Protein methyltransferases as transcriptional coregulators

描述(申请人提供)：核受体(NRs)和其他DNA结合转录因子通过招募辅助调节蛋白到目标基因的启动子来调节其目标基因的转录。根据启动子的调控背景，许多共同调节者可以作为辅助激活因子或辅助抑制因子来辅助NRs。然而，具体的辅调节因子是作为辅助激活因子还是辅助抑制因子发挥作用的机制尚不清楚，这将是本申请的中心焦点。转录抑制涉及辅阻遏子复合体的招募，该复合体通常包括去乙酰化的酶，并在组蛋白上形成抑制性甲基化标记。特别是，基因启动子中组蛋白H3(H3K9)的赖氨酸9的二甲基化和三甲基化与基因抑制有关。小鼠对常染色质相关的H3K9甲基转移酶G9a和GLP的研究表明，这两种酶负责细胞中H3K9的大部分单甲基化和去甲基化。基因敲除小鼠的结果加上额外的生化研究表明，G9a和GLP至少在部分功能上是异源二聚体伙伴。G9a还与辅阻遏物复合体有关，该复合体介导了几种抑制性转录因子的作用。G9a和GLP也可以作为NRS的共激活因子，这表明G9a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的调节环境。本项目的目的是了解G9a和GLP对辅激活子和辅阻遏子的作用机制。核心假设是，特定的蛋白质-蛋白质相互作用决定了G9a和GLP在给定启动子上是作为辅助激活因子还是辅助抑制因子发挥作用。在其他蛋白质-蛋白质相互作用中，G9a和GLP优先与组蛋白H3结合的能力将被研究，因为它在辅助调节功能中的作用是在赖氨酸9处被二甲基化的。此外，还将定义G9a和GLP功能的共同、不同和互补方面。为此，将确定G9a和GLP结构域的结构域和特定的蛋白质-蛋白质相互作用，这些结构域对于它们作为辅助激活因子和辅助抑制因子的功能是重要的。将对瞬时转染的报告基因和G9a和GLP的内源性靶基因进行分析。因此，这些研究将极大地扩展我们对协调控子和组蛋白修饰对基因转录调控的具体贡献的理解。此外，由于NRs在内分泌和代谢生理的正常和病理调节中发挥着许多关键作用，拟议的研究将提供对人类健康具有重要意义的新知识。项目简介拟议的项目将扩大我们对各种激素如何调节特定基因的活动，从而控制性发育、对压力的反应以及葡萄糖和脂肪酸新陈代谢等重要生理过程的了解。同样的激素在许多疾病的发生、发展和治疗中也发挥着重要作用，包括癌症、糖尿病和心脏病。

DESCRIPTION (provided by applicant): 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 demethylation 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. PROJECT NARRATIVE The proposed project will extend our knowledge of how a variety of hormones regulate the activities of specific genes and thereby control important physiological processes such as sexual development, response to stress, and glucose and fatty acid metabolism. The same hormones also play important roles in the onset, progression, and treatment of many diseases, including cancer, diabetes, and heart disease.