Core Histone Tall Interactions and Function in Chromatin

描述(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体。这种复合体带来了极大长度的DNA在微小的细胞核内的有序包装。此外，染色质复合体的元件直接参与了涉及DNA的核内多个过程的调节。事实上，细胞核内的很大一部分信号转导似乎最终指导了核心组蛋白的翻译后修饰，在某些情况下，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。在过去的3-5年里，许多工作都致力于阐明和生化纯化调节机制和酶，这些调控机制和酶介导了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区的特殊区域内。生物物理实验表明，尾部结构域是在多个水平上调节染色质结构和功能的关键成分。此外，这些结构域中的翻译后修饰被认为调节了这种组蛋白尾部定向调控。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。 这项工作的主要目标是阐明核心组蛋白尾部结构域决定染色质纤维结构和功能状态的分子细节和机制。纤维是由一串串核小体折叠而成，是受尾部结构域调控的关键结构。然而，关于尾部结构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。使用一种新的定位化学作图方法，我们将研究各种模型染色质复合体中的组蛋白尾部相互作用。我们将集中在潜在的核小体之间的相互作用，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方法和特殊标记的核心组蛋白的核磁共振来研究核小体中单个组蛋白尾巴的盐依赖结合稳定性。此外，我们将使用这些相同的方法来检验组蛋白乙酰化的特定模式和染色质重塑活动对组蛋白-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.