Function of PHD Domain Proteins in Chromatin Regulation

描述（由申请人提供）：PHD指（植物同源结构域）模块是一种特征性染色质相关蛋白基序。该模块存在于整个真核蛋白质组中，并且许多蛋白质的PHD指中的突变与癌症、免疫缺陷和精神发育迟滞综合征以及其他遗传疾病相关。我们以前证明，一个子集的PHD手指作为高亲和力结合模块组蛋白H3三甲基化赖氨酸4（H3 K4 me 3）。我们通过离散的PHD指状核蛋白识别H3 K4 me 3，将其与多种不同的功能联系起来，包括提供第一个证据，即破坏组蛋白修饰的读出可能导致遗传性人类疾病。我们的长期目标是全面了解含PHD结构域的蛋白质如何影响染色质动力学以及这些活动与基本核功能和人类疾病过程的关系。在这里，我们专注于多PHD结构域包含蛋白NSD 2（也称为MMSET和WHSC 1），组蛋白赖氨酸甲基转移酶牵连的血液恶性肿瘤多发性骨髓瘤的发病机制。然而，NSD 2调节染色质的分子机制及其酶活性与疾病发病机制的关系还不清楚。我们的初步工作表明，NSD 2在染色质处的主要生理活性是组蛋白H3在赖氨酸36（H3 K36 me 2）处的二甲基化，并且NSD 2-通过H3 K36 me 2催化-驱动骨髓瘤细胞中的致癌编程。 在目标1中，我们建议扩展我们的基因组研究，确定NSD 2在癌症和正常细胞中的全基因组分布，并研究NSD 2活性和染色质靶向在确定H3 K36 me 2染色质景观中的作用。我们还旨在阐明NSD 2 PHD结构域的分子作用模式及其在NSD 2细胞功能调节中的作用。在目标2中，我们将描述 H3 K36甲基化的作用模式。我们将鉴定优先识别H3 K36 me 2的蛋白质，并测试这些蛋白质将NSD 2在染色质上的活性转移到下游生物学结果的假设。我们还将探讨更广泛的假设，即精细水平的生物调控可以通过组蛋白甲基化的微妙变化来实现。目标3的目标是使用我们已经开发的用于功能相关的NSD 2底物的蛋白质组范围发现的新型化学生物蛋白质组学策略来鉴定NSD 2的新底物。最有前途的目标在核途径的调节作用将使用分子和细胞的方法相结合的研究。这些研究将确定由NSD 2调控的新的核信号通路，这些通路可能在人类疾病中发挥作用。这些研究将为组蛋白甲基化如何调节基本的核过程以及这些活动与人类疾病发病机制的关系提供重要的新见解。

DESCRIPTION (provided by applicant): The PHD finger (Plant Homeodomain) module is a signature chromatin-associated protein motif. This module is present throughout eukaryotic proteomes, and mutations in the PHD fingers of many proteins are associated with cancers, immunodeficiency and mental retardation syndromes, and other genetic disorders. We previously demonstrated that a subset of PHD fingers act as high affinity binding modules for histone H3 trimethylated at lysine 4 (H3K4me3). We linked H3K4me3 to multiple different functions via its recognition by discrete PHD finger nuclear proteins, including providing the firs evidence that disrupting the read-out of a histone modification can cause an inherited human disease. Our long-term goal is to develop a comprehensive understanding of how PHD domain-containing proteins impact on chromatin dynamics and the relationship of such activities to fundamental nuclear functions and human disease processes. Here we focus on the multiple PHD domain-containing protein NSD2 (also named MMSET and WHSC1), a histone lysine methyltransferase implicated in the pathogenesis of the hematologic malignancy multiple myeloma. However, the molecular mechanism by which NSD2 regulates chromatin and the relationship of its enzymatic activity to disease pathogenesis is not well understood. Our preliminary work indicates that the primary physiologic activity at chromatin of NSD2 is dimethylation of histone H3 at lysine 36 (H3K36me2), and that NSD2 - via H3K36me2 catalysis - drives oncogenic programming in myeloma cells. In Aim 1, we propose to extend our genomic studies and determine the genome-wide distribution of NSD2 in cancer and normal cells, and investigate the role of NSD2 activity and chromatin targeting in determining the H3K36me2 chromatin landscape. We also aim to elucidate the molecular mode of action for the NSD2 PHD domains and their role in the regulation of NSD2 cellular functions. In Aim 2, we will characterize the mode of action for H3K36 methylation. We will identify proteins that preferentially recognize H3K36me2 and test the hypothesis that these proteins transduce NSD2 activity at chromatin to downstream biological outcomes. We will also explore the broader hypothesis that exquisite level of biological regulation can be achieved by subtle changes in histone methylation. The goal of Aim 3 is to identify new substrates of NSD2 using a novel chemical biological-proteomic strategy we have developed for proteome-wide discovery of functionally-relevant NSD2 substrates. The role of the most promising targets in regulation of nuclear pathways will be investigated using a combination of molecular and cellular approaches. These studies will identify new nuclear signaling pathways that are regulated by NSD2 and that may play a role in human disease. Together these studies will provide important new insights into how histone methylation regulates fundamental nuclear processes and the relationship of these activities to the pathogenesis of human diseases.