Protein methyltransferases as transcriptional coregulators

核受体（NR）和其他DNA结合转录因子通过将辅调节蛋白募集到靶基因的启动子来调节其靶基因的转录。许多协同调节因子 可以作为共激活子或共阻遏子辅助NR，这取决于启动子的调控环境。然而，一个特定的辅调节子是否作为辅激活子或辅抑制子的功能的机制是未知的，将是本申请的中心焦点。转录 阻遏涉及辅阻遏物复合物的募集，辅阻遏物复合物通常包括使组蛋白去乙酰化并在组蛋白上产生阻遏性甲基化标记的酶。特别地，基因启动子中组蛋白H3（H3 K9）的赖氨酸9的二-和三甲基化与基因阻遏相关。 常染色质相关的H3 K9甲基转移酶G9 a和GLP的敲除小鼠研究表明，这两种酶负责H3的大部分单-和二甲基化 K9细胞敲除小鼠的结果加上额外的生化研究表明，G9 a和GLP作为异源二聚体伴侣发挥至少一些功能。G9 a也与 介导几种抑制性转录因子作用的辅阻遏物复合物。G9 a和GLP也可以作为NR的共激活因子，这表明G9 a可能作为转录激活和抑制之间的调节开关发挥关键作用，这取决于特定启动子上的调节环境。本研究的目的是了解G9 a和GLP对辅激活子和辅抑制子的作用机制。核心假设是特定的蛋白质- 蛋白质相互作用决定了G9 α和GLP在给定启动子上是作为共激活子还是作为共阻遏子起作用。在其他蛋白质-蛋白质相互作用中，将研究G9 a和GLP优先结合在赖氨酸9处二甲基化的组蛋白H3（本实验室最近发现）的能力，以确定其在辅助调节剂功能中的作用。此外，将定义G9 a和GLP功能的共同、不同和互补方面。为此，将确定G9 a和GLP结构域的结构域和特异性蛋白质-蛋白质相互作用，这对于它们作为共激活剂和共阻遏物的功能是重要的。将对两种瞬时 转染的报告基因和G9 a和GLP的内源性靶基因。因此，这些研究将显着扩展我们的理解的具体贡献的辅助调节和组蛋白修饰的基因转录调控。此外，由于NR在内分泌和代谢生理学的正常和病理调节中起着许多关键作用，因此拟议的研究将提供对人类健康具有重要意义的新知识。

Nuclear receptors (NRs) and other DNA-binding transcription factors regulate transcription of their target genes by recruiting coregulator proteins to the promoter of the target genes. Many coregulators can assist NRs as either coactivators or corepressors, depending on the regulatory context of the promoter. However, the mechanisms that govern whether a specific coregulator functions as coactivator or corepressor is unknown and will be a central focus of this application. Transcriptional repression involves recruitment of corepressor complexes which often include enzymes that deacetylate and make repressive methylation marks on histones. In particular, di- and trimethylation of lysine 9 of histone H3 (H3 K9) in gene promoters has been associated with gene repression. Knock-out mouse studies of the euchromatin-associated H3 K9 methyltransferases G9a and GLP indicated that these two enzymes are responsible for the majority of mono- and dimethylation of H3 K9 in cells. The knock-out mouse results plus additional biochemical studies indicate that G9a and GLP function as heterodimer partners for at least some of their functions. G9a is also associated with corepressor complexes that mediate the effects of several repressive transcription factors. G9a and GLP can also function as coactivators for NRs, suggesting that G9a may play a critical role as a regulatory switch between activation and repression of transcription, depending on the regulatory context on a particular promoter. The goal of this project is to understand the mechanisms of coactivator and corepressor function by G9a and GLP. The central hypothesis is that specific protein- protein interactions determine whether G9a and GLP function as coactivators or corepressors on a given promoter. Among other protein-protein interactions, the ability of G9a and GLP to bind preferentially to histone H3 that is dimethylated at lysine 9 (recently discovered in this laboratory) will be investigated for its role in coregulator function. In addition, common, distinct, and complementary aspects of G9a and GLP function will be defined. Toward that end, the domains and specific protein- protein interactions of the domains of G9a and GLP which are important for their functions as coactivators and corepressors will be determined. Analyses will be performed on both transiently transfected reporter genes and endogenous target genes of G9a and GLP. These studies will thus significantly extend our understanding of the specific contributions of coregulators and histone modifications to transcriptional regulation of genes. In addition, since NRs play many critical roles in normal and pathological regulation of endocrine and metabolic physiology, the proposed studies will provide new knowledge that has important implications for human health.