Identification of histone H4 methyl-R3 effector proteins in mammalian cells

描述（由申请人提供）：过去十年在我们对翻译后组蛋白修饰如何调节基因表达的理解方面取得了巨大进展。然而，我们对某些组蛋白修饰如何在分子/生物化学水平上发挥其生物学效应的认识要有限得多。精氨酸甲基转移酶（PRMT）对精氨酸3（H4甲基-R3）上的组蛋白H4的甲基化对几种核激素受体的激活至关重要，并且在早期胚胎中受到动态调节。目前，精氨酸甲基化如何导致转录激活尚不清楚，因为尚未鉴定出H4甲基-R3结合蛋白。我计划通过识别和表征特异性识别这种修饰的效应蛋白来确定精氨酸3上组蛋白H4的甲基化如何导致转录激活。我计划确定蛋白质效应器，在识别H4甲基-R3组蛋白尾部在人类胚胎癌（EC）细胞在一个公正的生化屏幕的主要球员。我将通过基于细胞和体外试验确定H4甲基-R3结合蛋白在转录激活中的功能作用。我建议首先确定H4甲基-R3结合蛋白，通过使用一个公正的生化筛选，以确定hovel组蛋白结合蛋白，从EC细胞提取物，使用修改后的组蛋白尾肽。我将确定如何H4甲基-R3结合蛋白（S）的功能，在转录激活的第一个确定的基因调控过程中EC细胞分化。针对H4甲基-R3结合蛋白的siRNA，随后通过微阵列将用于确定通过精氨酸甲基化激活的潜在基因。H4甲基-R3结合蛋白的染色质免疫沉淀，然后与基因组微阵列杂交，将确定效应蛋白直接结合的基因。一旦确定了候选基因，我将使用重组的体外转录系统来确定H4甲基-R3结合如何促进基因激活。基因表达途径的表观遗传调控在细胞命运决定以及正常和病理发育中起着核心作用。阐明组蛋白精氨酸甲基化如何影响胚胎癌细胞转录激活的机制将提高我们对基因调控机制的理解，管理分化和发展。这些研究可能会发现对参与发育的基因激活途径至关重要的新蛋白质，以及癌症治疗的可能治疗靶点。

DESCRIPTION (provided by applicant): The last decade has brought tremendous progress in our understanding of how post-translational histone modifications regulate gene expression. However, our knowledge of how certain histone modifications exert their biological effects on a molecular/biochemical level is far more limited. Methylation of histone H4 on arginine 3 (H4 methyl-R3) by arginine methyltransferases (PRMTs) is critical to the activation of several nuclear hormone receptors and is dynamically regulated in the early embryo. Currently, it is not known how arginine methylation leads to transcription activation as no H4 methyl-R3 binding proteins have yet been identified. I plan to identify how methylation of histone H4 on arginine 3 leads to transcription activation by identifying and characterizing the effector proteins that specifically recognize this modification. I plan to identify protein effectors that are major players in recognizing H4 methyl-R3 on histone tails in human embryonic carcinoma (EC) cells in an unbiased biochemical screen. I will determine the functional roles of the H4 methyl-R3 binding protein(s) in transcription activation through cell based and in vitro assays. I propose to first identify H4 methyl-R3 binding proteins through the use of an unbiased biochemical screen to identify hovel histone-binding proteins from EC cell extract using modified histone-tail peptides. I will determine how the H4 methyl-R3 binding protein(s) function in transcription activation by first identifying the genes regulated during the process of EC cell differentiation. siRNA against the H4 methyl-R3 binding protein followed by microarrays will be employed to determine potential genes activated through arginine methylation. Chromatin immunoprecipitation of the H4 methyl-R3 binding protein followed by hybridization to genomic microarrays will determine genes directly bound by the effector protein. Once candidate genes are identified I will use a reconstituted an in vitro transcription system to determine how the H4 methyl-R3 binding facilitates gene activation. Epigenetic regulation of gene expression pathways play a central role in cell fate determination, and in normal and pathological development. Elucidation of the mechanism of how histone arginine methylation affects transcription activation in embryonic carcinoma cells will improve our understanding of gene regulatory mechanisms governing differentiation and development. These studies may identify new proteins critical to gene activation pathways involved in development as well as possible therapeutic targets for cancer treatment.