Combinatorial Regulation of Gene Networks During Cardiac Development and Disease

项目总结/摘要 总体组件 心血管疾病是成人死亡的最常见原因，先天性心脏病（CHD） 是最常见的先天缺陷近年来出现的一个重要概念是， 心脏转录因子（TF）网络和相关染色质重塑机制的失调 会导致冠心病和心力衰竭TF网络核心成员的强制表达足以 将非肌细胞重新编程为心肌样细胞用于再生医学目的，这表明 用于确定细胞命运的组合代码。随着关键转录因子在全基因组范围内的作用被发现， 对它们在更高级DNA组织中的功能的概念性理解正在出现。证据表明 DNA的三维组织促进了基因组位点的激活或抑制， 关于TF和染色质重塑复合物如何协同蛋白-蛋白相互作用的问题 参与这个过程。我们整合了一个多学科的发育心脏病专家团队， 计算生物学家和系统生物学家，拥有CRISPR/Cas9基因组工程专业知识， 人类iPS细胞，以研究基因组如何被TF和染色质重塑物调节，以控制 心脏基因表达和命运。我们在本提案中测试的具体假设，其中涉及 相互作用以协调调节心脏基因的核心心脏TF的组合 1）心脏TFs GATA 4和TBX 5以谱系特异性方式与 核孔复合物调节3D基因组结构和随后的转录输出; 2） 特定的BAF染色质重塑复合物动态形成，以协调不同的 通过与心脏TF相互作用的心脏形态发生和谱系决定方面;以及3） MEF 2C-心肌蛋白复合物与GATA 4、TBX 5和BAF 60 c相互作用，募集转录因子。 复合物受上游信号传导和心肌蛋白二聚化的影响来调节心脏基因表达。 为了解决这些问题，我们在蛋白质-蛋白质相互作用的研究中整合了独特的专业知识 和翻译后修饰通过先进的蛋白质组学核心;分析复杂的能力， PPIs，DNA结合和转录输出相关的转录调节因子，通过 高级生物信息学核心;以及利用最先进的基因组工程方法的能力 通过基因组工程核心本建议中的问题将在人类的背景下进行研究。 致病突变，以揭示控制正常和异常的潜在机制和范式 心脏发生这里开发的综合知识将使转录的清晰阅读成为可能。 心脏细胞命运决定和分化的“代码”，可用于CHD和 用于再生医学

PROJECT SUMMARY/ABSTRACT OVERALL COMPONENT Cardiovascular disease is the most common cause of mortality in adults, and congenital heart defects (CHDs) are the most common form of birth defects. An important concept that has emerged in recent years is that dysregulation of cardiac transcription factor (TF) networks and related chromatin remodeling machinery contributes to CHDs and heart failure. Forced expression of the core members of the TF network is sufficient to reprogram non-myocytes into cardiomyocyte-like cells for regenerative medicine purposes, suggesting a combinatorial code for determining cell fate. As genome-wide roles for critical TFs are being discovered, a conceptual understanding of their function in higher order DNA organization is emerging. Evidence that the three-dimensional organization of DNA promotes activation or repression of genomic loci has raised the question of how cooperative protein-protein interactions involving TF and chromatin remodeling complexes participate in this process. We have integrated a multidisciplinary team of developmental cardiologists, computational biologists, and systems biologists, with expertise in CRISPR/Cas9 genome engineering in human iPS cells, to investigate how the genome is regulated by TFs and chromatin remodelers to control cardiac gene expression and fate. The specific hypotheses that we test in this proposal, which involve a combination of core cardiac TFs that interact with one another to coordinately regulate cardiac gene expression, are as follows: 1) that the cardiac TFs GATA4 and TBX5 interact in a lineage-specific fashion with the nuclear pore complex to regulate the 3D genomic architecture and subsequent transcriptional output; 2) that specific BAF chromatin remodeling complexes form dynamically to coordinate regulation of distinct aspects of cardiac morphogenesis and lineage decisions through interaction with cardiac TFs; and 3) that the MEF2C-myocardin complex, which interacts with GATA4, TBX5 and BAF60c, recruits a transcriptional complex influenced by upstream signaling and myocardin dimerization to regulate cardiac gene expression. To address these questions, we have integrated unique expertise in the study of protein-protein interactions and post-translational modifications through the Advanced Proteomics Core; the ability to analyze complex datasets of PPIs, DNA-binding, and transcriptional output related to transcriptional regulators through the Advanced Bioinformatics Core; and the ability to leverage state-of-the-art genome engineering approaches through the Genome Engineering Core. The questions in this proposal will be studied in the context of human disease-causing mutations to reveal underlying mechanisms and paradigms that control normal and abnormal cardiogenesis. The integrated knowledge developed here will enable a clear reading of the transcriptional “code” for cardiac cell fate determination and differentiation that may be leveraged for interventions in CHD and for regenerative medicine.