Epigenetic control of multipotent cardiac progenitor cell differentiation

描述（申请人提供）：最近心脏祖细胞（CPCs）的鉴定为研究和治疗心脏病提供了新的范例。为了充分发挥CPC的治疗潜力，有必要了解引导CPC分化为心肌细胞、平滑肌细胞或内皮细胞的遗传和表观遗传机制。目前，我们对谱系分化的表观遗传调控的基本知识对开发基于细胞的治疗提出了相当大的障碍。atp依赖性染色质重塑介导一个关键的表观遗传机制。这些大的多蛋白复合物打开染色质来调节转录因子进入DNA。SWI/SNF是染色质重塑的主要类型之一，在发育的各个方面发挥关键作用，包括心脏发育和疾病。为了破解SWI/SNF介导的CPC分化的表观遗传机制，我们将重点放在了一个关键的调控亚基BAF250a上，该亚基介导SWI/SNF的组装/募集，并控制核小体密度以及组蛋白甲基化和泛素化。除了心脏外，SWI/SNF复合物和BAF250a在多个组织中表达，SWI/SNF复合物在每个组织中都有独特的分布，以促进组织特异性分化程序。因此，了解一个通用因子BAF250a是如何被用来驱动心源性程序的，将指导我们指导多能细胞进入心肌细胞谱系的必要步骤。事实上，我们广泛的初步数据表明，第二心野（SHF）的BAF250a缺失导致E13左右的非小梁状右心室和室间隔缺陷和胚胎致死。我们已经建立了基于esc的体外系统，再现了体内SHF CPCs的形成和分化，并表明BAF250a消融在CPCs中特异性抑制心肌细胞的形成。消融BAF250a可选择性下调心脏关键转录因子Mef2c和Nkx2.5的表达，但不下调Isl1和Gata4的表达。因此，我们假设baf250a介导的染色质修饰能够使CPC向心肌细胞分化所必需的转录因子子集的适当表达。因此，我们建议确定BAF250a在调节SHF CPC分化中的功能（Aim 1），以验证BAF250a对cSWI/SNF复合物的组装和募集至关重要的假设（Aim 2），并验证BAF250a介导的表观遗传修饰通过调节关键心脏转录因子的启动子可及性来控制CPC分化的假设（Aim 3）。这些研究将对控制CPC分化的表观遗传机制产生新的见解，并可能对理解和治疗心脏病具有重要意义。

DESCRIPTION (provided by applicant): The recent identification of cardiac progenitor cells (CPCs) provides a new paradigm for studying and treating heart disease. To realize the full potential of CPCs for therapeutic purposes, it is essential to understand the genetic and epigenetic mechanisms guiding CPC differentiation into cardiomyocytes, smooth muscle, or endothelial cells. At present, our rudimentary knowledge about the epigenetic regulation of lineage differentiation presents a considerable roadblock to developing cell-based therapies. ATP-dependent chromatin remodelers mediate one critical epigenetic mechanism. These large multiprotein complexes open up chromatin to modulate transcription factor access to DNA. SWI/SNF, one of the major types of chromatin remodelers, plays a key role in various aspects of development, including heart development and disease. To decipher SWI/SNF-mediated epigenetic mechanisms in CPC differentiation, we have focused on a key regulatory subunit BAF250a that mediates SWI/SNF assembly/recruitment and controls nucleosome density as well as histone methylation and ubiquitylation. While the SWI/SNF complex and BAF250a are expressed in multiple tissues besides heart, the SWI/SNF complex is uniquely deployed in each tissue to propel tissue-specific differentiation programs. Thus, understanding how a general factor, BAF250a, is utilized to drive the cardiogenic program will instruct us about the steps necessary to direct a multipotent cell into the cardiomyocyte lineage. Indeed, our extensive preliminary data show that BAF250a deletion in second heart field (SHF) caused non-trabeculated right ventricle and ventricular septal defects and embryonic lethality around E13. We have established ESC-based in vitro systems that recapitulate the formation and differentiation of SHF CPCs in vivo and showed that BAF250a ablation in CPCs specifically inhibits cardiomyocyte formation. BAF250a ablation in CPCs also selectively down-regulated the expression of key cardiac transcription factors Mef2c and Nkx2.5 but not Isl1 and Gata4. Thus, we hypothesize that BAF250a-mediated chromatin modifications enable the proper expression of a subset of transcription factors essential for CPC differentiation into cardiomyocytes. Therefore, we propose to determine the function of BAF250a in regulating SHF CPC differentiation (Aim 1), to test the hypothesis that BAF250a is essential for the assembly and recruitment of cSWI/SNF complex to its targets (Aim 2) and to test the hypothesis that BAF250a-mediated epigenetic modifications control CPC differentiation by regulating the promoter accessibility of key cardiac transcription factors (Aim 3). These studies will generate novel insights into epigenetic mechanisms that govern CPC differentiation and may have significant implications in understanding and treating heart disease.