Regulation of cardiac patterning by the BAF complex subunit Smarcc1a

项目摘要 心脏模式是一个重要的过程，在胚胎中产生具有独特功能的不同区域。 心随着心脏的发育，它从一个简单的线性心管转变为一个有心房的图案结构， 以及心室腔和房室管（AVC）在它们的连接处。形成这些 形态上的差异伴随着基因表达模式的动态变化： 首先广泛地表达在整个心管中，变得限于腔室或AVC。尽管 AVC和腔室模式的功能重要性，我们不完全了解分子机制 促进特定基因限制在任何一个区域。 为了解决这个悬而未决的问题，我们的实验室在斑马鱼中进行了基因筛选， 破坏了主动脉瓣和心腔的形态在之前的筛选中，我们发现了一个突变体 AVC和心室形态发生严重缺陷，导致相对线性的心脏， 在心房和心室之间的边界处存在形态上不同的AVC。另外，变异心脏 未能表现出对AVC的基因表达的适当细化。定位克隆显示， 致病突变破坏了斑马鱼smarcc1a基因，编码SWI/SNF型ATP依赖性染色质 与哺乳动物BAF 155同源的重塑复合物亚基。这些结果提出了一种新的模型， 通过含Smarcc1a的BAF复合物的染色质重塑， 促进AVC和商会身份之间的区别。 为了测试这个模型，我将首先确定Smarcc1a在何时何地限制AVC的基因表达。 具体来说，我将研究smarcc1a影响基因表达区域模式的程度， 确定基因表达缺陷何时首次出现在smarcc1a突变体中，并利用转基因策略 smarcc1a突变体的组织特异性拯救。此外，我将确定作用于下游的效应基因， Smarcc1a驱动心脏模式。使用功能丧失、功能获得和上位性分析，我将测试 smarcc 1a是否通过其对tbx 2b表达的抑制的影响来控制心脏模式。我也会 使用转录组学方法来鉴定更广泛的受smarcc 1a调节的候选基因名册， 我将测试最佳候选人是否是执行心脏模式的关键效应器。 总之，这些研究很可能揭示smarcc1a在调节区分不同基因的机制中的新作用。 从相邻心腔的特征中提取AVC的特征。smarcc1a的这个角色将代表一个 BAF染色质重塑复合物在心脏模式化过程中以前未被认识到的功能。因此， 这项工作有可能产生更广泛的生物医学影响，因为它可能会提高我们对如何 编码染色质重塑复合物组分的基因突变可能与 先天性心脏病

PROJECT SUMMARY Cardiac patterning is an essential process that creates distinct regions with unique functions within the embryonic heart. As the heart develops, it transforms from a simple linear heart tube into a patterned structure with atrial and ventricular chambers and the atrioventricular canal (AVC) at their junction. The formation of these morphological distinctions is accompanied by dynamic changes in gene expression patterns: genes that were at first broadly expressed throughout the heart tube become restricted to either the chambers or the AVC. Despite the functional importance of AVC and chamber patterning, we do not fully understand the molecular mechanisms that promote the restriction of specific genes to either region. To address this open question, our laboratory has carried out genetic screens in zebrafish in search of mutations that disrupt the morphology of the AVC or the cardiac chambers. In a previous screen, we identified a mutant with severe defects in AVC and chamber morphogenesis, resulting in a relatively linear heart without a morphologically distinct AVC at the boundary between the atrium and the ventricle. In addition, the mutant heart fails to exhibit the appropriate refinement of gene expression to the AVC. Positional cloning revealed that the causative mutation disrupts the zebrafish smarcc1a gene, encoding a SWI/SNF-type ATP-dependent chromatin remodeling complex subunit homologous to mammalian BAF155. These results suggest a novel model in which chromatin remodeling by Smarcc1a-containing BAF complexes refines the gene expression patterns that promote the distinction between AVC and chamber identities. To test this model, I will first determine when and where Smarcc1a acts to restrict gene expression to the AVC. Specifically, I will examine the extent to which smarcc1a influences regional patterns of gene expression, determine when gene expression defects first emerge in smarcc1a mutants, and utilize transgenic strategies for tissue-specific rescue of smarcc1a mutants. Additionally, I will identify effector genes that act downstream of Smarcc1a to drive cardiac patterning. Using loss-of-function, gain-of-function, and epistasis analysis, I will test whether smarcc1a controls cardiac patterning through its influence on repression of tbx2b expression. I will also use transcriptomic approaches to identify a broader roster of candidate genes that are regulated by smarcc1a, and I will test whether top candidates are key effectors in executing cardiac patterning. Together, these studies are likely to reveal a novel role of smarcc1a in regulating the mechanisms that distinguish the traits of the AVC from the traits of the adjacent cardiac chambers. This role of smarcc1a would represent a previously unappreciated function for BAF chromatin remodeling complexes during cardiac patterning. Thus, this work has the potential to have a broader biomedical impact, as it may enhance our understanding of how mutations in genes encoding components of chromatin remodeling complexes could relate to the origins of congenital heart disease.