Epigenetic control of multipotent cardiac progenitor cell differentiation

描述（由申请人提供）：最近对心脏祖细胞（CPC）的鉴定为研究和治疗心脏病提供了新的范例。为了实现CPC的治疗目的的全部潜力，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为开发基于细胞的疗法提供了相当大的障碍。 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以调节转录因子与DNA的接触。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。为了解释SWI/SNF介导的CPC分化的表观遗传机制，我们集中在一个关键的调节亚基BAF 250 a，介导SWI/SNF组装/招募和控制核小体密度以及组蛋白甲基化和泛素化。 虽然SWI/SNF复合物和BAF 250 a在除心脏外的多种组织中表达，但SWI/SNF复合物在每个组织中独特地部署以推进组织特异性分化程序。因此，了解一般因子BAF 250 a如何用于驱动心源性程序将指导我们指导多能细胞进入心肌细胞谱系所需的步骤。事实上，我们广泛的初步数据表明，BAF 250 a在第二心脏领域（SHF）的缺失导致非小梁右心室和室间隔缺损和胚胎死亡E13左右。我们已经建立了基于ESC的体外系统，该系统概括了体内SHF CPC的形成和分化，并表明CPC中的BAF 250 a消融特异性抑制心肌细胞形成。BAF 250 a消融在CPC中也选择性下调关键心脏转录因子Mef 2c和Nkx2.5的表达，但不下调Isl 1和Gata 4的表达。因此，我们假设BAF 250 a介导的染色质修饰能够正确表达CPC分化为心肌细胞所必需的转录因子子集。因此，我们建议确定BAF 250 a在调节SHF CPC分化中的功能（目的1），以检验BAF 250 a对于cSWI/SNF复合物组装和募集至其靶点至关重要的假设（目的2），并检验BAF 250 a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（目的3）。这些研究将产生新的见解表观遗传机制，管理CPC分化，并可能在理解和治疗心脏病有重大意义。

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.