Function of ING PHD domains in chromatin regulation

描述(申请人提供)：PhD手指是一种标志性的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性疾病有关。我们的长期目标是建立一个全面的分子理解，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。在这项提议中要检验的一般假设是，PHD结构域识别特定的甲基化的组蛋白标记。初步工作表明，几个PHD结构域与组蛋白H3在赖氨酸4甲基化(H3K4me3)上具有高亲和力和特异性。通过模型PhD结构域表征H3K4me3识别的生理作用应该有助于阐明染色质动力学的破坏如何导致多种病理状态。在这里，我们提出了一系列的生化、细胞和蛋白质组学分析来研究PHD结构域的分子活性。(1)确定ING2 PHD结构域在H3K4me识别染色质中的分子功能。生物化学和功能研究被用来研究ING2抑癌蛋白的PHD结构域是H3K4me3的特异性效应分子的假设。我们将(I)阐明ING2 PhD结构域对H3K4me状态的专一性的分子基础，(Ii)确定H3K4me如何影响ING2相关的组蛋白去乙酰基酶活性，以及(Iii)表征肌醇磷脂对ING2-H3K4me3相互作用的调节。(2)阐明ING2 PhD结构域识别H3K4me的细胞功能。在前期工作的基础上，我们将研究ING2 PhD结构域与H3K4me3急性基因抑制有关的假设。我们将表征ING2 PHD结构域与H3K4me在目标基因上的生理相互作用，并确定这些相互作用如何影响基因表达程序。我们还将测试选定的核因子如何调节ING2-H3K4me的功能。(3)表征具有不同于ING2功能的特定PhD手指的H3K4Me识别活性。初步工作表明，除了ING2，其他ING蛋白的PHD结构域和RAG2重组酶的PHD结构域与H3K4me3结合，可能将H3K4me3与不同的核过程联系起来。提出了生化和功能的方法来研究这些含有PhD手指的蛋白质识别H3K4me3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.