Regulation of chromatin dynamics

染色质动力学的调节

• 批准号: 10405319
• 负责人: Craig L Peterson
• 金额: $ 97.29万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10405319
• 关键词: Aneuploidy; Area; Biological Assay; Buffers; Cell division; Centromere; Chromatin; Chromosome Structures; Chromosomes; Code; Complex; Cryoelectron Microscopy; DNA; DNA Repair; DNA biosynthesis; DNA replication fork; Deposition; Development; Disease; Enzymes; Equilibrium; Event; Fluorescence; G2 Phase; Gene Expression; Genes; Genetic; Genetic Transcription; Genome Stability; Genomics; Histones; Lead; Link; Maintenance; Malignant Neoplasms; Mammalian Cell; Mammals; Mass Spectrum Analysis; Methods; Molecular Genetics; Nuclear; Nucleosomes; Play; Principal Investigator; Process; RNA Polymerase II; Reaction; Regulation; Replication Origin; Research; Role; S phase; Structure; Transcript; Transcriptional Regulation; Untranslated RNA; Variant; Work; Yeasts; base; chromatin remodeling; dimer; experimental study; forkhead protein; genetic approach; in vivo; novel; prevent; programs; promoter; single molecule; stem cell function; stem cells; virtual

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

项目负责人/主要研究者（最后一名、第一名、中间名）：Peterson、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 重塑控制染色体动力学的机器值得注意的是，基因实验已经揭示了ATP- 依赖性染色质重塑酶作为几乎所有染色体过程的基本调节剂， 它们的失调导致包括癌症在内的多种疾病。我们的研究工作可以组织起来 分为三个相互关联的领域：（1）ATP依赖的染色质重塑酶的机制研究， 主要集中在14-亚基，SWR 1C重塑的结构和功能;（2）调查的作用， INO 80 C在DNA复制和基因组稳定性的维持中的重塑作用;（3）探索INO 80 C如何在DNA复制和基因组稳定性的维持中发挥作用。 新复制基因的表达在复制叉传代后受到抑制，这一过程称为 转录缓冲SWR 1C重塑酶催化一种新的ATP依赖性组蛋白 控制H2A.Z组蛋白变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，以及位于着丝粒侧翼的核小体， 复制起点SWR 1C和INO 80 C的哺乳动物同源物，包括p400/Tip 60和hINO 80 复合物是干细胞正常功能、基因组稳定性、发育和基因表达的关键。如何 SWR 1C催化H2A.Z的ATP依赖性沉积在很大程度上仍然是未知的，我们提出的 机制研究将包括基于整体和单分子荧光的测定，以确定 组蛋白二聚体交换反应，以及质谱和cryoEM方法的组合， 探讨SWR 1C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 基因组稳定性是编码和非编码RNA转录的一个重要因素，这种平衡的破坏会影响基因组的稳定性。 特别是，我们对INO 80 C的研究发现，它可以阻止普遍的非编码转录， 对酵母和哺乳动物细胞中复制体功能的影响。我们提出了多种基因组方法来探测 关键未解问题：INO 80 C如何阻断非编码转录？转录如何影响 分叉结构？INO 80 C是否与保守的叉头转录因子协作来组织起源 变成了一个核隔间我们的体内研究将扩展到S期的转录调控。我们 已经使用Nascent转录测序来证实新复制的基因被短暂抑制2- 直至G2期。这个过程被称为“转录缓冲”，在哺乳动物中是保守的 并且被认为防止S期期间的瞬时非整倍体状态。如何建立缓冲， removed是未知的，这里我们建议识别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测它们的功能。 OMB编号0925-0001/0002（2012年8月批准至2015年8月31日修订版）页码续页格式页码