Continuous requirement for the Clr4 complex but not RNAi for centromeric heterochromatin assembly in fission yeast harboring a disrupted RITS complex.
Continuous requirement for the Clr4 complex but not RNAi for centromeric heterochromatin assembly in fission yeast harboring a disrupted RITS complex.
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DOI：
10.1371/journal.pgen.1001174
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发表时间：
2010-10-28
期刊：
影响因子：
4.5
通讯作者：
Partridge JF
中科院分区：
文献类型：
--
作者：
Shanker S;Job G;George OL;Creamer KM;Shaban A;Partridge JF
在分裂酵母中形成着丝粒异染色质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。连接RNAi途径和CLR4/SUV39H1组蛋白H3K9甲基转移酶复合体(Clr-C)的正反馈机制导致了H3K9甲基化才能产生完整的siRNA，而siRNA产生则需要实现全组蛋白甲基化。尽管如此，已经有人提出，ArgAerte蛋白Ago1是异染色质组装的关键初始触发因素，它与DICER无关的“PriRNAs”相关联。RITS复合体在物理上将Ago1和H3-K9me结合蛋白Chp1联系在一起。在这里，我们开发了一种异染色质组装的方法，在这种方法中，RNAi或Clr-C成分的缺失可以通过重新引入缺失的基因来逆转。我们先前证明了RITS复合体的一个突变版本(Tas3WG)，它在生化上将Ago1从Chp1和Tas3蛋白中分离出来，允许维持异染色质，但当CLR4被移除并重新引入时，它的形成被阻止。在这里，我们表明，阻断发生在Clr-C中的突变体，但不是RNAi途径中的突变体。因此，当RITS的完整性被破坏时，ClR-C组件，而不是RNAi因子，在组装中发挥更关键的作用。与以前的报道一致，缺乏Clr-C成分的细胞在着丝粒DNA重复序列上完全缺乏H3K9me2，而RNAi途径突变体积累了低水平的H3K9me2。CLR4+在Δago1Δ细胞中的过表达进一步支持了建立着丝粒异染色质的RNAi非依赖机制的存在，导致了一些从头开始的H3K9me2在着丝粒上的积累。这些发现以及我们的观察结果表明，ago1Δ和dcr1Δ突变体显示出难以区分的低水平H3K9me2(与以前的报告相反)，挑战了PriRNAs触发异染色质形成的模型。相反，我们的结果表明，RNAi在异染色质的组装中与RNAi不依赖的因子合作。着丝粒是细胞分裂过程中促进染色体运动的染色体区域。它们由包装成异染色质的重复DNA序列组成。着丝粒异染色质的破坏会导致染色体丢失，从而导致流产和遗传疾病。我们试图定义导致异染色质组装的精确步骤，使用分裂酵母作为模型系统。为了实现这一点，我们使用了我们的新型Tas3WG突变株，它可以繁殖预先组装的异染色质，但不能支持其从头开始建立。目前的模型表明，小RNA通过靶向RNAi机制并随后将Clr-C染色质修饰复合体靶向着丝粒来启动异染色质组装。在这里，我们证明了产生或结合小RNA的RNAi途径组件的瞬时耗尽不会扰乱我们Tas3WG菌株中的异染色质组装。相反，Clr-C复合体的瞬时耗尽阻碍了异染色质的组装，这表明在Tas3WG细胞的异染色质组装过程中，持续的Clr-C活性起着关键作用。我们已经直接测试了当Clr-C在缺乏RNAi和Clr-C的细胞中表达时，是否可以靶向着丝粒。我们发现，ClR-C的RNAi非依赖性招募可以发生，并可能有助于异染色质组装的关键启动机制。
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 (Tas3WG) 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. Centromeres are the chromosomal regions that promote chromosome movement during cell division. They consist of repetitive DNA sequences that are packaged into heterochromatin. Disruption of centromeric heterochromatin leads to chromosome loss that can result in miscarriages and genetic disorders. We have sought to define the precise steps leading to heterochromatin assembly using fission yeast as the model system. To accomplish this we employed our novel Tas3WG mutant strain that can propagate preassembled heterochromatin but cannot support its de novo establishment. Current models suggest that small RNAs initiate heterochromatin assembly by targeting the RNAi machinery and subsequently the Clr-C chromatin-modifying complex to the centromere. Here, we demonstrate that transient depletion of components of the RNAi pathway that generate or bind small RNAs does not perturb heterochromatin assembly in our Tas3WG strain. Instead, transient depletion of the Clr-C complex blocks heterochromatin assembly, suggesting a critical role for continuous Clr-C activity during heterochromatin assembly in Tas3WG cells. We have directly tested whether Clr-C can target centromeres when expressed in cells deficient for RNAi and Clr-C. We find that RNAi–independent recruitment of Clr-C can occur and likely contributes to the critical initiating mechanisms of heterochromatin assembly.
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