Biochemistry and Genetics of Chromatin Assembly Factors in Drosophila

描述（由申请人提供）：体内染色质的组装由核心组蛋白伴侣和ATP依赖性核小体组装因子的协同作用介导。染色质组装是一个重要的反应，接口的所有主要过程的核DNA代谢。我们的长期目标是了解真核生物染色体是如何组装、复制和调控的。 果蝇CHD 1和ISWI复合物ACF、NoRC/ToRC和RSF在体内外均能以ATP依赖的方式介导核小体的组装。具体的假设是ACF和ACF样因子介导的重组的染色体丝以及高阶染色质，并可以调节核反应，如转录，DNA复制和修复。 在早期胚胎中，雄性原核中的染色质组装之前是精子染色质重塑（SCR）和驱逐父精蛋白。这种反应是由我们最近发现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介导的鱼精蛋白伴侣和ATP依赖的马达蛋白的协同作用。 具体目标是：1.研究ATP依赖的染色质体外组装机制。我们将确定ISWI和CHD 1的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级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之间的相互作用。2.研究果蝇体内ACF和ACF样因子的活性和靶点。我们将研究基因组，亚细胞和细胞周期依赖的染色质组装因子的定位。我们将探讨ACF样因子在核心组蛋白和连接组蛋白沉积到染色体中的功能。我们将分析一个新的果蝇装配因子NoRC/ToRC的具体生物学功能。3.分析介导SCR的因子的生化和生物学功能。我们将描述介导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）的基本理解。它将有助于设计诊断和治疗涉及这些核过程缺陷的人类疾病的方法。此外，我们的研究将有助于分子技术的发展，以重建功能的后生动物染色体。 公共卫生相关性：染色质是人体细胞核DNA的天然状态。将DNA包装到染色质中对于维持基因组完整性和调节DNA代谢是必不可少的。这个项目的目标是了解介导染色质组装的因子的功能。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.