Project 2: Regulation of cardiac gene regulation and differentiation by dynamic chromatin remodeling complexes

项目2：动态染色质重塑复合物对心脏基因调控和分化的调控

• 批准号: 10471990
• 负责人: Benoit Gaetan Bruneau
• 金额: $ 58.59万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10471990
• 关键词: ATP phosphohydrolase; Ablation; Acute; Adopted; Affect; Binding Sites; Cardiac; Cardiac Myocytes; Cardiac development; Cells; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Complex; Congenital Abnormality; Congenital Heart Defects; DNA; Data; Disease; Embryo; Etiology; Failure; GATA4 gene; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genomics; Heart; Heart Diseases; Histones; Human; Human Genetics; Immunoprecipitation; In Vitro; Knowledge; Lead; Link; Live Birth; Mass Spectrum Analysis; Mesoderm; Molecular; Morbidity - disease rate; Morphogenesis; Mutate; Mutation; Nucleosomes; Play; Positioning Attribute; Process; Proteins; Regulation; Role; SMARCA4 gene; Structure; System; Testing; Tissues; Transcriptional Regulation; cardiogenesis; chromatin remodeling; combinatorial; dosage; gene network; in vivo; inducible gene expression; insight; mortality; mouse genetics; programs; transcription factor

PROJECT SUMMARY/ABSTRACT PROJECT 2 Congenital heart defects (CHDs) are among the most common and devastating birth defects in humans, occurring in about 1% of live births and resulting in significant mortality and morbidity. We have very little understanding of the processes that, when gone awry, cause CHDs. Human genetics have uncovered mutations in transcription factors (TFs) and chromatin remodeling factors as the underlying etiology of several CHDs. Thus, understanding the function of these critical regulators of cell identity and function is crucial. The function of TFs is intimately related to and regulated by the status of the chromatin at their target binding sites. Chromatin structure affects the accessibility and activity of TFs. Therefore, the regulation of chromatin structure is critical for controlling transcription. Chromatin remodeling occurs largely via ATP-dependent chromatin remodeling complexes, which alter the structure of histone-associated DNA to allow access to the transcriptional machinery. By immunoprecipitation of complexes followed by mass spectrometry (IP-MS) we identified the stoichiometrically regulated components of dynamic BAF complexes during in vitro cardiac differentiation. BAF60c and BAF170 are preferably included in cardiac BAF complexes, as are newly identified subunits such as WDR5, which is mutated in human CHD. Genetic ablation of BRG1, BAF60c, or BAF170 revealed essential roles in activating and maintaining the cardiac program. We further examined BAF60c- and BAF170-containing complexes, and identified an additional subset of complexes that include the alternate ATPase, BRM. We find that BRM is strikingly essential for activation of the cardiogenic program: In the absence of BRM, CP transcriptional regulators fail to be activated, leading to catastrophic failure of cardiogenesis, with cells instead adopting alternate fates. We hypothesize that specific BAF complexes form dynamically to coordinate regulation of distinct aspects of cardiac morphogenesis and lineage decisions. We will test this hypothesis in three Specific Aims: 1. To uncover the molecular mechanisms by which dynamic BAF complex subunits regulate specific gene expression programs. 2. To determine the functional importance of the BAF complex subunit switch in cardiomyocyte differentiation. 3. To decipher the redundant and specific roles played by the interchangeable BAF complex subunits. The knowledge generated from this study, integrating the data generated in the other PPG projects, will be crucial to our understanding of the transcriptional regulation of cardiac development, and mechanisms underlying CHDs. Importantly, we will gain significant insight into the basis of the integration of cardiac TFs with chromatin remodeling complexes, and the broad set of essential interactions that lead to finely tuned regulation of cardiac gene expression.

项目总结/摘要 计划2 先天性心脏病（CHD）是人类最常见和最具破坏性的出生缺陷之一， 发生在约1%的活产中，并导致显著的死亡率和发病率。我们几乎没有 了解当出错时导致冠心病的过程。人类遗传学揭示了 转录因子（TF）和染色质重塑因子的突变是几种疾病的潜在病因， 冠心病因此，了解这些细胞身份和功能的关键调节因子的功能至关重要。的 TF的功能与其靶结合位点的染色质状态密切相关并受其调节。 染色质结构影响TF的可及性和活性。因此，染色质的调节 结构对于控制转录至关重要。染色质重塑主要通过ATP依赖的 染色质重塑复合物，它改变组蛋白相关DNA的结构，以允许进入细胞。 转录机制通过复合物的免疫沉淀，然后进行质谱分析（IP-MS），我们 确定了化学计量调节的成分动态BAF复合物在体外心脏 分化BAF 60 c和BAF 170优选包括在心脏BAF复合物中，如新鉴定的 亚基如WDR 5，其在人类CHD中突变。BRG 1、BAF 60 c或BAF 170的基因切除 揭示了激活和维持心脏程序的重要作用。我们进一步检查了BAF 60 c-和 BAF 170的复合物，并确定了一个额外的子集的复合物，包括替代 ATP酶、BRM。我们发现BRM对于心源性程序的激活是非常重要的： 由于缺乏BRM，CP转录调节因子不能被激活，导致了 心脏发生，细胞采用交替的命运。我们假设特定的BAF复合物形成 动态地协调心脏形态发生和谱系决定的不同方面的调节。我们 我们将在三个具体目标中检验这一假设：1。为了揭示分子机制， BAF复合物亚基调节特定的基因表达程序。2.确定功能重要性 BAF复合物亚基转换在心肌细胞分化中的作用。3.去破译那些冗余的和特定的 可互换的BAF复合物亚基所起的作用。从这项研究中产生的知识， 整合其他PPG项目中产生的数据，对于我们理解 心脏发育的转录调控和CHD的潜在机制。重要的是，我们将获得 对心脏TF与染色质重塑复合物整合的基础的重要见解， 广泛的基本相互作用，导致心脏基因表达的微调调节。