Roles of Chromatin-modifying Factors in Epigenetic Control of the Genome

我们在S.pombe上的研究表明，在异染色质沉默缺陷突变体的遗传筛选中发现的几个因素(如Clr3、Clr4和Clr6)参与了组蛋白尾部的修饰。其中，CLR4属于高度保守的组蛋白甲基转移酶Suv39家族，其特异性地使组蛋白H3在赖氨酸9(H3-K9)上甲基化，跨越与重复DNA元件相关的异染色域。生化分析表明，CLR4是多亚基复合体的一个组成部分，它包含一个cullin家族蛋白CUL4，它作为组装泛素连接酶的支架，以及一个WD蛋白Rik1，它通过RNA聚合酶II转录耦合过程将CLR4的活性重新聚集到靶重复基因座上。Clr3和Clr6是组蛋白十乙基化酶，与人类的第二类和第一类HDAC有很强的同源性。我们已经证明，Clr6至少存在于两个不同的核心络合物中。其中一个复合体(Clr6-C1)主要针对基因启动子，负责通过组蛋白的局部去乙酰化来调节基因的表达。第二个Clr6复合体(Clr6-CII)针对转录的染色体区域和着丝粒基因，负责组蛋白的整体脱乙酰化。我们的分析表明，Clr6-CII的缺陷破坏了染色质的整体保护功能，如抑制反义转录、链特异性抑制异染色质重复序列和保护DNA免受遗传毒性物质的损伤。我们还对Clr3进行了生化表征。Clr3存在于一种称为SHREC的多酶效应复合体中，除了与Clr3相关的组蛋白十乙基酶活性外，它还含有一个Snf2家族的染色质重塑因子Mit1。我们已经证明，SHREC的靶点是所有主要的异染色质结构域，它的活性对于正确定位核小体以组装高阶染色质结构是必不可少的，这对异染色质功能至关重要。我们正在继续研究这些和其他组蛋白修饰活动的功能。鉴于组蛋白修饰物在物种之间是保守的，并控制着基本的染色体过程，包括在发育过程中保持基因表达模式的稳定和保持基因组的完整性，因此对它们的深入了解对于开发治疗癌症和其他人类疾病的有效治疗措施非常重要。异染色质在特定位置成核的方式取决于组蛋白十乙基酶、异染色质蛋白的活性以及CLR4通过其染色域与甲基化的H3-K9以及与甲基化的H3尾巴结合的能力。此外，H3-K9的甲基化对于Swi6、Chp2和Chp1等HP1蛋白的招募是必不可少的。我们的研究揭示了一个新的主题，即与甲基化的H3-K9结合的HP1蛋白为参与许多细胞过程的因子提供了一个动态平台，包括参与细胞类型转换和染色体适当分离的蛋白质。Chp1是RITS复合体的一个组成部分，它将RNAi机制拴在异染色质基因座上，促进顺式基因中重复序列的转录后沉默。然而，Chp2和Swi6在异染色质组装中的确切功能以及它们与其他因素的关系尚不清楚。我们最近发现Swi6和Chp2与Clr6和SHREC组蛋白脱乙酰酶复合体有关，这两个复合体对异色着丝粒重复序列的转录沉默至关重要。这项工作进一步揭示了Swi6和Chp2蛋白及其相关的HDAC复合体在限制RNA聚合酶II跨着丝粒异染色质结构域占据方面具有重叠的功能。有趣的是，纯化的Swi6组分还含有参与多种染色体过程的因子，如染色质重塑和DNA复制。此外，Swi6共纯化了姐妹染色单体凝聚所必需的粘附素加载因子，并与着丝粒特异性组蛋白H3变体CENP-A共同纯化，CENP-A以异染色质依赖的方式整合到染色质中。这些分析表明，HP1蛋白与多种因素有关，包括对抑制染色质组装至关重要的组蛋白修饰因子。识别HP1相关因子及其在染色质组装中的作用可能有助于我们理解与HP1表达变化相关的乳腺癌的原因。尽管HP1蛋白对于组蛋白脱乙酰酶在异染色质结构域中重复序列的优先招募至关重要，但存在另一种机制来靶向分散在基因组中的重复序列。具体地说，我们发现了一种通过转座酶衍生的CENP-B同源物家族对逆转录转座子进行基因组监测的新机制。我们发现CENP-B定位于并募集组蛋白脱乙酰酶来沉默反转录转座子。这一机制也抑制了散布在S.pombe基因组中的反转录转座子残留物。CENP-B介导的监测是主动的，能够防止已灭绝的反转录转座子重新进入宿主基因组。这些结果揭示了一个可能的古老的反转录转座子监视途径，并表明真核细胞拥有一套抑制活性的工具箱，这些抑制活性要么通过HP1蛋白跨大区域靶向，要么被CENP-B和其他DNA结合因子以位点特异性的方式靶向。

The involvement of histone modifications in higher-order chromatin assembly has been highlighted by our studies in S. pombe showing that several factors identified in genetic screen for mutants defective in heterochromatic silencing (such as such as Clr3, Clr4 and Clr6) are involved in modifications of histone tails. Among these, Clr4 belongs to a highly conserved Suv39 family of histone methyltransferases, which specifically methylate histone H3 at lysine 9 (H3-K9) across heterochromatic domains associated with repetitive DNA elements. Biochemical analysis has shown that Clr4 is a component of multisubunit complex containing a cullin family protein Cul4 that serves as scaffold to assemble ubiquitin ligases, and a WD protein Rik1 which mediates recruitment of Clr4 activity to the target repeat loci via a RNA polymerase II transcription coupled process. Clr3 and Clr6 are histone decaetylases with strong homologies to class II and class I HDACs from humans. We have shown that Clr6 exists in at least two distinct core complexes. One of these complexes (Clr6-C1) predominantly targets gene promoters and is responsible for regulation of gene expression through local deacetylation of histones. The second Clr6 complex (Clr6-CII) that targets transcribed chromosomal regions and centromeric loci is responsible for global deacetylation of histones. Our analyses suggest that defects in Clr6-CII abrogate global protective functions of chromatin such as suppression of antisense transcripts, strand-specific repression of heterochromatic repeats and protection of DNA from damage by genotoxic agents. We have also performed biochemical characterization of Clr3. Clr3 exists in a multienzyme effector complex termed SHREC that in addition to histone decaetylase activity associated with Clr3 contains a Snf2 family chromatin remodeling factor Mit1. We have shown that SHREC is targeted across all major heterochromatic domains and its activities are essential for proper positioning of nucleosomes to assemble higher-order chromatin structures, critical for heterochromatin functions. We are continuing to investigate the functions of these and other histone modifying activities. Given that histone modifiers are conserved among species and control fundamental chromosomal processes including stable maintenance of gene expression patterns during development and maintenance of genomic integrity, their deeper understanding is important for the development of effective therapeutic measures for treatment of cancer and other human diseases. Heterochromatin nucleated at specific sites spread in a manner that depends upon the activities of histone decaetylases, heterochromatin proteins and the ability of Clr4 to both methylate H3-K9 as well as bind to methylated H3 tail via its chromodomain. Moreover, methylation of H3-K9 is essential for recruitment of HP1 proteins such as Swi6, Chp2 and Chp1. Our research has unraveled a new theme wherein HP1 proteins bound to methylated H3-K9 provide a dynamic platform for factors involved in many cellular processes, including proteins involved in cell-type switching and proper segregation of chromosomes. Chp1, a component of the RITS complex tethers RNAi machinery to heterochromatic loci, facilitating post-transcriptional silencing of repeats in cis. However, the exact functions of Chp2 and Swi6 in heterochromatin assembly and their associations with other factors were poorly understood. We recently showed that Swi6 and Chp2 associate with Clr6 and SHREC histone deacetylase complexes, which are critical for transcriptional silencing of the heterochromatic centromeric repeats. This work further revealed that Swi6 and Chp2 proteins and their associated HDAC complexes have overlapping functions in limiting RNA polymerase II occupancy across pericentromeric heterochromatin domains. Interestingly, purified Swi6 fraction also contains factors involved in a variety of chromosomal processes such as chromatin remodeling and DNA replication. In addition, Swi6 co-purifies a cohesin loading factor essential for sister chromatid cohesion, and with centromere-specific histone H3 variant CENP-A, which is incorporated into chromatin in a heterochromatin-dependent manner. These analyses suggest that HP1 proteins associate with a variety of factors including histone-modifying factors essential for the assembly of repressive chromatin. Identifications of HP1 associated factors and their role in chromatin assembly may help us understand the causes of breast cancer associated with altered HP1 expression. Although HP1 proteins are critical for the preferential recruitment of histone deacetylases to repeat elements within heterochromatin domains, alternative mechanisms exist to target these activities to repeats dispersed across the genome. Specifically, we have uncovered a novel genome surveillance mechanism for retrotransposons by a family of transposase-derived CENP-B homologs. We found that CENP-Bs localize at and recruit histone deacetylases to silence retrotransposons. This mechanism also represses retrotransposon relics scattered throughout the S. pombe genome. CENP-B-mediated surveillance is proactive, capable of preventing an extinct retrotransposon from reentering the host genome. These results reveal a likely ancient retrotransposon surveillance pathway and suggest that eukaryotic cells have a toolkit of repressor activities that are either targeted across large domains via HP1 proteins or in a site-specific manner by CENP-B and other DNA binding factors.