Molecular and Genetic Analysis of Castor in Cardiac Development

描述（由申请人提供）：了解心肌细胞命运决定所需的分子机制对于揭示先天性心脏病的病理和治疗至关重要。为了解决这些问题，我们克隆并鉴定了锌指转录因子Castor （Cst）的脊椎动物同源物。我们已经证明，在非洲爪蟾中，Cst是心肌细胞分化所必需的；在没有Cst的情况下，腹中线的细胞保留了早期心脏祖细胞的命运，但被阻止分化为心肌细胞。最近的全基因组关联研究进一步强调了Cst的作用，表明Cst与高血压和高血压之间存在遗传联系。本提案的总体目标是阐明CST功能的细胞和分子机制。为了解决这些问题，我们在小鼠中产生了一组独特的Cst等位基因，现在将使用这些等位基因来确定心脏发育中Cst表达细胞的需求和命运。此外，为了解决CST在心脏发育中的分子机制，我们的实验室已经采取了一套方法来识别CST转录复合物。从这些研究中，我们已经证明CST直接与先天性心脏病相关蛋白（CHD5）相互作用，CHD5是一种最初从包含唐氏综合征患者先天性心脏病基因的最小区域克隆和鉴定的蛋白质。此外，我们使用了一种基于定向蛋白质组学的方法来证明CST和CHD5直接与核小体重塑和去乙酰化酶（NuRD）复合物相关，包括组蛋白去乙酰化酶-1和2 （HDAC1/2）。基于我们的发现，我们假设Cst作为一种转录抑制因子在早期心脏细胞命运决定中起作用。为了验证这一假设，我们将a)确定CST表达细胞对发育中的心脏的命运和需求，b)定义CST - nurd转录复合物的核心成分，c)确定CHD5-CST相互作用在调节CST活性中的作用。

DESCRIPTION (provided by applicant): An understanding of the molecular mechanisms that are required for cardiomyocyte cell fate decisions is critical for uncovering the pathologies and treatments for congenital heart disease. To address these issues, we have cloned and characterized the vertebrate orthologues of the zinc finger transcription factor, Castor (Cst). We have gone on to show that in Xenopus Cst is in required for cardiomyocyte differentiation; in the absence of Cst, cells at the ventral midline retain early cardiac progenitor fate but are blocked from differentiating into cardiomyocytes. The role of Cst is further emphasized by recent genome-wide association studies showing a genetic link between Cst and high blood pressure and hypertension. The overall goal of this proposal is to elucidate the cellular and molecular mechanism by which CST functions. To address these issues we have generated a set of unique alleles of Cst in mouse and will now use these alleles to determine the requirement and fate of Cst expressing cells in cardiac development. In addition, to address the molecular mechanisms by which CST functions in heart development, our lab has undertaken a set of approaches to identify the CST transcriptional complex. From these studies we have demonstrated that CST directly interacts with the congenital heart disease associated protein (CHD5), a protein initially cloned and identified from the minimal region containing the gene responsible for congenital heart disease in Down Syndrome patients. Moreover, we have used a directed proteomic-based approach to show that CST and CHD5 directly associate with the Nucleosome Remodeling and Deacetylase (NuRD) complex including histone deacteylase-1 and 2 (HDAC1/2). Based on our findings, we hypothesize that Cst functions as a transcriptional repressor which is required for early cardiac cell fate decisions. To test this hypothesis we will a) determine the fate and requirement of Cst-expressing cells to the developing heart, b) define the core components of the Cst-NurD transcriptional complex and c) determine the role of CHD5-CST interaction in regulating CST activity. PUBLIC HEALTH RELEVANCE: Clinical and genetic studies in model organisms have provided direct evidence for the role of CASTOR (CST) in heart development and human disease. However, almost nothing is known about how CST functions. This proposal will provide insight into the general mechanisms of CST function, define the mode of action of CST in human disease, and identify additional potential candidate genes associated with human congenital heart disease.