Mechanisms of chromatin remodeling at yeast promoters

描述(申请人提供)：基因表达的时间和程度最终控制着人类和其他生物的正常细胞增殖和细胞分化。将含有组蛋白的真核DNA包装到染色质中，实际上可以抑制转录的所有步骤。因此，染色质不仅是调节转录的平台，也是防止异常转录的过滤器。染色质结构的重塑是转录的所有阶段所必需的。染色质重塑在人类中的生物学重要性体现在对染色质重塑至关重要的基因突变与许多癌症有关的事实上。这个项目的目标是通过阐明重塑染色质结构的分子步骤来了解基因表达是如何在染色质的背景下发生的。组蛋白变异体H_2A.Z在染色质重塑中起关键作用。H_2A.Z标记基因启动子，并已被提出通过形成易于拆卸的核小体来帮助平衡基因转录。然而，H2A.Z核小体被分解的机制仍然不清楚。此前，我使用了一种涉及酵母遗传学、基因组学和生物化学的组合方法来剖析H2A.Z沉积的机制。在本项目中，我将扩展该方法来研究H_2A.Z核小体的分解途径。一个目标是通过筛选在H2A.Z驱逐中有缺陷的突变来识别涉及的基因。然后，这些突变体将被用来研究H2A.Z驱逐在转录激活中的作用。在第二个目标中，这些基因编码的蛋白质将在核小体驱逐试验中进行生化表征。最后，将应用动力学方法来理解H_2A.Z核小体组装和拆解的相反路径是如何保持动态平衡的，以准备用于转录的基因。在人类中，H2A.Z控制着发育和细胞周期调节因子的表达，而编码H2A.Z的基因的过度表达与癌症的进展有关。鉴于染色质结构及其重塑途径的保守性，为酵母系统开发的原理很可能对人类是可翻译的。这项研究可能会导致对 抗癌药物开发的新靶点。

DESCRIPTION (provided by applicant): The timing and the extent of gene expression ultimately control normal cell proliferation and cellular differentiation in humans and other organisms. The packaging of eukaryotic DNA with histone proteins into chromatin inhibits virtually all steps in transcription. As such, chromatin functions not only as a platform to regulae transcription, but also as a filter to prevent aberrant transcription. The remodeling of chromatin structure is integral to all stages of transcription. The biological importance of chromatin remodeling in humans is manifested by the fact that mutations in genes important for chromatin remodeling are associated with numerous cancers. The goal of this project is to understand how gene expression occurs in the context of chromatin by elucidating the molecular steps that remodel chromatin structure. The histone variant H2A.Z is a key player in chromatin remodeling. H2A.Z marks gene promoters and has been proposed to help poise genes for transcription by forming nucleosomes that are predisposed for disassembly. However, the mechanism by which H2A.Z nucleosomes are disassembled remains obscure. Previously, I used a combinatorial approach that involves yeast genetics, genomics and biochemistry to dissect the mechanism of H2A.Z deposition. In this project, I will extend the approach to study the disassembly pathway of H2A.Z nucleosomes. One aim is to identify the genes involved by screening mutants that are defective in H2A.Z eviction. These mutants will then be used to study the role of H2A.Z eviction in transcriptional activation. In the second aim, the proteins encoded by these genes will be characterized biochemically in nucleosome eviction assays. Finally, a kinetic approach will be applied to understand how opposing pathways of assembly and disassembly of H2A.Z nucleosomes are maintained in a dynamic equilibrium to prepare genes for transcription. In humans, H2A.Z controls the expression of developmental and cell cycle regulators, and the overexpression of the gene encoding H2A.Z is linked to cancer progression. Given the conserved nature of chromatin structure and its remodeling pathways, the principles developed for the yeast system are likely translatable to humans. This study could lead to the identification of new targets for anticancer drug development.