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.

心脏病是全世界死亡的主要原因，心肌细胞 (CM) 转录的破坏会导致心脏病的进展。描述了转录因子 (TF) 对某些单个基因的转录调控。然而，对于功能相关基因或基因表达程序如何在健康和患病的 CM 中以协调的方式共同调控，人们知之甚少。 Mediator Complex作为连接转录机器和转录因子来控制转录的桥梁，但Mediator转录调控的分子机制尚不清楚。 Mediator 是一个大型多蛋白复合物，组织为子模块，其中 CDK8 激酶子模块调节 RNA 聚合酶 II 转录活性。 MED12 是一种重要的介体成分，在激酶子模块内，是 CDK8 激酶活性所必需的。尽管传统上被认为是通过 CDK8 激酶子模块实现的转录抑制因子，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，人类和小鼠衰竭心脏中的 MED12 水平升高，支持这种新的 MED12 功能在心脏病转录错误调节中的临床相关性。我们发表的针对 CM 特异性 Med12 缺失的小鼠的研究表明，MED12 通过与 TF MEF2 直接相互作用来调节钙处理基因表达程序。我们鉴定了与 MED12 相互作用的其他 TF，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。为了确定 MED12 对心脏病转录失调的影响，定义其在 CM 中的分子相互作用和功能并确定其细胞特异性作用至关重要。其他细胞中也报道了激酶子模块的亚群，但它们的功能尚未确定。此外，尚未研究单个激酶子模块蛋白在替代激酶子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 敲除模型 (cKO) 和新生成的 CM Med12 转基因模型 (cTg) 均会导致心力衰竭，强调了 MED12 对 CM 转录稳态的关键贡献。我们将使用 Med12 遗传模型来描述 MED12 在 CM 中的功能。我们将鉴定正常 CM 和 MED12 过表达的 CM 中的 MED12 激酶亚群、与 TF 的相互作用以及基因组相互作用。我们还将使用 hi-PSC 评估人类 MED12 错义突变结合 TF 和激酶子模块蛋白的能力。我实验室的长期目标是揭示控制心脏协调转录程序的机制。在本提案中，我们的目标是 1) 确定 CM 中 MED12 调节的转录机制，2) 确定 MED12 驱动的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疾病期间恢复心脏功能的治疗策略提供新的见解。