Function of ING PHD domains in chromatin regulation

描述（由申请人提供）：PHD指是一种特征性染色质相关蛋白基序，其突变与癌症、免疫缺陷综合征和其他遗传疾病相关。我们的长期目标是从分子水平全面了解PHD结构域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。在这个提议中要测试的一般假设是PHD结构域识别特定的甲基化组蛋白标记。初步工作表明，几个PHD结构域结合具有高亲和力和特异性的组蛋白H3甲基化赖氨酸4（H3 K4 me 3）。H3 K4 me 3-识别模型PHD域的生理作用的表征应该有助于阐明如何在染色质动力学的中断可以有助于许多病理状态。在这里，一系列的生化，细胞和蛋白质组学分析提出了调查的PHD结构域的分子活性。(1)确定ING 2 PHD结构域在染色质上识别H3 K4 me中的分子功能。生物化学和功能的研究提出了调查的假说，PHD结构域的ING 2肿瘤抑制蛋白是一个特定的效应分子H3 K4 me 3。我们将（i）阐明ING 2 PHD结构域对H3 K4 me状态特异性的分子基础，（ii）确定H3 K4 me如何影响ING 2相关的组蛋白脱乙酰酶活性，（iii）表征磷酸肌醇对ING 2-H3 K4 me 3相互作用的调节。(2)阐明ING 2 PHD结构域识别H3 K4 me的细胞功能。基于前期工作，我们将研究ING 2 PHD结构域将H3 K4 me 3与急性基因阻遏联系起来的假设。我们将表征ING 2 PHD结构域与H3 K4 me在靶基因处的生理相互作用，并确定这些相互作用如何影响基因表达程序。我们还将测试选择的核因子如何调节ING 2-H3 K4 me功能。(3)表征具有不同于ING 2的功能的特定PHD指状物的H3 K4 me识别活性。初步研究表明，除了ING 2，其他ING蛋白和RAG 2重组酶的PHD结构域结合H3 K4 me 3，可能将H3 K4 me 3连接到不同的核过程。提出了生物化学和功能的方法来研究H3 K4 me 3-识别这些PHD指蛋白的生物学。

DESCRIPTION (provided by applicant): The PHD finger is a signature chromatin-associated protein motif, mutations in which are associated with cancers, immunodeficiency syndromes, and other genetic disorders. Our long-term goal is to develop a comprehensive molecular understanding of how PHD domains impact on chromatin dynamics and the relationship of such activities to fundamental nuclear functions and human disease processes. The general hypothesis to be tested in this proposal is that PHD domains recognize specific methylated histone marks. Preliminary work indicates that several PHD domains bind with high affinity and specificity to histone H3 methylated at lysine 4 (H3K4me3). Characterization of the physiologic role of H3K4me3-recognition by model PHD domains should be instrumental for elucidating how disruption in chromatin dynamics can contribute to numerous pathologic states. Here, a series of biochemical, cellular, and proteomic analyses are proposed to investigate the molecular activities of PHD domains. (1) To determine the molecular function of the ING2 PHD domain in H3K4me recognition at chromatin. Biochemical and functional studies are proposed to investigate the hypothesis that the PHD domain of the ING2 tumor suppressor protein is a specific effector molecule of H3K4me3. We will (i) elucidate the molecular basis of ING2 PHD domain specificity for H3K4me states, (ii) determine how H3K4me impacts on ING2-associated histone deacetylase activity, and (iii) characterize regulation of the ING2-H3K4me3 interaction by phosphoinositides. (2) To elucidate the cellular functions of H3K4me recognition by the ING2 PHD domain. Based on preliminary work, we will investigate the hypothesis that the ING2 PHD domain links H3K4me3 to acute gene repression. We will characterize the physiologic interaction of the ING2 PHD domain with H3K4me at target genes, and determine how these interactions impact on gene expression programs. We will also test how select nuclear factors regulate ING2-H3K4me functions. (3) To characterize the H3K4me-recognition activity of specific PHD fingers with functions distinct from ING2. Preliminary work indicates that besides ING2, the PHD domains of the other ING proteins and that of the RAG2 recombinase bind H3K4me3, potentially linking H3K4me3 to diverse nuclear processes. Biochemical and functional approaches are proposed to study the biology of H3K4me3-recognition by these PHD fingers-containing proteins.