Transcriptional Coordination and Gene Regulation by MED12 in the Cardiomyocyte

心肌细胞中 MED12 的转录协调和基因调控

• 批准号: 10735555
• 负责人: KEDRYN K BASKIN
• 金额: $ 58.86万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735555
• 关键词: Architecture; Binding; Biological Assay; CREB1 gene; Calcium; Cardiac; Cardiac Myocytes; Cells; ChIP-seq; Chromatin; Complex; Coupled; Data; Development; Disease; Gel Chromatography; Gene Expression; Gene Expression Regulation; Genes; Genetic Models; Genetic Transcription; Genomics; Goals; Heart; Heart Diseases; Heart failure; Homeostasis; Human; Individual; Knock-out; Link; Mediating; Mediator; Missense Mutation; Modeling; Molecular; Multiprotein Complexes; Mus; Mutation; Phosphotransferases; Population; Proteins; Publishing; RNA Polymerase II; Reporter; Reporter Genes; Reporting; Repression; Role; Serum Response Factor; Testing; Therapeutic; Transcription Repressor; Transcriptional Regulation; Transgenic Mice; Transgenic Model; Transgenic Organisms; Work; chromosome conformation capture; clinically relevant; cohesin; experimental study; heart function; insight; mortality; novel; overexpression; programs; transcription factor; transcriptome sequencing

Heart disease is the leading cause of mortality worldwide and disruption of transcription in cardiomyocytes (CMs) contributes to the progression of heart disease. Transcriptional regulation of some individual genes by transcription factors (TFs) is described. However, much less is known about how functionally related genes, or gene expression programs, are collectively regulated in a coordinated manner in healthy and diseased CMs. The Mediator Complex serves as a bridge to link transcriptional machinery and TFs to control transcription, but the molecular mechanisms of transcriptional regulation by Mediator are not well-understood. Mediator is a large multiprotein complex organized as submodules, with the CDK8 kinase submodule regulating RNA Polymerase II transcriptional activity. MED12 is an essential Mediator component, and within the kinase submodule, is required for CDK8 kinase activity. Although traditionally considered a transcriptional repressor via the CDK8 kinase submodule, our published data demonstrates that increased MED12 is also able to directly activate transcription. Our preliminary studies indicate that MED12 levels are increased in failing hearts from humans and mice, sup- porting the clinical relevance of this novel MED12 function in mis-regulation of transcription in heart disease. Our published studies in mice with CM-specific Med12 deletion demonstrate that MED12 regulates a calcium-handling gene expression program, through direct interaction with the TF MEF2. We identified additional TFs that interact with MED12, and these findings suggest that MED12 has multiple transcriptional functions depending on its interactions, but its functions have not been delineated. To determine the contribution of MED12 to transcriptional mis-regulation in heart disease, it is crucial to define its molecular interactions and functions in CMs and identify its cell-specific roles. Subpopulations of kinase submodules have been reported in other cells, but their functions have not been defined. Additionally, functions of individual kinase submodule proteins have not been investigated for their roles in alternative kinase subcomplexes or for functions independent of the kinase submodule. Our published CM-specific Med12 knockout model (cKO) and newly generated CM Med12 transgenic model (cTg) both develop heart failure, emphasizing the critical contribution of MED12 to transcriptional homeostasis in CMs. We will use our Med12 genetic models to delineate the functions of MED12 in CMs. We will identify MED12 kinase subpopulations, interactions with TFs, and genomic interactions in normal CMs and those with MED12 overexpression. We will also assess the ability of a human MED12 missense mutations to bind TFs and kinase submodule proteins using hi-PSCs. The long-term goal of my lab is to uncover mechanisms that control coordinated transcriptional programs in the heart. In this proposal we aim to 1) determine the mechanisms of MED12-regulated transcription in CMs, and 2) identify the transcriptional mechanisms of MED12- driven heart failure. Consequently, a better understanding of the molecular mechanisms governing cardiac gene expression will provide new insight into therapeutic strategies to restore cardiac function during disease.

心脏病是全球死亡率的主要原因，心肌细胞（CMS）转录的破坏促进了心脏病的发展。描述了通过转录因子（TFS）对某些单个基因的转录调节。然而，关于与健康和患病的CMS以协调的方式共同调节功能相关的基因或基因表达程序的知之甚少。介体复合物是将转录机械和TF连接以控制转录的桥梁，但是调解器的转录调节的分子机制并不理解。介体是一种大型多蛋白络合物，以子模型组织，CDK8激酶子模块调节RNA聚合酶II转录活性。 Med12是必不可少的介体成分，在激酶子模块中是CDK8激酶活性所必需的。尽管传统上通过CDK8激酶子模块视为转录阻遏物，但我们已发布的数据表明，增加的Med12也能够直接激活转录。我们的初步研究表明，在人类和小鼠的心脏失败的心脏中，MED12水平升高，这支持了这种新型MED12在心脏病中转录错误调节中的临床相关性。我们在CM特异性MED12缺失的小鼠中发表的研究表明，Med12通过与TF MEF2直接相互作用来调节钙处理基因表达程序。我们确定了与Med12相互作用的其他TF，这些发现表明Med12取决于其相互作用的多个转录功能，但尚未描述其功能。为了确定MED12对心脏病中转录错误调节的贡献，定义其分子相互作用和功能至关重要，并确定其细胞特异性作用。在其他细胞中已经报道了激酶子模型的亚群，但尚未定义它们的功能。此外，尚未研究单个激酶子复核蛋白的功能，因为它们在替代激酶亚复合物中的作用或独立于激酶子模块的功能。我们发表的CM特异性MED12基因敲除模型（CKO）和新生成的CM Med12转基因模型（CTG）都会发展为心力衰竭，强调了Med12对CMS转录稳态的关键贡献。我们将使用MED12遗传模型来描述Med12在CMS中的功能。我们将确定MED12激酶亚群，与TFS的相互作用以及正常CMS中的基因组相互作用以及MED12过表达的基因组相互作用。我们还将评估使用HI-PSC结合TFS和激酶子模块蛋白的人类Med12错义突变的能力。我实验室的长期目标是发现控制心脏中协调转录程序的机制。在此提案中，我们的目的是1）确定CMS中Med12调节转录的机制，以及2）确定Med12驱动心力衰竭的转录机制。因此，更好地了解控制心脏基因表达的分子机制将为恢复疾病期间心脏功能的治疗策略提供新的见解。