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

描述(由申请人提供)：在过去的十年中，我们在理解翻译后组蛋白修饰如何调节基因表达方面取得了巨大的进步。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效应的了解要有限得多。精氨酸甲基转移酶(PRMTs)通过精氨酸甲基转移酶(PRMTs)将组蛋白H4甲基化到精氨酸3(H4甲基-R3)上，对多种核激素受体的激活起关键作用，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，因为还没有鉴定出H4甲基-R3结合蛋白。我计划通过识别和表征识别这种修饰的效应蛋白，来确定组蛋白H4在精氨酸3上的甲基化是如何导致转录激活的。我计划在一个无偏见的生化筛选中，确定在人类胚胎癌细胞(EC)的组蛋白尾部识别H4甲基-R3的主要蛋白质效应物。我将通过细胞和体外实验来确定H4甲基-R3结合蛋白(S)在转录激活中的功能作用。我建议首先通过使用无偏见的生化筛选来鉴定H4甲基-R3结合蛋白，以使用改良组蛋白尾肽从EC细胞提取液中鉴定HOVELL组蛋白结合蛋白。我将首先确定EC细胞分化过程中调节的基因，以确定H4甲基-R3结合蛋白(S)在转录激活中的作用。针对H4甲基-R3结合蛋白的siRNA将被用来确定通过精氨酸甲基化激活的潜在基因。染色质免疫沉淀H4甲基-R3结合蛋白，然后与基因组微阵列杂交，将确定与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。基因表达通路的表观遗传调控在决定细胞命运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有助于我们更好地理解调控分化和发育的基因调控机制。这些研究可能会确定对参与发育的基因激活途径至关重要的新蛋白质，以及可能的癌症治疗靶点。

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.