Histone Tail Interactions and Functions in Chromatin

描述（由申请人提供）：真核细胞细胞核内的DNA与组蛋白和其他蛋白质组装，形成一个复杂的，多方面的复合体，称为染色质。染色质不仅用于包装基因组，而且染色质结构的元素与基因控制机制密切相关。核心组蛋白尾部结构域对于染色质内多层结构的形成至关重要，并且细胞核内的大部分信号转导最终指导这些结构域的翻译后修饰，以促进转录等核过程。在一些情况下，执行这些修饰的酶的突变与包括人类癌症在内的各种疾病有关。然而，尾结构域定义染色质结构的分子机制——以及最终潜在DNA的功能——仍然知之甚少。这项工作的主要目标是阐明核心组蛋白尾部结构域决定高阶染色质结构形成的分子机制，以及这些结构域内特定赖氨酸的乙酰化如何改变尾部结构和相互作用以允许基因表达。我们的目标是1)表征模型核小体和核小体阵列中H3和H4尾结构域的短程和远程核小体间相互作用，2)确定连接蛋白H1、结构转录因子HMGN或染色质重塑活性是否特异性地改变尾结构域的相互作用，3)定量评估选定尾结构域的结合以及乙酰化对这些相互作用在重组和天然染色质中的影响。我们将使用几种新方法，包括尾部dna相互作用的定向化学作图，紫外激光交联方法和其他化学探测方法来研究尾部结构域的结构和相互作用。此外，我们将使用化学保护方法和特定标记的核心组蛋白的NMR来定量评估核小体内单个组蛋白尾部的盐依赖性结合稳定性。这项工作将促进我们对影响基因表达控制和最终影响癌症等疾病的关键分子机制的理解。在真核细胞细胞核内，DNA与核心组蛋白和其他蛋白质相关，形成称为染色质的多面复合体。这种复合物在细胞核的微小体积内有序地包装了巨大长度的DNA，并直接整合到包括基因表达调节在内的多个过程中。该项目的结果将阐明被称为组蛋白尾部结构域的关键元素定义和调节染色质结构和基因表达的分子机制，从而使人们更好地了解与癌症和遗传疾病相关的基本过程。

DESCRIPTION (provided by applicant): DNA within the eukaryotic cell nucleus is assembled with histones and other proteins to form a complicated, multifaceted complex known as chromatin. Chromatin not only serves to package the genome but elements of chromatin structure have been intimately integrated into gene control mechanisms. The core histone tail domains are essential for the formation of multiple levels of structure within chromatin and a large portion of signal transduction within the nucleus ultimately directs posttranslational modification of these domains in order to facilitate nuclear processes such as transcription. In several cases, mutations in enzymes that carry out these modifications have been linked to various diseases including cancers in humans. However, the molecular mechanisms by which the tail domains define chromatin structures - and ultimately the functionality of the underlying DNA - remain poorly understood. The primary goal of the work described in this proposal is to elucidate the molecular mechanisms by which the core histone tail domains dictate the formation of higher order chromatin structures and how acetylation of specific lysines within these domains alters tail structures and interactions to allow for gene expression. Our aims are to 1) characterize short-range and long-range inter- nucleosomal interactions of the H3 and H4 tail domains in model nucleosomes and nucleosome arrays, 2) Determine whether linker histone H1, the architectural transcription factor HMGN or a chromatin remodeling activity specifically alters interactions of the tail domains and to 3) quantitatively assess binding of selected tail domains and the effect of acetylation on these interactions in reconstituted and native chromatin. We will use several novel approaches including site-directed chemical mapping of tail-DNA interactions, a UV laser crosslinking approach, and other chemical probing approaches to investigate structures and interactions of the tail domains. Further, we will use a chemical protection approach and NMR of specifically labeled core histones to quantitatively assess the salt-dependent binding stability of individual histone tails within nucleosomes. This work will advance our understanding of critical molecular mechanisms that impinge upon control of gene expression and ultimately diseases such as cancer. PUBLIC HEALTH RELEVANCE Within the eukaryotic cell nucleus, DNA is associated with core histones and other proteins to form a multi-faceted complex known as chromatin. This complex brings about the orderly packaging of the immense length of DNA within the tiny volume of the nucleus and is directly integrated into multiple processes including regulation of gene expression. The results of this project will illuminate the molecular mechanisms by which critical elements known as the histone tail domains define and regulate chromatin structure and gene expression and thus allow a greater understanding of fundamental processes related to cancer and genetic diseases.