Department of Biochemistry, St Jude Children's Research Hospital, Memphis, Tennessee, United States of America.
PLoS Genet. 2010 Oct 28;6(10):e1001174. doi: 10.1371/journal.pgen.1001174.
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.
裂殖酵母着丝粒异染色质的形成需要染色质修饰酶和来自着丝粒转录本的小 RNA 的共同作用。RNAi 途径和 Clr4/Suv39h1 组蛋白 H3K9 甲基转移酶复合物（Clr-C）之间的正反馈机制导致 RNAi 途径和 Clr-C 复合物需要 H3K9 甲基化才能完全产生 siRNA，并需要 siRNA 产生才能实现组蛋白甲基化完全。尽管如此，有人提出 Argonaute 蛋白 Ago1 通过与其与 Dicer 无关的“priRNAs”的结合，成为异染色质组装的关键初始触发因素。RITS 复合物将 Ago1 与 H3-K9me 结合蛋白 Chp1 物理连接起来。在这里，我们利用一种异染色质组装测定法，其中通过删除 RNAi 或 Clr-C 成分而导致的沉默丧失可以通过重新引入缺失的基因来逆转。我们之前曾表明，RITS 复合物的突变体（Tas3(WG)）在生化上使 Ago1 与 Chp1 和 Tas3 蛋白分离，允许维持异染色质，但在 Clr4 被删除并重新引入时阻止其形成。在这里，我们表明该阻断发生在 Clr-C 突变体中，而不是在 RNAi 途径突变体中。因此，当 RITS 的完整性被破坏时，Clr-C 成分而不是 RNAi 因子在组装中发挥更关键的作用。与之前的报道一致，缺乏 Clr-C 成分的细胞完全缺乏着丝粒 DNA 重复序列上的 H3K9me2，而 RNAi 途径突变体积累低水平的 H3K9me2。进一步支持存在 RNAi 独立机制来建立着丝粒异染色质，在 clr4Δago1Δ 细胞中过量表达 clr4(+) 导致着丝粒处有一些新的 H3K9me2 积累。这些发现以及我们观察到 ago1Δ 和 dcr1Δ 突变体显示出相同的低水平 H3K9me2（与之前的报告相反），这对 priRNAs 触发异染色质形成的模型提出了挑战。相反，我们的结果表明，RNAi 与 RNAi 独立因子在异染色质的组装中协同作用。
