Molecular Mechanism of Histone H3/H4 Chaperone Function

描述(由申请人提供)：将基因组打包成染色质是正常生长、发育和分化所必需的。染色质是一种动态结构，它严格调控所有以DMA为底物的过程，包括转录、DMA复制、DMA修复和重组。此外，染色质的组装和拆解过程在人类疾病中很重要，正如在多种遗传畸形以及与改变染色质结构的蛋白质的异常有关的癌症和白血病亚型中所看到的那样。负责染色质分解和染色质组装的关键蛋白是组蛋白H3和H4伴侣抗沉默功能1(ASF1)和染色质组装因子(CAF-1)复合体。这些蛋白在真核生物进化过程中高度保守，是本研究的重点，因为它们在组蛋白H3/H4沉积和核小体分解活动中起核心作用。该项目的长期目标是对组蛋白H3/H4伴侣之间的关系以及它们在体外和体内染色质组装和拆解的机制有一个统一的认识。第一个目标是用分子遗传学和结构方法确定Asf1介导组蛋白H3/H4伴侣功能的决定因素。第二个目的是用生物物理分析的方法研究Asf1分子伴侣活性的分子机制。第三个目标是深入了解中心DNA复制依赖的染色质组装因子-三亚单位CAF-1复合体的功能及其与Asf1的相互作用，以建立染色质组装的分子机制。这些研究将利用我们最近的ASF1-H3/H4晶体结构，我们已经产生的重组染色质组装因子，以及在萌芽酵母模型系统中的生理分析。通过将PI和Co-PI在生物物理学、结构生物学、遗传学和生物化学方面的个人专业知识结合并应用于组蛋白H3/H4伴侣的合作研究，我们处于独特的地位，在详细了解组蛋白伴侣活性的分子基础方面取得了重要进展。这项工作将填补目前对染色质组装和拆解这些基本过程的理解的关键空白，这些过程是所有依赖DMA的细胞功能的关键和核心。与公众健康的相关性。许多疾病都是基因表达不正确的结果。从这项工作中对染色质组装和分解的分子和结构的理解将进一步促进我们修改人类疾病中涉及的表观遗传密码的能力，以达到治疗干预的目的。

DESCRIPTION (provided by applicant): The packaging of the genome into chromatin is essential for normal growth, development, and differentiation. Chromatin is a dynamic structure that tightly regulates all of the processes that use DMA as a substrate, including transcription, DMA replication, DMA repair, and recombination. Furthermore, chromatin assembly and disassembly processes are important in human disease, as seen in the multiple genetic malformations, as well as cancer and leukemia subtypes that have been linked to aberrations in proteins that modify chromatin structure. The key proteins responsible for chromatin disassembly and chromatin assembly are the histone H3 and H4 chaperones Anti-silencing function 1 (Asf1) and the Chromatin Assembly Factor (CAF-1) complex. These proteins are highly conserved throughout eukaryotic evolution and are the focus of this study because of their central role in histone H3/H4 deposition and nucleosome disassembly activities. The long-term goal of this project is to gain a unified understanding of the relationship among histone H3/H4 chaperones and their mechanism of chromatin assembly and disassembly in vitro and in vivo. The first Aim is to define the determinants of Asfl -mediated histone H3/H4 chaperone function using molecular genetics and structural approaches. The second Aim is to investigate the molecular mechanism of the H3/H4 chaperone activity of Asfl using biophysical analyses. The third Aim is to gain key insight into the function of the central DNA replication-dependent chromatin assembly factor - the trisubunit CAF-1 complex, and its interactions with Asfl to establish the molecular mechanism of chromatin assembly. These studies will take advantage of our recent Asf1-H3/H4 crystal structure, recombinant chromatin assembly factors that we have already generated, and physiological analyses in the budding yeast model system. By combining and applying the individual expertise of the PI and Co-Pi in biophysics, structural biology, genetics and biochemistry to this collaborative study of histone H3/H4 chaperones, we are in a unique position to make important strides toward a detailed understanding of the molecular basis of histone chaperone activity. This work will fill critical gaps in the current understanding of these fundamental processes of chromatin assembly and disassembly, which are essential and central to all DMA-dependent cellular functions. Relevance to the public health. Many diseases are the result of incorrect gene expression. The molecular and structural understanding of chromatin assembly and disassembly that will come from this work will further our ability to modify the epigenetic codes involved in human diseases for the purpose of therapeutic intervention.