Project 3: Control of cardiac transcription by MEF2 and myocardin

项目摘要/摘要 项目3 包括心力衰竭在内的心血管疾病是成年人死亡的最常见原因， 先天性心脏病是美国最常见的出生缺陷形式。一个重要的概念，具有 近年来出现的问题是，心脏转录因子网络的破坏在 先天性心脏病和成人心力衰竭。MEF2C是心脏核心转录因子之一 并且是心脏发育、出生后心脏基因表达和体内平衡所必需的。MEF2C 作为信号响应转录因子，与众多辅助调节蛋白相互作用，控制 然而，关于MEF2C在心脏中的功能仍有许多有待确定的问题。最多的 到目前为止所描述的MEF2C的有效转录辅助激活因子是myocardin。MEF2C专门与 肌钙蛋白的一种长亚型(肌钙蛋白-935)，可协同激活心脏转录。初步 研究发现了一种新的桥接机制，两个Myocardin-935分子与MEF2C和MEF2C相互作用 通过亮氨酸拉链(LZ)二聚基序相互作用以协同激活成对的MEF2位点， 支持肌钙蛋白二聚体在激活依赖于MEF2的心脏基因中的中心作用。 此外，在对心脏增强剂的计算机分析中，配对的MEF2位点是普遍存在的 比偶然预测的频率更高，特别是在心脏增强剂中。然而，这些是否 增强剂是MEF2C-myocardin复合体的真正靶点以及myocardin二聚化如何影响 这些增强子的活性和体内基因表达仍有待确定。其他，未发布 初步研究确定了一个可能由框架内畸形引起的先天性心脏缺陷家庭 导致肌钙蛋白LZ二聚化基序丢失的微缺失。同样，未发表的作品显示 肌钙蛋白亮氨酸拉链结构域类似缺失的小鼠死于先天性心脏缺陷。 该项目将检验这一假设，即MEF2C-myocardin复合体招募更大的转录 共同调节复合体，受上游信号和肌钙蛋白二聚化影响进行调节 心脏基因表达。为了检验这一总体假设，该项目将定义 MEF2C和Myocardin在胚胎和成人心脏中的表达，并将确定磷酸化位点和其他 对MEF2C和Myocardin的修饰。这将为翻译后监管提供关键的洞察力 这些关键的心脏转录因子。该项目还将利用RNA-SEQ、CHIP-SEQ和其他基因组-- 胚胎和内源组织识别MEF2C转录靶点的广泛方法- 肌钙蛋白复合体，将决定肌钙蛋白二聚化如何影响复合体和下游 基因表达。最后，本项目将确定心脏对肌钙蛋白二聚化的要求。 检测肌钙蛋白亮氨酸拉链突变体致死心脏发育表型的体内发育 并将确定与肌钙蛋白二聚体丢失相关的基因表达变化。

PROJECT SUMMARY/ABSTRACT PROJECT 3 Cardiovascular disease, including heart failure, is the most common cause of mortality in adults, and congenital heart defects are the most common form of birth defects in the US. An important concept that has emerged in recent years is that disruptions of cardiac transcription factor networks play important roles in congenital heart defects and in heart failure in adults. MEF2C is one of the core cardiac transcription factors and is required for cardiac development and for postnatal cardiac gene expression and homeostasis. MEF2C functions as signal responsive transcription factor that interacts with numerous co-regulator proteins to control gene expression, yet much remains to be determined about how MEF2C functions in the heart. The most potent transcriptional coactivator for MEF2C described to date is myocardin. MEF2C specifically interacts with a long isoform of myocardin (myocardin-935) to synergistically activate cardiac transcription. Preliminary studies identified a novel bridging mechanism whereby two myocardin-935 molecules interact with MEF2C and with each other via a leucine zipper (LZ) dimerization motif to cooperatively activate paired MEF2 sites, supporting a central role for myocardin dimerization for activation of MEF2-dependent cardiac genes. Furthermore, in silico analyses of cardiac enhancers suggests that paired MEF2 sites are prevalent and occur more frequently than predicted by chance specifically in cardiac enhancers. However, whether these enhancers are bona fide targets of the MEF2C-myocardin complex and how myocardin dimerization influences activity of these enhancers and gene expression in vivo remains to be determined. Additional, unpublished preliminary studies have identified a family with congenital heart defects likely caused by an in-frame microdeletion that results in loss of the myocardin LZ dimerization motif. Similarly, unpublished work shows that mice with an analogous deletion the leucine zipper domain of myocardin die with congenital heart defects. This project will test the hypothesis that the MEF2C-myocardin complex recruits a larger transcriptional coregulatory complex, which is influenced by upstream signaling and myocardin dimerization to regulate cardiac gene expression. To test this overall hypothesis, this project will define the interaction partners of MEF2C and myocardin in the embryonic and adult heart and will identify the phosphorylation sites and other modifications on MEF2C and myocardin. This will provide critical insight into the post-translational regulation of these key cardiac transcription factors. This project will also utilize RNA-seq, ChIP-seq, and other genome- wide approaches from embryos and endogenous tissues to identify transcriptional targets of the MEF2C- myocardin complex and will determine how myocardin dimerization influences the complex and downstream gene expression. Finally, this project will determine the requirement for myocardin dimerization for heart development in vivo by examining the lethal heart development phenotype in myocardin leucine zipper mutant mice and will identify gene expression changes associated with loss of myocardin dimerization.