromeres following reintegration of the missing Clr-C component. In contrast, Tas3WG cells lacking RNAi components retain residual H3K9me, but lack siRNAs. On reintegration of the missing RNAi component, these cells convert to allow full heterochromatin assembly on centromeric repeats. These results suggest that Clr-C can function independently of the RNAi pathway in the assembly of centromeric heterochromatin. To further test this hypothesis, H3K9me was withdrawn from RNAi deficient cells to determine whether Clr-C can target centromeres de novo independently of the RNAi pathway. Overexpression of Clr4 in clr4Δago1Δ cells supports recruitment of Clr-C activity to centromeric repeats to allow initiation of heterochromatin. These data suggest that normally RNAi-independent and RNAi-dependent mechanisms cooperate for full heterochromatin assembly.
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Related In: Results  -  Collection

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pgen-1001174-g009: Table summarizing results from this study.Tas3WG cells lacking Clr-C components lack H3K9me, but retain heterochromatin independent siRNAs and priRNAs. These cells cannot support recruitment of Clr-C to centromeres following reintegration of the missing Clr-C component. In contrast, Tas3WG cells lacking RNAi components retain residual H3K9me, but lack siRNAs. On reintegration of the missing RNAi component, these cells convert to allow full heterochromatin assembly on centromeric repeats. These results suggest that Clr-C can function independently of the RNAi pathway in the assembly of centromeric heterochromatin. To further test this hypothesis, H3K9me was withdrawn from RNAi deficient cells to determine whether Clr-C can target centromeres de novo independently of the RNAi pathway. Overexpression of Clr4 in clr4Δago1Δ cells supports recruitment of Clr-C activity to centromeric repeats to allow initiation of heterochromatin. These data suggest that normally RNAi-independent and RNAi-dependent mechanisms cooperate for full heterochromatin assembly.
Mentions: Our demonstration that heterochromatin can assemble following transient depletion of RNAi components in tas3WG cells is suggestive that the RNAi pathway is acting downstream of Clr-C. However, given that low levels of centromeric H3K9me2 are maintained in RNAi-defective cells, it is difficult to assess whether RNAi is required for the initial step in heterochromatin initiation. To address this question, we removed residual H3K9me2 from RNAi defective cells by introduction of the clr4Δ allele. We then tested whether H3K9me2 could be deposited at centromeres following expression of Clr4 in these cells that lack both Clr4 and Ago1 or Clr4 and Dcr1 (Figure 8A). Following overexpression of clr4+ in ago1Δclr4Δ cells, H3K9me2 could be detected on centromeric repeats above the background observed in clr4 cells, and similar to levels found normally in ago1Δ cells. Similar results were obtained following overexpression of clr4+ in dcr1Δclr4Δ cells (Figure 8B). Thus, when overexpressed, Clr4 can target centromeric repeats to initiate H3K9me2 deposition in the absence of a functional RNAi pathway. We note, however, that reintroduction of clr4+ into its normal locus in these cells is not sufficient, in the absence of the RNAi pathway, for accumulation of detectable centromeric H3K9me2 (data not shown). Together, these experiments strongly indicate that Clr-C can initiate H3K9me deposition at centromeres via RNAi-independent mechanisms, but that cooperation between RNAi-dependent and RNAi-independent factors normally results in full heterochromatin assembly (summarized in Figure 9).

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