Biochemistry and genetics of chromatin assembly factors in Drosophila

果蝇染色质装配因子的生物化学和遗传学研究 染色质在体内的组装是由核心组蛋白伴侣和ATP的协同作用介导的。 依赖的核小体组装因子。染色质组装是一个重要的反应， 核DMA代谢的所有主要过程。我们的长期目标是了解真核生物 染色体被组装、复制和调节。 果蝇ACF是一种典型的ATP依赖性染色质装配因子，它包括两个亚基， ACFL和ISWI。除了ACF之外，其他因子可以介导核小体组装成ATP依赖性的核小体。 在体外和体内的方式。 具体的假设是ACF和ACF样因子介导了溶酶体的重构 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）研究核小体组装的活性碳纤维在体外，（ii） 果蝇细胞中ACF功能免疫细胞学研究及（iii）遗传和分子分析 编码ACF和其他组装因子的基因突变。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。我们会寻找 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 并描述了其他果蝇ATP依赖的染色质组装因子。我们将研究 在染色质组装中ATP依赖因子和组蛋白伴侣之间的相互作用。 3.研究果蝇体内ACF和ACF样因子的活性和靶点。我们会调查 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的基本理解 （转录、复制和修复）。这将有助于设计人类疾病的诊断和治疗方法 这些核过程中的缺陷引起的疾病。此外，我们的研究将有助于 发展分子技术来重建功能性后生动物染色体。

iochemistry 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. Wewill 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.