Core Histone Tall Interactions and Function in Chromatin

描述（由申请人提供）：在真核细胞核内，DNA与核心组蛋白结合形成核小体;核小体与辅助蛋白进一步组装成称为染色质的多面复合物。这种复合体使得巨大长度的DNA在细胞核的微小体积内有序地包装。此外，染色质复合物的元件直接参与调节细胞核内涉及DNA的多个过程。事实上，细胞核内的大部分信号转导似乎最终指导核心组蛋白的翻译后修饰，并且在一些情况下，进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。在过去的3-5年里，许多努力致力于阐明和生化纯化的调控机制和酶介导的核心组蛋白翻译后修饰。有趣的是，几乎所有这些修饰都发生在被称为核心组蛋白尾部结构域的专门区域内。生物物理实验表明，尾区是在多个水平上调节染色质结构和功能的关键组分。此外，这些结构域中的翻译后修饰被认为调节这种组蛋白尾导向调节。然而，它们定义染色质结构的机制-以及最终潜在DNA的功能-仍然未知。 在这个建议中描述的工作的主要目标是阐明的分子细节和机制，核心组蛋白尾部结构域决定的染色质纤维的结构和功能状态。纤维由核小体串折叠而成，是受尾域调控的关键结构。然而，关于尾域的分子相互作用知之甚少。最近的研究表明，尾巴在核小体内进行精确和局部的相互作用。使用一种新的定点化学作图方法，我们将研究组蛋白尾部的相互作用，在各种模型染色质复合物。我们将集中在潜在的核小体间的相互作用可能是专门参与稳定凝聚的染色质纤维。此外，我们将使用化学保护的方法和专门标记的核心组蛋白的NMR研究核小体内的单个组蛋白尾部的盐依赖性结合稳定性。此外，我们将使用这些相同的方法来检查组蛋白乙酰化的特定模式和染色质重塑活性对组蛋白-DNA相互作用的影响。

DESCRIPTION (provided by applicant): Within the eukaryotic cell nucleus, DNA is associated with core histones to form nucleosomes; which are further assembled with ancillary proteins into 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. In addition, elements of the chromatin complex are directly involved in the regulation of multiple processes within the nucleus that involve DNA. Indeed, a large portion of signal transduction within the cell nucleus appears to ultimately direct the post-translational modification of the core histone proteins and, in several cases, mutations in enzymes that carry out these modifications have been linked to various diseases including cancers in humans. In the last 3-5 years, much effort has been devoted to the elucidation and biochemical purification of the regulatory machinery and enzymes that mediate core histone posttranslational modifications. Interestingly, nearly all of these modifications occur within specialized regions known as the core histone tail domains. Biophysical experiments have shown that the tail domains are key components in regulating the structure and function of chromatin at multiple levels. Moreover, posttranslational modifications in these domains are thought to modulate this histone tail-directed regulation. However, the mechanism by which they define chromatin structures - and ultimately the functionality of the underlying DNA - remains unknown. The primary goal of the work described in this proposal is to elucidate the molecular details and the mechanisms by which the core histone tail domains dictate the structural and functional state of the chromatin fiber. The fiber is formed by the folding up of strings of nucleosomes and is a key structure regulated by the tail domains. However little is known regarding molecular interactions of the tail domains. Recently have shown that the tails make precise and localized interactions within nucleosomes. Using a novel site-directed chemical mapping approach, we will examine histone tail interactions in a variety of model chromatin complexes. We will focus on potential inter-nucleosomal interactions likely to be specifically involved in stabilizing the condensed chromatin fiber. Further, we will use a chemical protection approach and NMR of specifically labeled core histones to study the salt-dependent binding stability of individual histone tails within nucleosomes. In addition, we will use these same methods to examine the effects of specific patterns of histone acetylation and a chromatin remodeling activity on histone-DNA interactions.