Combinatorial Regulation of Gene Networks During Cardiac Development and Disease

项目摘要/摘要 整体组件 心血管疾病是成年人最常见的死亡原因，先天性心脏病(CHDS) 是最常见的出生缺陷形式。近年来出现的一个重要概念是 心脏转录因子网络的失调及其相关的染色质重塑机制 会导致冠心病和心力衰竭。强制表达TF网络的核心成员足以 出于再生医学的目的，将非心肌细胞重新编程为心肌细胞样细胞，表明 用于确定细胞命运的组合码。随着关键因子在全基因组范围内的作用被发现，一个 对它们在高级DNA组织中的作用的概念性理解正在形成。有证据表明 DNA的三维组织促进了基因组座位的激活或抑制，提高了 涉及转铁蛋白和染色质重塑复合体的蛋白质-蛋白质相互作用的问题 参与这一进程。我们整合了一个由发育心脏病专家组成的多学科团队， 计算生物学家和系统生物学家，在CRISPR/Cas9基因组工程方面具有专业知识 人iPS细胞，研究基因组如何受转录因子和染色质重构体调控 心脏基因表达与命运。我们在此提案中测试的特定假设，涉及 相互作用以协调调控心脏基因的核心心脏转录因子的组合 表达如下：1)心脏转录因子GATA4和TBX5以谱系特异性的方式与 核孔复合体，调节3D基因组结构和随后的转录输出；2) 特定的BAF染色质重塑复合体动态形成，以协调不同的 通过与心脏功能因子的相互作用，心脏形态发生和血统决定的方面；以及3) MEF2C-myocardin复合体，与GATA4，TBX5和BAF60c相互作用，招募转录 受上游信号和肌钙蛋白二聚体影响的复合体，调节心脏基因的表达。 为了解决这些问题，我们整合了研究蛋白质-蛋白质相互作用的独特专业知识。 通过高级蛋白质组学核心进行翻译后修饰；分析复杂结构的能力 与转录调节因子相关的PPI、DNA结合和转录输出的数据集 先进的生物信息学核心；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。本提案中的问题将在人类的背景下进行研究 致病突变揭示控制正常和异常的潜在机制和范式 心脏生成。这里开发的综合知识将使我们能够清楚地阅读转录 决定心脏细胞命运和分化的“密码”，可用于冠心病和冠心病的干预 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.