Biochemistry and Genetics of Chromatin Assembly Factors in Drosophila

描述(申请人提供)：染色质在体内的组装是由核心组蛋白伴侣蛋白和依赖于ATP的核小体组装因子的协同作用所介导的。染色质组装是连接核DNA代谢所有主要过程的重要反应。我们的长期目标是了解真核细胞染色体是如何组装、繁殖和调控的。果蝇CHD1和ISWI型复合体ACF、NORC/TORC和RSF在体内和体外均能以ATP依赖的方式介导核小体组装。具体的假设是ACF和ACF样因子介导寡核小体细丝和高阶染色质的重组，并调节核反应，如转录、DNA复制和修复。在早期胚胎中，在男性原核染色质组装之前，精子染色质重塑(SCR)和父亲的精蛋白被驱逐。这一反应是由我们最近确定的一组单独的因素介导的。具体的假设是，类似于核小体组装，SCR是由鱼精蛋白伴侣蛋白和ATP依赖的运动蛋白协同作用介导的。其具体目的是：1.研究依赖于ATP的染色质体外组装的机制。我们将确定ISWI和CHD1内在核小体组装活性的分子基础。我们将分析核小体组装和高阶染色质纤维形成的前体。我们将研究在染色质组装过程中ATP依赖因子和组蛋白伴侣蛋白之间的相互作用。2.在果蝇体内检测ACF和ACF样因子的活性和靶点。我们将研究依赖于基因组、亚细胞和细胞周期的染色质组装因子的定位。我们将探索ACF样因子在核心和连接子组蛋白沉积到染色体中的作用。我们将分析一种新的果蝇组装因子NORC/TORC的特定生物学功能。3.分析影响SCR的因素的生化和生物学功能。我们将描述调节SCR的因素及其作用的分子机制。我们将阐明SCR介导因子在体内的生物学功能。我们的工作将增加对染色质组装和相关核过程(转录、复制和修复)的基本了解。它将有助于设计诊断和治疗涉及这些核过程中缺陷的人类疾病的方法。此外，我们的研究将有助于发展重建功能后生动物染色体的分子技术。 与公共健康相关：染色质是人类细胞中核DNA的天然状态。将DNA包装到染色质中对于维持基因组的完整性和调节DNA代谢是必不可少的。这个项目的目标是了解调节染色质组装的因素的功能。我们的工作将有助于设计诊断和治疗涉及核反应缺陷的人类疾病的方法。

DESCRIPTION (provided by applicant): The assembly of chromatin in vivo is mediated by a concerted action of core histone chaperones and ATP-dependent nucleosome assembly factors. The chromatin assembly is an important reaction that interfaces all major processes of the nuclear DNA metabolism. Our long-term goal is to understand how eukaryotic chromosomes are assembled, reproduced and regulated. Drosophila CHD1 and ISWI-containing complexes ACF, NoRC/ToRC and RSF can mediate nucleosome assembly in an ATP-dependent manner in vitro and in vivo. The specific hypothesis is that ACF and ACF-like factors mediate reconstitution of oligonucleosome filament as well as higher-order chromatin and can regulate nuclear reactions, such as transcription, DNA replication and repair. In the early embryo, chromatin assembly in the male pronucleus is preceded by sperm chromatin remodeling (SCR) and eviction of paternal protamines. This reaction is mediated by a separate set of factors that we recently identified. The specific hypothesis is that, similar to nucleosome assembly, SCR is mediated by a concerted action of protamine chaperones and ATP-dependent motor proteins. The specific aims are to: 1. Investigate mechanisms of ATP-dependent chromatin assembly in vitro. We will determine the molecular basis for intrinsic nucleosome assembly activity of ISWI and CHD1. We will analyze precursors of nucleosome assembly and formation of higher-order chromatin fiber. We will study the interplay between ATP-dependent factors and histone chaperones during chromatin assembly. 2. Examine activities and targets of ACF and ACF-like factors in vivo in Drosophila. We will examine genomic, subcellular and cell-cycle dependent localization of chromatin assembly factors. We will explore the function of ACF-like factors in the deposition of core and linker histones into chromosomes. We will analyze specific biological functions of a novel Drosophila assembly factor NoRC/ToRC. 3. Analyze biochemical and biological functions of factors that mediate SCR. We will characterize factors that mediate SCR and molecular mechanisms of their action. We will elucidate biological functions of SCR-mediating factors in vivo. Our work will add to the fundamental understanding of chromatin assembly and related nuclear processes (transcription, replication and repair). It will help to design methods of diagnosis and treatment of human diseases that involve defects in these nuclear processes. Furthermore, our studies will contribute to the development of molecular techniques to reconstitute functional metazoan chromosomes. PUBLIC HEALTH RELEVANCE: Chromatin is the native state of nuclear DNA in human cells. Packaging of DNA into chromatin is essential for the maintenance of genome integrity and regulation of DNA metabolism. The goal of this project is to understand the function of factors that mediate the assembly of chromatin. Our work will help to design methods of diagnosis and treatment of human diseases that involve defects in nuclear reactions.