Reconstituting heterochromatin and gene silencing in vivo

项目总结 异染色质在维持基因组稳定和转录基因沉默(TGS)中起着关键作用。它 已经变得越来越清楚，影响异染色质完整性的通路的错误调节会导致或 导致许多人类疾病，包括许多癌症。的组成和功能 异染色质结构域在裂殖酵母Pombe-to中很大程度上是保守的 人类和其他后生动物。异染色质的建立、表观遗传维持和TGS 生物体是受众多染色质相关因子调控的复杂过程。尽管如此 复杂性，异染色质生物学的核心原理已经被提出，但仍然是推测的。本研究 将通过提取异染色质的基本特征来实验测试和阐明这些核心原理 一个高度受控和垂直的环境。本研究方案由两个目标组成。目标1是 组蛋白3赖氨酸9甲基化(H3K9me)依赖的异染色质在酵母中的重组 天然缺乏H3K9Me的酿酒细胞，其目标是定义对 抑制和可遗传的染色质状态从分裂酵母到人类都是保守的。成功地建设了一个 酿酒酵母细胞中H3K9me依赖的异染色质结构域将提供一个独特的系统 研究异染色质是如何表观遗传的，以及它是如何沉默转录和限制其他 DNA交易。因此，目标2是利用体内重组的依赖于H3K9me的异染色质来 探讨TGS的作用机制。依赖H3K9me的异染色质将在酿酒酵母中重组 通过将庞氏葡萄球菌和人类异染色质相关蛋白依次募集到特定的S. 酿酒酵母基因组基因座。然后，这个最小的异染色质结构域将被用来测试三种模型 TGS，并评估组蛋白去乙酰化和核小体重构在沉默形成中的作用 染色质结构域。合成生物学、下一代测序和基于蛋白质组学的组合 将利用实验方法来执行这项研究计划。这项工作有可能改变我们的 了解异染色质形成的机制，从而为未来旨在逆转的研究提供信息 人类疾病潜在的异染色质相关过程中的缺陷。此外，重组 体内异染色质结构域与庞氏葡萄球菌和人类细胞中发现的因子的结合将有助于 适合于高通量筛选小分子调制的基于细胞的分析的发展 参与异染色质形成和疾病进展的蛋白质和蛋白质复合体的功能。 因此，拟议的研究将加深我们对人类潜在基本过程的理解 并开辟了治疗疾病的新途径。

PROJECT SUMMARY Heterochromatin plays critical roles in maintaining genome stability and transcriptional gene silencing (TGS). It has become increasingly clear that misregulation of pathways influencing heterochromatin integrity cause or contribute to many human maladies, including numerous cancers. The composition and function of heterochromatin domains are largely conserved from the fission yeast Schizosaccharomyces pombe to humans and other metazoans. Heterochromatin establishment, epigenetic maintenance, and TGS in these organisms are complex processes regulated by numerous chromatin-associated factors. Despite such complexity, core principles of heterochromatin biology have been proposed but remain speculative. This study will experimentally test and articulate these core principles by distilling essential features of heterochromatin in a highly-controlled and orthogonal environment. This research proposal is composed of two aims. Aim 1 is to reconstitute histone 3 lysine 9 methylation (H3K9me)-dependent heterochromatin in Saccharomyces cerevisiae cells, which naturally lack H3K9me, with the goal of defining the minimal requirements for a repressive and heritable chromatin state conserved from fission yeast to human. Successful construction of an H3K9me-dependent heterochromatin domain in S. cerevisiae cells will provide a unique system for investigating how heterochromatin is epigenetically inherited and how it silences transcription and limits other DNA transactions. Accordingly, Aim 2 is to utilize in vivo reconstituted H3K9me-dependent heterochromatin to investigate the mechanism of TGS. H3K9me-dependent heterochromatin will be reconstituted in S. cerevisiae cells by sequentially recruiting S. pombe and human heterochromatin-associated proteins to a specific S. cerevisiae genomic locus. This minimal heterochromatin domain will then be utilized to test three models of TGS and assess the contribution of histone deacetylation and nucleosome remodeling to the formation of silent chromatin domains. A combination of synthetic biology-, next generation sequencing-, and proteomics-based experimental approaches will be utilized to execute this research plan. This work has potential to transform our mechanistic understanding of heterochromatin formation and thereby inform future studies aimed at reversing defects in heterochromatin-associated processes underlying human diseases. Furthermore, the reconstitution of heterologous heterochromatin domains in vivo with factors found in S. pombe and human cells will facilitate the development of cell-based assays amenable to high-throughput screens for small molecules that modulate the function of proteins and protein complexes involved in heterochromatin formation and disease progression. The proposed studies will thus deepen our understanding of fundamental processes underlying human diseases and open new avenues to their treatment.