Regulation of chromatin dynamics

项目总监/首席研究员(最后、第一、中间)：彼得森、克雷格、刘易斯 我们研究的总体目标是确定染色体结构如何影响基因 转录、DNA复制和修复，特别强调识别和表征染色质 控制染色体动态的重塑机器。值得注意的是，基因实验揭示了ATP- 依赖染色质重塑酶作为几乎每个染色体过程的基本调节器，以及 它们的失调会导致多种疾病，包括癌症。我们的研究工作可以被组织起来 分为三个相互关联的领域：(1)依赖于ATP的染色质重塑酶的机制研究， 主要关注14亚基SWR1C重构体的结构和功能；(2)研究 INO80C重构体在DNA复制和维持基因组稳定性中的作用；以及(3)探索 在复制分叉通路之后，新复制基因的表达受到抑制，这一过程称为 转录缓冲。SWR1C重塑酶催化一种新的、依赖于ATP的组蛋白 控制H2A.Z组蛋白变异体在侧翼核小体内沉积的交换事件 由RNA聚合酶II转录的基因的启动子，以及着丝粒和着丝粒两侧的核小体。 复制源。SWR1C和INO80C的哺乳动物同源物，包括P400/Tip60和hINO80 复合体是干细胞正常功能、基因组稳定、发育和基因表达的关键。多么 SWR1C催化依赖于ATP的H2A.Z沉积在很大程度上仍不清楚，我们提出的 机理研究将包括基于整体和单分子荧光的分析，以确定以下步骤 组蛋白二聚体交换反应，以及质谱学和低温EM方法的组合 探索SWR1C如何区分不同的核小体底物。 我们和其他人的研究表明，染色质动力学在调节中发挥着重要作用。 编码和非编码RNA的转录，以及这种平衡的破坏都会影响基因组的稳定性。 特别是，我们在INO80C上的工作发现，它防止了普遍存在的非编码转录 关于酵母和哺乳动物细胞的复制体功能。我们提出了多种基因组方法来探索 关键悬而未决的问题：INO80C如何阻止非编码转录？转录如何影响 叉子结构？INO80C是否与保守的叉头转录因子协作组织起源 进入核弹舱？我们的体内研究将扩展到S阶段的转录调控。我们 已经使用新的转录测序来证实新复制的基因被瞬时抑制2- 折叠，直到下一个G2阶段。这一过程被称为“转录缓冲”，在哺乳动物中是保守的。 并被认为可以防止S期出现短暂的非整倍体状态。如何建立缓冲以及 移除是未知的，在此我们建议标识建立缓冲的复制链接程序集 并使用基因组和分子遗传学方法来探索它们的功能。 OMB编号0925-0001/0002(08/12版批准至2015年8月31日)页面续格式页面

Program Director/Principal Investigator (Last, First, Middle): Peterson, Craig, Lewis The overall objective of our research is to determine how chromosome structure influences gene transcription, DNA replication and repair, with special emphasis on identifying and characterizing the chromatin remodeling machines that control chromosome dynamics. Notably, genetic experiments have revealed ATP- dependent chromatin remodeling enzymes as essential regulators of virtually every chromosomal process, and their dysregulation leads to a variety of diseases, including cancer. Our research efforts can be organized into three inter-related areas: (1) Mechanistic studies of ATP-dependent chromatin remodeling enzymes, focusing primarily on the structure and function of the 14-subunit, SWR1C remodeler; (2) Investigating roles for the INO80C remodeler in DNA replication and the maintenance of genome stability; and (3) Probing how the expression of newly replicated genes is repressed following replication fork passage, a process termed transcriptional buffering. The SWR1C remodeling enzyme catalyzes a novel, ATP-dependent histone exchange event that controls the deposition of the H2A.Z histone variant within nucleosomes that flank promoters of genes transcribed by RNA polymerase II, as well as nucleosomes that flank centromeres and replication origins. Mammalian homologs of SWR1C and INO80C, including the p400/Tip60 and hINO80 complexes, are key for proper stem cell function, genome stability, development, and gene expression. How SWR1C catalyzes ATP-dependent deposition of H2A.Z remains largely unknown, and our proposed mechanistic studies will include ensemble and single molecule fluorescence-based assays to define steps of the histone dimer exchange reaction, as well as a combination of mass spectrometry and cryoEM methods to probe how SWR1C distinguishes different nucleosomal substrates. Studies from us and others have demonstrated that chromatin dynamics play a large role in regulating transcription of both coding and noncoding RNAs, and disruption of this balance can impact genomic stability. In particular, our work on INO80C has found that it prevents pervasive noncoding transcription from impinging on replisome function in both yeast and mammalian cells. We propose a variety of genomic methods to probe key unanswered questions: How does INO80C block noncoding transcription? How does transcription impact fork structure? Does INO80C collaborate with the conserved forkhead transcription factors to organize origins into a nuclear compartment? Our in vivo studies will extend to transcriptional regulation during S phase. We have used Nascent transcript sequencing to confirm that newly replicated genes are transiently repressed 2- fold until the subsequent G2 phase. Termed “transcriptional buffering” this process is conserved in mammals and is believed to prevent transient aneuploid states during S phase. How buffering is established and removed is not known, and here we propose to identify replication-linked assemblies that establish buffering and to use genomic and molecular genetic approaches to probe their function. OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015) Page Continuation Format Page