Function of PHD Domain Proteins in Chromatin Regulation

描述(由申请人提供)：PHD Finger(植物同源结构域)模块是一个标志性的染色质相关蛋白基序。这个模块存在于真核蛋白质组中，许多蛋白质在PhD手指中的突变与癌症、免疫缺陷和智力低下综合征以及其他遗传疾病有关。我们以前证明了PHD指的一个子集作为组蛋白H3的高亲和力结合模块，组蛋白H3在赖氨酸4位三甲基化(H3K4me3)。我们通过离散的PhD手指核蛋白的识别将H3K4me3与多种不同的功能联系起来，包括提供了首次证据表明干扰组蛋白修饰的读出会导致遗传性人类疾病。我们的长期目标是全面了解包含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，我们关注的是含有多个PHD结构域的蛋白NSD2(也称为MMSET和WHSC1)，它是一种组蛋白赖氨酸甲基转移酶，与血液系统恶性多发性骨髓瘤的发病机制有关。然而，NSD2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尚不清楚。我们的初步工作表明，NSD2在染色质上的主要生理活性是组蛋白H3在36位赖氨酸(H3K36me2)的二甲基化，并且NSD2通过H3K36me2催化驱动骨髓瘤细胞的致癌编程。在目标1中，我们建议扩大我们的基因组研究，确定NSD2在肿瘤和正常细胞中的全基因组分布，并研究NSD2活性和染色质靶向在确定H3K36me2染色质景观中的作用。我们还旨在阐明NSD2 PhD结构域的分子作用模式及其在调节NSD2细胞功能中的作用。在目标2中，我们将描述 H3K36甲基化的作用模式。我们将识别优先识别H3K36me2的蛋白质，并测试这些蛋白质将染色质上的NSD2活性转导到下游生物结果的假设。我们还将探索更广泛的假设，即精细的生物调节水平可以通过组蛋白甲基化的细微变化来实现。目标3的目标是使用我们为在蛋白质组范围内发现功能相关的NSD2底物而开发的一种新的化学、生物-蛋白质组学策略来鉴定NSD2的新底物。将使用分子和细胞相结合的方法来研究最有希望的靶标在核途径调控中的作用。这些研究将确定受NSD2调控并可能在人类疾病中发挥作用的新的核信号通路。总之，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.