A high-throughput approach towards deciphering the histone code

描述(申请人提供)：当前分子生物学的一个主要挑战是了解组蛋白翻译后修饰如何协同工作来调节细胞中的DNA模板化过程。许多实验室的研究已经证实，染色质结构在DNA功能的各个方面发挥着基础性的作用，包括基因转录、DNA复制、重组和修复。染色质结构的核心是核小体核心颗粒，它由DNA和组蛋白组成。值得注意的是，组蛋白上存在着大量的共价修饰，如乙酰化、甲基化、泛素化和磷酸化。目前还不清楚它们是如何发挥作用的，但越来越多的证据表明，它们以组蛋白密码的形式发挥作用，调节基于染色质的事件。“组蛋白密码”假说的提出是为了解释在组蛋白上发现的各种修饰是如何发挥作用的(Strahl&Allis，2000)。它指出，单一和/或组合修饰，在一个或多个组蛋白上，招募包含结合修饰的特定结构域的效应蛋白(S)。虽然这一想法是在2000年正式提出的，但在破译组蛋白密码及其结合蛋白的全部范围方面进展有限。这一有限的进展主要是由于缺乏一种允许高通量筛选与修饰的组蛋白结合的效应蛋白的技术方法。为了解决这个问题，我们将开发一个全面的修饰组蛋白多肽文库，可以用来快速有效地筛选结合独特修饰模式的蛋白质。我们的方法将是制造高含量的多肽阵列，这些多肽阵列将被已知与染色质相关的人类蛋白质探测。蛋白质/多肽阵列的一般技术已经存在；然而，我们的特殊方法以前从未被尝试过--但如果我们要“破解”组蛋白密码的基本基础，这种方法是必不可少的。这些研究如果成功，对生物和生物医学界的影响将是巨大的。这是因为组蛋白修饰会影响所有需要DNA的过程。因此，组蛋白修饰酶的突变或放松调控会导致包括癌症在内的许多人类疾病。因此，我们对人类疾病的基本理解可能取决于对组蛋白密码的复杂性和语言的理解。染色质组织、DNA包装及其可及性方面的缺陷是人类疾病的主要原因，包括癌症和许多发育缺陷。这些研究将揭示基于DNA的活动是如何受到监管的，这将解决这些公共卫生问题的根本原因。

DESCRIPTION (provided by applicant): A major challenge in current molecular biology is to understand how histone post- translational modifications work in concert to regulate DNA-templated processes in the cell. Work from many laboratories has established that chromatin structure plays a fundamental role in every aspect of DNA function, including gene transcription, DNA replication, recombination and repair. At the heart of chromatin structure is the nucleosome core particle, which is comprised of DNA and histone proteins. Significantly, a vast number of covalent modifications such as acetylation, methylation, ubiquitylation and phosphorylation exist on histones. How they all function is still not clear, but growing evidence suggests that they work in the form of a 'histone code' to regulate chromatin-based events. The 'histone code' hypothesis was proposed to explain how the variety of modifications found on histones function (Strahl & Allis, 2000). It stated that single and/or combinatorial modifications, on one or more histones, recruits effector proteins containing specialized domains that bind the modification(s). While the idea was formally proposed in 2000, there has been limited progress in deciphering the full extent of the histone code and the proteins that bind to them. This limited progress is primarily due to a lack of a technical approach that allows for high-throughput screening of effector proteins that bind to modified histones. To address this problem, we will develop a comprehensive library of modified histone peptides that can be used to rapidly and efficiently screen for proteins which bind unique modification patterns. Our approach will be to make high-content peptide arrays that will be probed with human proteins known to associate with chromatin. The general technology of protein/peptide arrays already exists; however, our particular approach has not been tried before - but is essential to do if we are to "crack" the underlying basis of the histone code. The impact of these studies, if successful, would be enormous on the biological and biomedical community. This is due to the fact that histone modifications impact all processes requiring access to DNA. As such, mutations or deregulation of histone modifying enzymes cause a number of human diseases including cancer. Thus, our fundamental understanding of human disease may depend on understanding the complexity and language of the histone code. Defects in chromatin organization, DNA packaging and its accessibility is a major cause of human disease, including cancer and numerous developmental defects. These studies will reveal how DNA-based activities are regulated, which will address the underlying cause of these public health concerns.