Role of RAF1 in human cardiogenesis and congenital heart defects

项目摘要 这个项目的目标是发现RAF1依赖的基本机制，这些机制调节早期的 进一步阐明先天性心脏病(CHDS)和心肌病的分子基础。 RAF1(或CRAF)是一种普遍表达并控制细胞的丝氨酸/苏氨酸特异性蛋白激酶 增殖、凋亡、细胞分化和致癌转化。患有RAF1生殖系缺失的新生儿- 功能失调性心脏病最近被发现并存在严重的冠心病。此外，患有Noonan的婴儿 与综合征(NS)相关的RAF1突变表现为梗阻性肥厚型心肌病和多种 先心病的风险。目前，对于患有先天性心脏病或心肌病的NS儿童还没有专门的治疗方法，因此 迫切需要了解心脏发育缺陷的分子机制以确定 具体的治疗策略。使用hPSCs和心脏定向分化作为发育模型，我们 发现RAF1是人类心脏发生所必需的，通过调节心脏中胚层的规格和 结果表明，NS RAF1突变影响了这一过程。RAF1是如何调节早期人类心脏发生的 程序和NS突变，如在RAF1基因中发现的那些，如何损害心脏发育仍然存在 难以捉摸。因此，这项提议的总体目标是发现信号、遗传和表观遗传网络 在早期心脏发生过程中受RAF1调控，并受NS RAF1突变的影响。我们寻求执行一项 对RAF1或NS缺失改变的信号和表观遗传网络进行前所未有的深入调查 RAF1在人类心脏分化早期的突变。为了实现这个目标，我们编制了一个“工具箱” 通过基因组编辑生成的hiPSC系(CRISPR-Cas9)，以实现对增益和 功能丧失表型，有可靠的此类分析记录。我们的建议将在更大范围内取得进展 目前的知识通过实现以下目标：目标1：发现RAF1在早期人类中的功能 心脏生成。目的2：探讨Noonan综合征RAF1基因突变对人心脏的影响 发展。目的3：阐明核RAF1在人类早期心脏发生中的作用。成功完成 将阐明RAF1在人类心脏发生和分子生物学中的基础作用 NS RAF1中CHDS和心肌病的潜在机制以前所未有的分辨率进行了研究。它还将 提供有关转录、表观遗传和蛋白质网络控制的基本新知识 并将为后续研究为NS儿童设计新的治疗方法铺平道路 有心脏缺陷。最后，我们预计我们的研究将为研究其他 在NS中观察到的发育缺陷，并将突出HiPSCs作为一个强大的模型系统来破译 先天性心脏病的分子机制。

Project Summary The goal of this project is to discover fundamental RAF1-dependent mechanisms that regulate early steps of cardiogenesis to further delineate the molecular basis of congenital heart defects (CHDs) and cardiomyopathies. RAF1 (or CRAF) is a serine/threonine-specific protein kinase that is ubiquitously expressed and controls cell proliferation, apoptosis, cell differentiation, and oncogenic transformation. Newborns with RAF1 germline loss- of-function have been recently identified and present with severe CHDs. Moreover, infants with Noonan syndrome (NS) associated RAF1 mutations present with obstructive hypertrophic cardiomyopathy and a variety of CHDs. Currently, no specific treatment exists for NS children with CHDs or cardiomyopathy, therefore there is an urgent need to understand the molecular mechanisms underlying cardiac developmental defects to identify specific therapeutic strategies. Using hiPSCs and cardiac-directed differentiation as a developmental model, we discovered that RAF1 was required for human cardiogenesis by regulating cardiac mesoderm specification and showed that NS RAF1 mutations impacted this process. How RAF1 regulates early human cardiogenesis programs and how NS mutations, such as those found in the RAF1 gene, impair cardiac development remains elusive. Hence, the overall goal of this proposal is to discover signaling, genetic and epigenetic networks modulated by RAF1 and impacted by NS RAF1 mutations during early cardiogenesis. We seek to perform an unprecedented in-depth investigation into the signaling and epigenetic networks altered by loss of RAF1 or NS RAF1 mutations at early stages of human cardiac differentiation. Toward that goal, we have compiled a “toolbox” of hiPSC lines generated by genome editing (CRISPR-Cas9) to enable a comprehensive analysis of gain-and loss-of-function phenotypes, with a solid track record for such analyses. Our proposal will break ground beyond current knowledge by achieving the following aims: Aim 1: Discover the function of RAF1 in early human cardiogenesis. Aim 2: To interrogate the impact of Noonan syndrome RAF1 mutations on human cardiac development. Aim 3: Delineate the role of nuclear RAF1 in early human cardiogenesis. Successful completion of our proposal will illuminate the fundamental role of RAF1 in human cardiogenesis and the molecular mechanisms underlying CHDs and cardiomyopathy in NS RAF1 at an unprecedented resolution. It will also provide fundamental new knowledge regarding the transcriptional, epigenetic and protein networks controlling human cardiac development and will pave the way for follow up studies to design new therapies for NS children with heart defects. Finally, we anticipate that our study will open new avenues of investigation of other developmental defects observed in NS and will highlight hiPSCs as a powerful model system to decipher the molecular mechanisms underlying CHDs.