Mediator complex in cardiac development and function

介体(MED)是一种多亚基复合体，通过整合在转录启动过程中发挥核心作用 从基因特异性转录激活子到RNA聚合酶II的调控信号。遗传学研究已经 确定特定MED亚单位的突变与人类心脏病有关。具体来说， MED15基因缺失在DiGeorge/心面部畸形综合征患者中很常见，通常包括 先天性心脏缺陷。MED30中的一个错义突变会导致小鼠心肌病。新出现的证据 提示特殊的MED亚基在时间和/或空间特异性的调节中起着关键作用 基因表达。然而，对于心肌细胞(CMS)中MED的亚单位组成知之甚少。 不同的发育阶段，没有研究发现富含MED复合体 位于不同基因组位置的个体“特化”的MED亚基，以及特定的转录因子(TF) 它们直接相互作用，在心脏过程中将它们锚定在功能关键的增强子/启动子元件上 在心脏疾病的发展过程中，或在疾病进展过程中。我们发现MED亚基的转录水平 在发育过程中在CMS内波动。为了进一步了解MED亚基在 CMS，我用选定的MED亚基的心脏特异性敲除(CKO)产生了新的小鼠模型。 MED15、MED25和MED30 CKO小鼠表现出不同的表型。MED15 CKO小鼠在死后立即死亡 出生，从E12.5观察到心脏缺陷，而MED30 CKO小鼠在E10-10.5死亡，扩张 红心。相反，MED25 CKO小鼠存活到成年，没有表现出心脏缺陷，这表明 并不是所有的MED亚单位对心脏功能都是不可或缺的。因此，我的假设是MED15 而MED30亚基表现出独特的功能活动，这些活动塑造了CM转录调控中的关键事件， 形态发生和心脏功能。我的具体目标是：目标1.(K99)阐明特定阶段和地点- 从MED15的心脏表型分析MED15或MED30亚基在CMS发病中的特殊作用 和MED30 CKO小鼠，鉴定丰富的MED15或MED30的独特转录调控元件 在心脏早期发育过程中，识别与MED15或MED30特异相互作用的转录因子；以及 2.(R00)了解MED15和MED30亚基在衰老过程中的病理生理功能。 利用诱导型MED15研究成人心脏病的进展，包括扩张型心肌病和病理性肥厚 和MED30 CM特异性基因敲除(IcKO)小鼠模型。我有很强的背景和培训记录 老鼠遗传学和分子心脏病学。我在K99阶段的培训将包括由 我的主要导师和补充合作导师/顾问，正规课程，附加培训 实验技能(RNASEQ、芯片SEQ和生物信息学)和职业过渡计划。这份职业生涯规划 和研究项目将确保我成功地过渡到独立研究，以完成我的终极职业生涯 目的了解人类心血管疾病的发展、进展和分子基础。

Mediator (MED) is a multi-subunit complex that plays a central role in transcription initiation by integrating regulatory signals from gene-specific transcriptional activators to RNA polymerase II. Genetic studies have identified mutations in specific MED subunits to be associated with human cardiac diseases. Specifically, deletions in MED15 are common in patients with DiGeorge/velocardiofacial syndrome, which typically includes congenital heart defects. A missense mutation in MED30 causes cardiomyopathy in mice. Emerging evidence suggests a critical role for “specialized” MED subunits in the regulation of temporal- and/or spatial-specific gene expression. However, little is known as to the subunit composition of MED in cardiomyocytes (CMs) at distinct developmental stages, and no studies have examined enrichment of MED complexes harboring individual “specialized” MED subunits at distinct genomic loci, as well as the specific transcription factors (TFs) they directly interact with to anchor them to functionally critical enhancer/promoter elements during heart development, or during the progression of cardiac disease. We found that transcript levels of MED subunits fluctuate within CMs during development. To gain further understanding into MED subunit specific roles in CMs, I generated novel mouse models with cardiac-specific knockout (cKO) of selected MED subunits. MED15, MED25, and MED30 cKO mice showed distinct phenotypes. MED15 cKO mice died immediately after birth, with cardiac defects observed from E12.5, whereas MED30 cKO mice died at E10-10.5 with dilated hearts. Conversely, MED25 cKO mice survived to adulthood and displayed no cardiac defects, demonstrating that not all MED subunits are indispensable for cardiac function. Accordingly, my hypothesis is that MED15 and MED30 subunits exhibit unique functional activities that shape key events in CM transcriptional regulation, morphogenesis, and heart function. My Specific Aims are: Aim 1. (K99) To elucidate stage-specific and locus- specific roles of MED15 or MED30 subunits in developing CMs by analyzing cardiac phenotypes of MED15 and MED30 cKO mice, identifying unique transcriptional regulatory elements enriched for MED15 or MED30 during early heart development, and identifying TFs that specifically interact with MED15 or MED30; and Aim 2. (R00) To understand the pathophysiological functions of MED15 and MED30 subunits during the progression of adult heart disease, including DCM and pathological hypertrophy, by utilizing inducible MED15 and MED30 CM-specific knockout (icKO) mouse models. I have a strong background and training record in mouse genetics and molecular cardiology. My training in the K99 phase will consist of structured mentorship by my primary mentor and complementary co-mentors/consultants, formal coursework, additional training in experimental skills (RNAseq, ChIP-seq and bioinformatics) and a program of career transition. This career plan and research project will ensure my successful transition to independent research, to fulfill my ultimate career goal of understanding the development, progression, and molecular basis of human cardiovascular diseases.