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

我们研究的长期目标是研究染色质动力学的分子机制。 在分子细节上理解转录、DNA复制和DNA 修复是在高度紧密的染色质的背景下进行的，以及染色质的错误调节 会导致人类疾病，如癌症。这项拟议研究的总体目标是确定如何 保守的组蛋白变异体H2A.Z的沉积受染色质重塑因子的调节 哺乳动物细胞。H_2A.Z沉积在基因启动子两侧的核小体中，并发挥重要作用 在基因表达、基因组稳定性和适当的胚胎干细胞(ESC)分化方面。此外，误导- H2A.Z沉积的调节与癌症和心脏肥大有关。在酵母中，SWR1，其中一口井- 具有SWR1/INO80重构酶亚家族成员的特征，具有独特的二聚体交换 从核小体上移除H_2A/H_2B二聚体并用H_2A/H_2B二聚体取代它们的活性。P400 和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H_2A.Z沉积。有趣的是，在H2A.Z上调的肺癌细胞中，抑制 P400的抑制不影响H_2A.Z的沉积，而抑制SRCAP则导致H_2A.Z的减少 证词。此外，尽管P400是维持ESC身份所必需的，例如自我更新和 多发性，H_2A.Z是ESC分化所必需的，但不是维持ESC特性所必需的。这些 观察表明，P400和SRCAP具有不同的细胞类型和独特的功能。我们在这方面的总体战略 建议是利用生化和生物物理技术的强大组合，以及ES的基因组学 细胞解剖P400和SRCAP调节H2A.Z沉积的分子机制并确定 这些重塑酶不同的生化和生物学功能。这项建议有两个具体的 目标。在目标1中，我们将剖析P400和SRCAP重塑导致H2A.Z沉积的机制 复合体。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获得性有限，因为P400和SRCAP形成大的多蛋白质 复合体。为了解决这个问题，我们重新构建了P400和SRCAP复合体， 使用Multibac杆状病毒表达系统的重组亚基。我们将定义详细的动力学 二聚体交换反应中P400和SRCAP络合物的速率和底物特异性。我们会 使用各种二聚体交换分析，包括基于FRET的分析。此外，我们还将利用 P400和SRCAP复合体的ART-ART EM分析 这些复合体。我们还将探索P400和SRCAP在小鼠胚胎干细胞中的功能 (ESCS)。我们将研究抑制P400和/或SRCAP如何改变H2 A.Z的表观遗传格局 并影响ESC的同一性和差异性。在目标2中，我们将研究H_2A.Z沉积是如何通过 P400和SRCAP复合体的亚基和组蛋白乙酰化。我们将定义不同的角色 P400和SRCAP复合体在二聚体交换反应中的亚基，最初集中在 保守的RUBVL1/2亚基。我们将剖析RUVBL1/2如何管理 利用体外重组系统构建P400和SRCAP复合体。我们还将调查ATPase是如何 RUVLBL1/2的活性有助于这些络合物的二聚体交换活性。此外，Tip60 组蛋白乙酰转移酶是P400复合体的组成部分。我们将研究二聚体是如何交换的 P400的活性与Tip60的组蛋白乙酰化反应相协调。此外，我们最近确定了一部小说 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 H3-K56Ac调节哺乳动物P400和SRCAP二聚体交换活性的假说 细胞。

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.