Biochemistry and genetics of chromatin assembly factors in Drosophila

描述（由申请人提供）：果蝇中染色质装配因子的生物化学和遗传学体内染色质的装配由核心组蛋白伴侣和ATP依赖性核小体装配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DMA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ACF是一种典型的ATP依赖性染色质装配因子，它由Acfl和ISWI两个亚基组成。除ACF外，其他因子也可以在体外和体内以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。我们的假设是基于（i）核小体组装的研究ACF在体外，（ii）免疫细胞学研究ACF功能在果蝇细胞和（iii）基因突变的遗传和分子分析编码ACF和其他组装因子。具体目标是：1.探讨ACF在体外依赖ATP的染色质组装机制。我们将确定赋予ACF核小体组装活性的Acf 1和ISWI的结构特征。我们将探讨ACF染色质进行性组装的分子机制。我们将寻找核小体组装反应的活化中间体。2.在体外进行果蝇染色质组装因子的生化分析。我们将确定和表征其他果蝇ATP依赖的染色质组装因子。我们将研究ATP依赖因子和组蛋白伴侣之间的相互作用在染色质组装中的作用。3.研究果蝇体内ACF和ACF样因子的活性和靶点。我们将研究果蝇中编码（假定的）染色质装配因子的基因之间的相互作用。我们将分析它们在体内和细胞培养中在核小体组装和染色体维持中的功能。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。

DESCRIPTION (provided by applicant): Biochemistry and Genetics of Chromatin Assembly Factors in Drosophila 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 DMA metabolism. Our long-term goal is to understand how eukaryotic chromosomes are assembled, reproduced and regulated. Drosophila ACF is a prototype ATP-dependent chromatin assembly factor, which comprises two subunits, Acfl and ISWI. In addition to ACF, other factors 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 the reconstitution of oligonucleosome filament as well as higher-order chromatin and can regulate nuclear reactions, such as transcription, DMA replication and repair. Our hypothesis is based on (i) studies of nucleosome assembly by ACF in vitro, (ii) immunocytological investigation of ACF function in Drosophila cells and (iii) genetic and molecular analyses of mutations in genes that encode ACF and other assembly factors. The specific aims are to: 1. Investigate the mechanisms of ATP-dependent chromatin assembly by ACF in vitro. We will determine the structural features of Acf1 and ISWI that endow ACF with the nucleosome assembly activity. We will explore molecular mechanisms of processive chromatin assembly by ACF. We will search for activated intermediates of the nucleosome assembly reaction. 2. Perform biochemical analysis of Drosophila chromatin assembly factors in vitro. We will identify and characterize additional Drosophila ATP-dependent chromatin assembly factors. We will study the role of interactions between ATP-dependent factors and histone chaperones in chromatin assembly. 3. Examine activities and targets of ACF and ACF-like factors in vivo in Drosophila. We will investigate interactions between genes that encode (putative) chromatin assembly factors in Drosophila. We will analyze their function in nucleosome assembly and chromosome maintenance in vivo and in cell culture. 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.