RNAi and Epigenetic Control of Higher-Order Chromatin Assembly

我们实验室的研究重点是高阶染色质组装的表观遗传控制。高阶染色体结构的动态调节控制着多种细胞过程，从基因表达模式的稳定遗传到维持基因组完整性所必需的全球染色体结构的其他方面。我们早期的研究揭示了导致裂殖酵母异色结构组装的分子事件序列。我们发现，组蛋白尾部的脱乙酰酶和甲基转移酶活性的共价修饰共同作用于建立组蛋白密码，这是组装异色结构所必需的。此外，我们还表明，不同的部位特异性组蛋白H3甲基化模式决定了染色体的组织成离散的结构和功能区域。组蛋白H3在赖氨酸9处甲基化，严格定位于沉默的异染色质区域，而H3在赖氨酸4处甲基化，仅有几个氨基酸残基，专用于周围活跃的常染色质区域。我们继续关注组蛋白修饰的作用以及识别特定组蛋白修饰模式的因子(例如，在赖氨酸9处甲基化的与组蛋白H3特异结合的染色域蛋白Swi6)，并在理解高阶染色质组装的机制方面取得了重大进展。更重要的是，我们提供的证据表明，RNA干扰(RNAi)，即双链RNAs沉默同源基因，在将异染色质复合体靶向到基因组中的特定位置方面发挥着关键作用。我们最近的工作导致了一种自我实施环机制的发现，通过这种机制，RNAi机制作为异染色质结构域的稳定组成部分(通过RNAi复合体与异染色质标记的连接)来破坏逃脱异染色质介导的转录沉默的重复转录。在这个环机制中，RNAi机制对转录本的处理产生了小干扰RNAs(SiRNAs)，这些小干扰RNAs被用于进一步靶向异染色质复合体，因此该机制仍在继续。在一项全面的研究中，我们还开发了整个分裂酵母基因组中异染色质和常染色质分布的高分辨率图谱。这些分析，加上RNAi成分的定位和与参与异色沉默的RNAi效应复合体相关联的siRNAs的大规模测序，为这一模式真核生物基因组的表观遗传学特征提供了新的见解。我们的工作使我们认识到，异染色质是基因组的一个动态平台，参与招募与不同染色体过程有关的各种调节蛋白(效应器)。在其他因素中，这些效应物包括与组蛋白和核小体重塑蛋白(如我们实验室最近描述的SHREC复合体)脱乙酰化有关的因子，它们促进高阶染色质结构的组装。RNAi、异染色质和染色质修饰因子之间的联系在包括哺乳动物在内的高等真核生物中是保守的，对人类生物学和包括癌症在内的疾病具有广泛的意义。我们还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。反转录转座子能够对宿主产生不同的影响，深刻地影响宿主基因组和宿主转录组的组织、完整性和进化。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。CENP-B还抑制散布在分裂酵母基因组中的反转录转座子残留物，这些残留物通常位于基因启动子附近，对基因的表达产生影响。令人惊讶的是，分散在整个基因组中的逆转录元件聚集成专门的机构，其组织依赖于CENP-B。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，对宿主基因组组织和维持基因组完整性很重要。

Research in our laboratory is focused on the epigenetic control of higher-order chromatin assembly. The dynamic regulation of higher-order chromosome structure governs diverse cellular processes ranging from stable inheritance of gene expression patterns to other aspects of global chromosome structure essential for preserving genomic integrity. Our earlier studies revealed sequence of molecular events leading to the assembly of heterochromatic structures in the fission yeast Schizosaccharomyces pombe. We found that covalent modifications of histone tails by deacetylase and methyltransferase activities act in concert to establish the histone code essential for the assembly of heterochromatic structures. Moreover, we showed that distinct site-specific histone H3 methylation patterns dictate the organization of chromosomes into discrete structural and functional domains. Histone H3 methylated at lysine 9 is strictly localized to silent heterochromatic regions whereas H3 methylated at lysine 4, only a few amino acids away, is specific to the surrounding active euchromatic regions. We have continued to focus on the role of histone modifications and the factors that recognize specific histone modifications patterns (such as a chromodomain protein Swi6 that specifically binds histone H3 methylated at lysine 9) in the assembly of higher-order chromatin structures and have made significant progress in understanding the mechanism of higher-order chromatin assembly. More importantly, we provided evidence showing that RNA interference (RNAi), whereby double-stranded RNAs silence cognate genes, plays a critical role in targeting of heterochromatin complexes to specific locations in the genome. Our recent work has led to discovery of a self-enforcing loop mechanism though which RNAi machinery operates as a stable component of the heterochromatic domains (via tethering of RNAi complexes to heterochromatin marks) to destroy repeat transcripts that escape heterochromatin-mediated transcriptional silencing. In this loop mechanism, the processing of transcripts by RNAi machinery generate small interfering RNAs (siRNAs) that are utilized for further targeting of heterochromatin complexes, so the mechanism continues. In a comprehensive study, we have also developed a high-resolution map of the heterochromatin and euchromatin distribution across the entire fission yeast genome. These analyses together with mapping of RNAi components and large scale sequencing of siRNAs assocaited with an RNAi effector complex involved in heterochromatic silencing have yielded novel insights into the epigenetic profile of this model eukaryotic genome. Our work led to realization the heterochromatin serves as a dynamic platform of the genome involved in recruitment of diverse regulatory proteins (effectors) implicated in different chromosomal processes. Among other factors, these effectors include factors involved in deacetylation of histones and nucleosome remodeling proteins (such as SHREC complex described recently by our lab), which facilitate assembly of higher-order chromatin structures. The link between RNAi, heterochromatin, and chromatin-modifying factors is conserved in higher eukaryotes including mammals and has broad implications for human biology and disease including cancer. We have also uncovered a novel genome surveillance mechanism for retrotransposons by a family of transposase-derived CENP-B homologs. Retrotransposons are capable of exerting diverse effects on their hosts, profoundly influencing the organization, integrity and evolution of the host genome, and the host transcriptome. We discovered that CENP-Bs localize at and recruit histone deacetylases to silence retrotransposons. CENP-Bs also repress retrotransposon relics scattered throughout the fission yeast genome and often located near gene promoters to exert influence on expression of genes. Surprisingly, retroelements dispersed throughout the genome are clustered into specialized bodies, the organization of which depends on CENP-Bs. 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 important for host genome organization and maintenance of genomic integrity.