Molecular Mechanism of histone variant H2A.Z deposition by chromatin remodeling enzymes

染色质重塑酶沉积组蛋白变体 H2A.Z 的分子机制

• 批准号: 9803434
• 负责人: Shinya Watanabe
• 金额: $ 35.18万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-02 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9803434
• 关键词: ATP phosphohydrolase; Address; Affect; Baculovirus Expression System; Biochemical; Biological Assay; Biological Process; Chromatin; Chromatin Remodeling Factor; Chromatin Structure Alteration; Complex; DNA Repair; DNA biosynthesis; Deposition; Development; Disease; Enzymes; Epigenetic Process; Fluorescence Resonance Energy Transfer; Gene Expression; Genes; Genetic Transcription; Genome Stability; Genomics; Goals; Heart Hypertrophy; Histone Acetylation; Histones; In Vitro; Individual; Kinetics; Lead; Link; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Mammalian Cell; Molecular; Multiprotein Complexes; Mus; Nucleosomes; Play; Proteins; Reaction; Recombinants; Regulation; Research; Role; Substrate Specificity; System; Testing; Variant; Yeasts; base; biophysical techniques; cancer cell; cell type; chromatin remodeling; developmental disease; dimer; drug development; embryonic stem cell; histone acetyltransferase; human disease; insight; member; novel; promoter; reconstitution; self-renewal; stem cell differentiation; therapy development; treatment strategy

The long-term goal of our research is to investigate the molecular mechanisms of chromatin dynamics for understanding in molecular detail the fundamental questions of how transcription, DNA replication, and DNA repair take place within the context of highly compacted chromatin, and how mis-regulation of chromatin causes human diseases such as cancer. The overall objective of this proposed research is to determine how the deposition of the conserved histone variant H2A.Z is regulated by chromatin remodeling factors in mammalian cells. H2A.Z is deposited within nucleosomes that flank gene promoters, and plays essential roles in gene expression, genome stability, and proper embryonic stem cell (ESC) differentiation. Furthermore, mis- regulation of H2A.Z deposition is linked to cancer and cardiac hypertrophy. In yeast, SWR1, one of the well- characterized members of the SWR1/INO80 subfamily of remodeling enzymes, has a unique dimer exchange activity to remove H2A/H2B dimers from a nucleosome and replace them with H2A.Z/H2B dimers. The p400 and SRCAP chromatin remodeling enzymes are mammalian homologs of yeast SWR1 that are thought to be responsible for H2A.Z deposition. Interestingly, in lung cancer cells where H2A.Z is upregulated, suppression of p400 does not affect H2A.Z deposition while suppression of SRCAP leads to a decrease in H2A.Z deposition. Moreover, although p400 is required for maintenance of ESC identity such as self-renewal and pluripetency, H2A.Z is required for ESC differentiation, but not for maintenance of ESC identity. These observations suggest cell-type specific, distinct functions of p400 and SRCAP. Our overall strategy in this proposal is to exploit a powerful combination of biochemical and biophysical techniques, and genomics in ES cells to dissect the molecular mechanisms by which p400 and SRCAP regulate H2A.Z deposition and define the distinct biochemical and biological functions of these remodeling enzymes. This proposal has two specific aims. In Aim 1, we will dissect the mechanisms of H2A.Z deposition by the p400 and SRCAP remodeling complexes. The molecular mechanisms by which p400 and SRCAP catalyzes H2A.Z deposition are largely unknown, mainly due to the limited protein availability, as p400 and SRCAP form large multi-protein complexes. To address this, we have reconstituted the p400 and SRCAP complexes from individual, recombinant subunits using the Multibac baculovirus expression system. We will define the detailed kinetic rates and substrate specificities of the p400 and SRCAP complexes in the dimer exchange reactions. We will employ various dimer exchange assays including FRET-based assays. Furthermore, we will exploit state-of- the-art EM analysis of the p400 and SRCAP complexes to dissect the structural and functional relationship of these complexes. We will also explore the functions of p400 and SRCAP in mouse embryonic stem cells (ESCs). We will investigate how suppression of p400 and/or SRCAP alters the epigenetic landscape of H2A.Z and affects ESC identity and differentiation. In Aim 2, we will investigate how H2A.Z deposition is regulated by subunits of the p400 and SRCAP complexes and histone acetylations. We will define the role of different subunits of the p400 and SRCAP complexes in the dimer exchange reaction, focusing initially on the conserved RUBVL1/2 subunits. We will dissect how RUVBL1/2 govern the assemblies and functions of the p400 and SRCAP complexes using in vitro reconstitution system. We will also investigate how the ATPase activity of RUVLBL1/2 contributes to the dimer exchange activities of these complexes. In addition, the Tip60 histone acetyltransferase is a component of the p400 complex. We will investigate how the dimer exchange activity of p400 coordinates with the histone acetylation by Tip60. Furthermore, we recently identified a novel functional interaction between SWR1 and H3-K56Ac that regulates H2A.Z dynamics in yeast. We will test the hypothesis that the H3-K56Ac regulates the dimer exchange activities of p400 and SRCAP in mammalian cells.

我们研究的长期目标是研究染色质动力学的分子机制， 了解转录、DNA复制和DNA如何在分子细节上的基本问题 修复发生在高度致密的染色质的背景下，以及染色质的错误调节是如何发生的。 导致人类疾病，如癌症。这项研究的总体目标是确定如何 保守的组蛋白变体H2A.Z的沉积受染色质重塑因子的调节， 哺乳动物细胞H2A.Z沉积在基因启动子侧翼的核小体中，并在基因启动子的转录中起重要作用。 基因表达、基因组稳定性和胚胎干细胞（ESC）分化。此外，mis- H2A.Z沉积的调节与癌症和心脏肥大有关。在酵母中，SWR 1，一种很好的- SWR 1/INO 80亚家族的重塑酶，具有独特的二聚体交换 从核小体中除去H2 A/H2 B二聚体并用H2 A. Z/H2 B二聚体替换它们的活性。P400 和SRCAP染色质重塑酶是酵母SWR 1的哺乳动物同源物， 负责H2A.Z沉积。有趣的是，在H2A.Z上调的肺癌细胞中， p400的抑制不影响H2A.Z的沉积，而SRCAP的抑制导致H2A.Z的减少 证词此外，虽然p400是维持ESC身份所必需的，如自我更新和 由于H2A.Z是ESC分化所需的，但不是维持ESC身份所需的。这些 观察结果提示p400和SRCAP的细胞类型特异性的、不同的功能。我们在这方面的总体战略 一个建议是利用生物化学和生物物理技术的强大组合，以及ES中的基因组学 细胞，以剖析p400和SRCAP调节H2A.Z沉积的分子机制，并定义 这些重塑酶独特的生化和生物学功能。该提案有两个具体的 目标。在目的1中，我们将通过p400和SRCAP重塑来剖析H2 A. Z沉积的机制 配合物p400和SRCAP催化H2A.Z沉积的分子机制主要是 未知，主要是由于蛋白质可用性有限，因为p400和SRCAP形成大的多蛋白质 配合物为了解决这个问题，我们已经重建了p400和SRCAP复合物， 使用Multibac杆状病毒表达系统表达重组亚基。我们将定义详细的动力学 二聚体交换反应中p400和SRCAP复合物的速率和底物特异性。我们将 使用各种二聚体交换测定，包括基于FRET的测定。此外，我们将利用国家- p400和SRCAP复合物的最先进的EM分析，以剖析结构和功能的关系， 这些复合物。我们还将探讨p400和SRCAP在小鼠胚胎干细胞中的功能 （ESC）。我们将研究p400和/或SRCAP的抑制如何改变H2A.Z的表观遗传景观。 并影响ESC的身份和分化。在目标2中，我们将研究H2A.Z沉积是如何通过 p400和SRCAP复合物的亚基和组蛋白乙酰化。我们将定义不同角色 在二聚体交换反应中，p400和SRCAP复合物的亚基，最初集中在 保守的RUBVL 1/2亚基。我们将剖析RUVBL 1/2如何控制 p400和SRCAP复合物。我们还将研究ATP酶 RUVLBL 1/2的活性有助于这些复合物的二聚体交换活性。此外，Tip 60 组蛋白乙酰转移酶是p400复合物的组分。我们将研究二聚体交换 p400的活性与Tip 60对组蛋白的乙酰化作用相协调。另外，我们最近发现了一本小说 SWR 1和H3-K56 Ac之间的功能性相互作用，其调节酵母中的H2A.Z动力学。我们将测试 H3-K56 Ac调节哺乳动物p400和SRCAP二聚体交换活性的假说 细胞