Transcriptional Coordination and Gene Regulation by MED12 in the Cardiomyocyte

心脏病是世界范围内死亡的主要原因，心肌细胞（CM）中转录的破坏有助于心脏病的进展。描述了转录因子（TF）对某些单个基因的转录调控。然而，关于功能相关的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的知之甚少。介体复合物是连接转录机器和转录因子以控制转录的桥梁，但介体调控转录的分子机制还不清楚。介体是一个大的多蛋白复合物，由CDK 8激酶亚模块组成，调节RNA聚合酶II的转录活性。MED 12是一种重要的介体成分，在激酶子模块中，是CDK 8激酶活性所必需的。虽然传统上认为是通过CDK 8激酶子模块的转录抑制因子，但我们发表的数据表明，增加的MED 12也能够直接激活转录。我们的初步研究表明，MED 12水平在来自人类和小鼠的衰竭心脏中增加，支持这种新的MED 12功能在心脏病中转录的错误调节中的临床相关性。我们在CM特异性Med 12缺失小鼠中发表的研究表明，MED 12通过与TF MEF 2直接相互作用调节钙处理基因表达程序。我们发现了与MED 12相互作用的其他TF，这些发现表明MED 12具有多种转录功能，这取决于其相互作用，但其功能尚未被描述。为了确定MED 12在心脏病中对转录错误调节的贡献，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。激酶亚模块的亚群已在其他细胞中报道，但其功能尚未确定。此外，还没有研究单个激酶亚模块蛋白在替代激酶亚复合物中的作用或独立于激酶亚模块的功能。我们发表的CM特异性Med 12敲除模型（cKO）和新生成的CM Med 12转基因模型（cTg）均发生心力衰竭，强调了MED 12对CM转录稳态的关键贡献。我们将使用我们的MED 12遗传模型来描述MED 12在CM中的功能。我们将鉴定MED 12激酶亚群，与TF的相互作用，以及正常CM和MED 12过表达的CM中的基因组相互作用。我们还将使用hi-PSC评估人MED 12错义突变结合TF和激酶亚模块蛋白的能力。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。在这项提案中，我们的目标是1）确定CM中MED 12调节转录的机制，以及2）确定MED 12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。

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.