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

项目摘要/摘要 项目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%的活产婴儿中，并导致显著的死亡率和发病率。我们只有很少的 理解当出错时会导致先心病的过程。人类遗传学发现了 转录因子(TF)和染色质重塑因子突变是几个 CHDS。因此，了解这些细胞特性和功能的关键调节因子的功能是至关重要的。这个 转录因子的功能与其靶结合部位的染色质状态密切相关，并受其调控。 染色质结构影响因子的可及性和活性。因此，染色质的调节 结构是控制转录的关键。染色质重塑在很大程度上是通过依赖于ATP 染色质重塑复合体，它改变组蛋白相关DNA的结构，使其能够进入 转录机器。通过免疫沉淀和质谱联用(IP-MS)，我们 体外心脏动态BAF复合体化学计量学调控成分的鉴定 差异化。如新鉴定的，BAF60c和BAF170优选地包括在心脏BAF复合体中 如WDR5亚基，它在人类CHD中发生突变。BRG1、BAF60c或BAF170的遗传消融 揭示了在激活和维持心脏程序方面的重要作用。我们进一步研究了BAF60c-和 含有BAF170的络合物，并鉴定了包括替代的络合物的另一个子集 ATPase、BRM.我们发现BRM对于心源性程序的激活是非常重要的： 缺乏BRM、CP转录调控因子不能被激活，导致灾难性的失败 心脏发生，细胞采用另一种命运。我们假设特定的生物滤池复合体形成 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 我将在三个具体目标上检验这一假说：1.揭示 BaF复合体亚基调节特定的基因表达程序。2.确定功能重要性 BAF复合亚单位开关在心肌细胞分化中的作用。3.破译多余的和具体的 可互换的BAF复合体亚基所起的作用。从这项研究中产生的知识， 整合在其他PPG项目中生成的数据，将对我们理解 心脏发育的转录调控，以及导致CHDS的机制。重要的是，我们将获得 对心脏转录因子与染色质重塑复合体整合的基础的重大洞察，以及 广泛的基本相互作用，导致心脏基因表达的微调调节。

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.