Mediator complex in cardiac development and function

介体 (MED) 是一种多亚基复合物，通过整合在转录起始中发挥核心作用 从基因特异性转录激活剂到 RNA 聚合酶 II 的调节信号。遗传学研究已 确定了与人类心脏病相关的特定 MED 亚基的突变。具体来说， MED15 缺失在 DiGeorge/velocardiofacial 综合征患者中很常见，该综合征通常包括 先天性心脏缺陷。 MED30 的错义突变会导致小鼠心肌病。新出现的证据 表明“专门的”MED 亚基在时间和/或空间特异性调节中发挥着关键作用 基因表达。然而，对于心肌细胞 (CM) 中 MED 的亚基组成知之甚少。 不同的发育阶段，并且没有研究检查 MED 复合物的富集情况 不同基因组位点的单个“专门”MED 亚基以及特定转录因子 (TF) 它们在心脏过程中直接相互作用，将其锚定到功能关键的增强子/启动子元件上 发展或心脏病进展期间。我们发现 MED 亚基的转录水平 CM 在发育过程中会发生波动。为了进一步了解 MED 亚基的具体作用 CM，我生成了新型小鼠模型，对选定的 MED 亚基进行了心脏特异性敲除 (cKO)。 MED15、MED25 和 MED30 cKO 小鼠表现出不同的表型。 MED15 cKO 小鼠立即死亡 出生时，从 E12.5 开始观察到心脏缺陷，而 MED30 cKO 小鼠在 E10-10.5 时因扩张而死亡 心。相反，MED25 cKO 小鼠存活至成年并且没有表现出心脏缺陷，这表明 并非所有 MED 亚基对于心脏功能都是不可或缺的。因此，我的假设是 MED15 和 MED30 亚基表现出独特的功能活动，塑造 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) 阐明特定阶段和基因座- 通过分析 MED15 的心脏表型，了解 MED15 或 MED30 亚基在 CM 发展中的具体作用 和 MED30 cKO 小鼠，鉴定富含 MED15 或 MED30 的独特转录调控元件 在早期心脏发育期间，并识别与 MED15 或 MED30 特异性相互作用的 TF；和目标 2. (R00)了解MED15和MED30亚基在疾病发生过程中的病理生理功能 利用诱导型 MED15 治疗成人心脏病（包括 DCM 和病理性肥厚）的进展 和 MED30 CM 特异性敲除 (icKO) 小鼠模型。我在以下方面拥有丰富的背景和培训记录 小鼠遗传学和分子心脏病学。我在 K99 阶段的培训将包括以下人员的结构化指导： 我的主要导师和补充共同导师/顾问、正式课程、额外培训 实验技能（RNAseq、ChIP-seq 和生物信息学）和职业转型计划。这个职业规划 研究项目将确保我成功过渡到独立研究，以实现我的最终职业生涯 了解人类心血管疾病的发生、进展和分子基础的目标。

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.