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

基本信息

批准号：
10399479
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
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-02 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10399479
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 Mammalian Cell Molecular Multiprotein Complexes Mus Nucleosomes Play Proteins Reaction Recombinants Regulation Research Role Substrate Specificity System Testing Variant Yeasts base biophysical techniques cell type chromatin remodeling developmental disease dimer drug development embryonic stem cell histone acetyltransferase human disease insight lung cancer cell member novel promoter reconstitution self-renewal stem cell differentiation therapy development treatment strategy

项目摘要

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从分子细节上理解转录、DNA 复制和 DNA 是如何产生的基本问题修复是在高度压缩的染色质的背景下进行的，以及染色质的错误调节是如何发生的导致癌症等人类疾病。这项研究的总体目标是确定如何保守组蛋白变体 H2A.Z 的沉积受到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内，并发挥重要作用基因表达、基因组稳定性和适当的胚胎干细胞 (ESC) 分化。此外，误 H2A.Z 沉积的调节与癌症和心脏肥大有关。在酵母中，SWR1 是一种重塑酶 SWR1/INO80 亚家族的特征成员，具有独特的二聚体交换从核小体中去除 H2A/H2B 二聚体并用 H2A.Z/H2B 二聚体替换它们的活性。 p400 SRCAP 染色质重塑酶是酵母 SWR1 的哺乳动物同源物，被认为是负责H2A.Z沉积。有趣的是，在 H2A.Z 上调的肺癌细胞中，抑制 p400 的减少不影响 H2A.Z 沉积，而抑制 SRCAP 会导致 H2A.Z 减少沉积。此外，尽管 p400 是维持 ESC 身份（例如自我更新和多能性，H2A.Z 是 ESC 分化所必需的，但不是维持 ESC 同一性所必需的。这些观察结果表明 p400 和 SRCAP 具有细胞类型特异性、不同的功能。我们在这方面的总体策略提议是在 ES 中利用生化和生物物理技术以及基因组学的强大组合细胞剖析 p400 和 SRCAP 调节 H2A.Z 沉积的分子机制并定义这些重塑酶具有独特的生化和生物学功能。该提案有两个具体内容目标。在目标 1 中，我们将通过 p400 和 SRCAP 重塑来剖析 H2A.Z 沉积的机制复合物。 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 形成大型多蛋白质复合物。为了解决这个问题，我们从个体中重建了 p400 和 SRCAP 复合物，使用 Multibac 杆状病毒表达系统重组亚基。我们将定义详细的动力学 p400 和 SRCAP 复合物在二聚体交换反应中的速率和底物特异性。我们将采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state-of- 对 p400 和 SRCAP 复合物进行最先进的 EM 分析，以剖析其结构和功能关系这些复合物。我们还将探讨p400和SRCAP在小鼠胚胎干细胞中的功能（ESC）。我们将研究抑制 p400 和/或 SRCAP 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 沉积是如何受调节的 p400 和 SRCAP 复合物的亚基以及组蛋白乙酰化。我们将定义不同的角色 p400 和 SRCAP 复合物的亚基在二聚体交换反应中，最初关注保守的 RUBVL1/2 亚基。我们将剖析 RUVBL1/2 如何控制组件和功能使用体外重建系统的 p400 和 SRCAP 复合物。我们还将研究 ATPase 如何 RUVLBL1/2 的活性有助于这些复合物的二聚体交换活性。此外，Tip60 组蛋白乙酰转移酶是 p400 复合物的组成部分。我们将研究二聚体交换如何 p400 的活性与 Tip60 的组蛋白乙酰化相协调。此外，我们最近发现了一本小说 SWR1 和 H3-K56Ac 之间的功能相互作用调节酵母中的 H2A.Z 动态。我们将测试假设 H3-K56Ac 调节哺乳动物中 p400 和 SRCAP 的二聚体交换活性细胞。