Molecular and Genetic Analysis of Castor in Cardiac Development

描述(由申请人提供)：了解决定心肌细胞命运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，我们克隆并鉴定了脊椎动物锌指转录因子Castor(CST)的同源基因。我们进一步证明，在非洲爪哇，CST是心肌细胞分化所必需的；在没有CST的情况下，位于腹中线的细胞保留了早期的心脏祖细胞命运，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，研究表明CST与高血压和高血压之间存在遗传联系。这项建议的总体目标是阐明CST发挥作用的细胞和分子机制。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表达CST的细胞在心脏发育过程中的需求和命运。此外，为了解决CST在心脏发育中发挥作用的分子机制，我们的实验室采取了一系列方法来识别CST转录复合体。从这些研究中，我们已经证明CST与先天性心脏病相关蛋白(CHD5)直接相互作用，CHD5是一种最初从唐氏综合症患者中包含导致先天性心脏病的基因的最小区域克隆和鉴定的蛋白质。此外，我们还使用定向蛋白质组学的方法证明了CST和CHD5与核小体重塑和脱乙酰酶(NuRD)复合体(包括组蛋白脱乙酰酶-1和2(HDAC1/2))直接相关。根据我们的发现，我们假设CST作为转录抑制因子发挥作用，这是早期心脏细胞命运决定所必需的。为了验证这一假设，我们将a)确定CST表达细胞对发育心脏的命运和要求，b)定义CST-NuRD转录复合体的核心成分，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.