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.

心脏病是世界范围内导致死亡的主要原因，心肌细胞(CMS)转录中断导致心脏病的进展。介绍了转录因子对某些基因的转录调控作用。然而，对于健康和患病的CMS中，功能相关的基因或基因表达程序是如何以协调的方式集体调控的，我们知道的要少得多。介体复合体是连接转录机制和转录因子调控转录的桥梁，但介体调控转录的分子机制还不是很清楚。介体是一个大的多蛋白复合体，以亚模块的形式组织，CDK8激动子模块调节RNA聚合酶II的转录活性。MED12是一种重要的介体成分，在激活子模块中，是CDK8激活所必需的。虽然传统上被认为是通过CDK8激动子模块的转录抑制物，但我们发表的数据表明，增加的MED12也能够直接激活转录。我们的初步研究表明，在人类和小鼠衰竭的心脏中，MED12的水平增加，这支持了这一新的MED12功能在心脏病转录错误调节中的临床意义。我们发表的对CM特异性MED12缺失的小鼠的研究表明，MED12通过与转铁蛋白MEF2直接相互作用来调节钙处理基因的表达程序。我们发现了更多与MED12相互作用的转录因子，这些发现表明MED12根据其相互作用具有多种转录功能，但其功能尚未被描述。要确定MED12在心脏病转录失控中的作用，关键是要确定它在CMS中的分子相互作用和功能，并确定它在细胞中的特异性作用。在其他细胞中已经报道了不同亚群的激酶，但其功能尚未确定。此外，还没有研究单独的激酶子模块蛋白在替代的激酶子复合体中的作用或独立于激酶子模块的功能。我们发表的CM特异的MED12基因敲除模型(CKO)和新产生的CM MED12转基因模型(CTG)都发生了心力衰竭，强调了MED12对CMS转录动态平衡的关键贡献。我们将使用我们的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，与转录因子的相互作用，以及正常CMS和MED12过表达的CMS的基因组相互作用。我们还将使用hi-PSCs评估人类MED12错义突变结合TF和激活子模块蛋白的能力。我的实验室的长期目标是发现心脏中控制协调转录程序的机制。在这项建议中，我们的目标是1)确定MED12在CMS中调节转录的机制，以及2)确定MED12驱动的心力衰竭的转录机制。因此，更好地了解心脏基因表达的分子机制将为疾病期间恢复心脏功能的治疗策略提供新的见解。