Biochemistry and genetics of chromatin assembly factors in Drosophila

描述（由申请人提供）：果蝇染色质组装因子的生物化学和遗传学体内染色质的组装是由核心组蛋白伴侣和ATP依赖的核小体组装因子协同作用介导的。染色质组装是连接核DMA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生物染色体是如何组装、繁殖和调节的。果蝇ACF是一种原型atp依赖性染色质组装因子，由Acfl和ISWI两个亚基组成。除ACF外，其他因子在体内和体外均以atp依赖的方式介导核小体组装。具体假设是ACF及ACF样因子介导寡核小体丝和高阶染色质的重构，并调节转录、DMA复制和修复等核反应。我们的假设是基于(i)体外ACF对核小体组装的研究，（ii）果蝇细胞中ACF功能的免疫细胞学研究，以及（iii）编码ACF和其他组装因子的基因突变的遗传和分子分析。具体目标是：1。探讨体外ACF对atp依赖性染色质组装的机制。我们将确定使ACF具有核小体组装活性的Acf1和ISWI的结构特征。我们将探讨ACF在染色质组装过程中的分子机制。我们将寻找核小体组装反应的活化中间体。2. 对果蝇染色质组装因子进行体外生化分析。我们将鉴定和表征额外的果蝇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在染色质组装中的相互作用。3. 在果蝇体内检测ACF和ACF样因子的活性和靶标。我们将研究果蝇中编码（假定的）染色质组装因子的基因之间的相互作用。我们将在体内和细胞培养中分析它们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，以重建功能性后生动物染色体。

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.