Identification of Histone Modification Interaction Networks

描述(由申请人提供)： 真核DNA是通过形成一种称为染色质的核蛋白复合体来压缩的。该复合体的主要蛋白质成分是核心组蛋白H_2A、H_2B、H_3和H_4。核心组蛋白形成一个八聚体复合体，DNA包裹在它周围形成核小体，核小体是基本的重复结构，是高阶染色质折叠的基础。由于将DNA包装到染色质中对DNA的可及性造成了严重的限制，因此调控染色质结构的能力对于需要获得DNA的细胞过程至关重要，如转录、DNA复制和DNA修复。核心组蛋白的翻译后修饰已成为细胞内染色质结构调控的重要机制。因此，核心组蛋白是许多修饰的位置。过去几年出现的组蛋白修饰的一个有趣的方面是，它们不是孤立地发挥作用的。现在有几个不同修改之间的串扰的例子。例如，组蛋白H_2B赖氨酸123的泛素化是组蛋白H_3赖氨酸4和79甲基化所必需的。因此，对一个组蛋白的修饰可以影响其他修饰的存在。我们的建议是结合酵母遗传学、稳定同位素标记和质谱学来全面和定量地鉴定存在于组蛋白修饰之间的相互作用网络。我们将系统地突变酵母核心组蛋白中所有的组蛋白修饰位点。然后，我们将使用稳定同位素标记和质谱学来量化突变菌株中相对于野生型对照的所有组蛋白修饰。通过这种方式，将获得核心组蛋白修饰之间存在的所有串扰的不偏不倚的图像。与公共卫生相关：由于其巨大的线性长度，真核生物的基因组DNA需要高度浓缩才能适应细胞内。DNA通过与被称为组蛋白的蛋白质包装而浓缩成一种名为染色质的复合体。细胞还必须能够调节DNA特定区域的浓缩程度，以便在必要时可以访问DNA。调节染色质缩合的一个重要机制是通过对组蛋白进行修饰。这项提议试图确定组蛋白修饰的不同位置之间存在的相互作用。了解组蛋白修饰的功能对人类健康很重要，因为组蛋白修饰和染色质结构的调节缺陷在癌症等疾病中发挥着关键作用。

DESCRIPTION (provided by applicant): Eukaryotic DNA is compacted through the formation of a nucleoprotein complex known as chromatin. The primary protein components of this complex are the core histones H2A, H2B, H3 and H4. The core histones form an octameric complex around which DNA wraps to form the nucleosome which is the basic repeating structure that serves as the foundation for higher order chromatin folding. As the packaging of DNA into chromatin places severe constraints on the accessibility of DNA, the ability to regulate chromatin structure is critical for cellular processes that require access to DNA such as transcription, DNA replication and DNA repair. The post-translational modification of the core histones has emerged as an important mechanism by which chromatin structure is modulated in cells. Accordingly, the core histones are the sites of numerous modifications. An intriguing aspect of histones modifications, which has emerged in the past few years, is that they do not function in isolation. There are now several examples of cross-talk between different modifications. For example, the ubiquitylation of histone H2B lysine 123 is required for the methylation of histone H3 lysines 4 and 79. Hence, modifications on one histone can influence the presence of other modifications. Our proposal is to combine yeast genetics, stable isotope labeling and mass spectrometry to comprehensively and quantitatively identify the network of interactions that exist between histone modifications. We will systematically mutate all sites of histone modification in the yeast core histones. We will then use stable isotope labeling and mass spectrometry to quantitate all of the histone modifications in the mutant strain relative to a wild type control. In this way, an unbiased picture will be obtained of all the cross-talk that exists between modifications in the core histones. PUBLIC HEALTH RELEVANCE: Due to its enormous linear length, the genomic DNA of eukaryotes needs to be highly condensed to fit inside cells. The DNA is condensed through packaging with proteins known as histones into a complex called chromatin. Cells must also be able to regulate the degree to which specific regions of DNA are condensed so that the DNA can become accessible when necessary. One important mechanism to regulate chromatin condensation is through modifications to the histone proteins. This proposal seeks to identify interactions that exist between different sites of histone modification. An understanding of how histone modifications function is important for human health as defects in the regulation of histone modifications and chromatin structure play critical roles in diseases such as cancer.