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

描述（由申请人提供）：在过去十年中，我们对翻译后组蛋白修饰如何调节基因表达的理解取得了巨大进展。然而，我们对某些组蛋白修饰如何在分子/生化水平上发挥其生物学作用的了解远远有限。精氨酸甲基转移酶（PRMTs）对精氨酸3上的组蛋白H4 （H4甲基化- r3）的甲基化对几种核激素受体的激活至关重要，并在胚胎早期受到动态调节。目前还不清楚精氨酸甲基化是如何导致转录激活的，因为还没有发现H4甲基化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上的组蛋白H4甲基化如何导致转录激活。我计划在无偏倚的生化筛选中鉴定在识别人类胚胎癌（EC）细胞组蛋白尾部的H4甲基- r3中起主要作用的蛋白质效应物。我将通过细胞和体外实验确定H4甲基- r3结合蛋白在转录激活中的功能作用。我建议首先通过使用无偏倚的生化筛选来鉴定H4甲基- r3结合蛋白，使用修饰的组蛋白尾部肽从EC细胞提取物中鉴定hovel组蛋白结合蛋白。我将通过首先识别EC细胞分化过程中调控的基因来确定H4甲基- r3结合蛋白在转录激活中的作用。针对H4甲基- r3结合蛋白的siRNA，随后采用微阵列来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，将确定直接与效应蛋白结合的基因。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的转录激活的机制，将提高我们对分化和发育的基因调控机制的理解。这些研究可能会发现在发育过程中对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.