Biochemistry and genetics of chromatin assembly factors in Drosophila

果蝇染色质组装因子的生物化学和遗传学

• 批准号: 7487936
• 负责人: DMITRY V FYODOROV
• 金额: $ 27.54万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7487936
• 关键词: ATP Hydrolysis; Address; Alleles; Biochemical; Biochemical Genetics; Biochemical Reaction; Biochemistry; Biological Process; Cell Cycle Progression; Cell Nucleus; Cells; Chromatin; Chromatin Modeling; Chromosome Structures; Chromosomes; Classification; Commit; Complex; Coupled; Cultured Cells; DNA; DNA biosynthesis; Defect; Deposition; Development; Diagnosis; Drosophila genus; Embryo; Enzymes; Filament; Genes; Genetic; Genetic Transcription; Goals; Histones; Human; ISWI; In Vitro; Investigation; Knowledge; Maintenance; Mediating; Metabolism; Methods; Molecular; Molecular Chaperones; Molecular Genetics; Motor; Mutation; Nuclear; Nucleosomes; Numbers; Outcome Study; Play; Process; Property; Reaction; Regulation; Role; Techniques; Testing; Transcription Process; Work; base; chromatin assembly factor I; design; fly; human disease; in vivo; mutant; novel; prototype; reconstitution; repaired; research study

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.

果蝇染色质组装因子的化学和遗传学研究 染色质在体内的组装是由核心组蛋白伴侣蛋白和三磷酸腺苷协同作用介导的。 依赖的核小体组装因子。染色质组装是一个重要的界面反应 核内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是一个依赖于ATP的染色质组装因子的原型，它由两个亚基组成， ACFL和ISWI。除ACF外，其他因素也可介导ATP依赖的核小体组装 在体外和体内的方式。 具体的假设是ACF和ACF样因子介导了寡核小体的重建 还有细丝以及高阶染色质，并能调节核反应，如转录、DMA 复制和修复。我们的假设是基于(I)ACF体外组装核小体的研究，(Ii) 果蝇细胞ACF功能的免疫细胞学研究及(III)遗传和分子分析 编码ACF和其他组装因子的基因的突变。具体目标是： 1.探讨ACF体外依赖ATP组装染色质的机制。我们会 确定赋予ACF核小体组装活性的Acf1和ISWI的结构特征。 我们将探索ACF进行染色质组装的分子机制。我们将寻找 核小体组装反应的活性中间体。 2.对果蝇染色质组装因子进行体外生化分析。我们将确定 以及其他果蝇依赖于ATP的染色质组装因子的特征。我们将研究这一角色 染色质组装中ATP依赖因子和组蛋白伴侣蛋白相互作用的研究。 3.在果蝇体内检测ACF和ACF样因子的活性和靶点。我们将进行调查 果蝇中编码(可能)染色质组装因子的基因之间的相互作用。我们会 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作用。 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的诊断和治疗方法。 涉及这些核过程缺陷的疾病。此外，我们的研究将有助于 重建功能性后生动物染色体的分子技术的发展。