The Role of YAP1:TEAD Enhancers in the Retinoic Acid-Regulatory Network of Cardiac Progenitors

摘要 破译引导心脏祖细胞（CPC）分化的分子电路将有助于 了解先天性心脏病（CHD）的病因。维生素A/视黄酸（RA）信号传导是一个主 心脏细胞规格的调节器。RA诱导心脏祖细胞后向分化 第二心脏场（pSHF）的中央处理器（CPC），填充心房和静脉窦结构的池。 因此，维生素A/RA缺乏和过量，在这个发展窗口导致结构性 心脏异常因此，了解调节RA信号稳态的机制是一个重要的研究课题。 心脏领域的主要目标。在这里，我们采用了单细胞分辨率的功能基因组方法， 研究人胚胎干细胞衍生的CPC（hESC-CPC）中的视黄酸调控网络。 我们的初步数据表明，Hippo效应子YAP 1和TEAD 4是RA的非典型效应子 心房心肌细胞分化的关键信号。我们的研究表明，雅普缺失影响了 RA靶基因的表达和心房心肌细胞（CM）的表型特性。而且我们的 WT与雅普KO CPC中单细胞ATAC-seq分析表明，雅普促进关键 心房转录因子（TF）的染色质，包括MEIS 2，HOXA 1和GATA 6，支持一个关键的 雅普在心房增强子（Aim 1）TF组成中的作用。有趣的是，我们正在进行的研究表明，RA 信号传导诱导雅普：TEAD 4的全基因组从头募集到CPC中的心房增强子， 独立于Hippo激酶的活性。我们的研究结果表明，RA招募雅普的染色质 通过RA激活的类固醇核受体NR 2F 2。NR 2F 2是一个关键的RA效应子，它调节 在hESC-CPC和pSHF祖细胞中获得心房谱系。此外，人NR 2F 2突变导致 冠心病因此，我们将研究作为NR 2F 2辅因子的雅普和TEAD 4的一种新的非正则函数， 调节心房特化，强调雅普/TEAD作为可能的CHD调节剂的潜力（Aim 2）。 因此，我们正在进行的小鼠模型和iPSC的研究表明，22q11.2基因缺失或 DiGeorge综合征（DGS）导致雅普信号通路失活。DGS是人类疾病的主要原因之一， CHD，这在很大程度上归因于TBX 1与下游途径的单倍不足，例如 维生素A/RA。已经表明，在SHF中TBX 1的损失导致不平衡的维生素A信号传导， 引起这些细胞和CHD的不稳定规格。因此，DGS中的雅普失活可能有助于 SHF细胞中异常的RA信号传导，然而，CHD。因此，我们将研究恢复雅普的活性是否 在DGS小鼠模型中改善CHD表型。最后，与加州大学洛杉矶分校CHD Biocore合作，我们 将验证维甲酸和雅普途径在患有以下疾病的婴儿活检表型中的影响： CHD和DGS（目标3）。这些目标的发现将为正常CPC提供新的机制见解 分化和改变，导致CHD中CPC分化缺陷。

ABSTRACT Deciphering the molecular circuitries that guide cardiac progenitor cell (CPC) differentiation will contribute to understand the causes of congenital heart disease (CHD). Vitamin A/Retinoic Acid (RA) signaling is a master regulator of cell specification in the heart. RA induces posterior specification of the cardiac progenitor cells (CPCs) of the Second Heart Field (pSHF), the pool that populates the atria and sinus venosus structures. Consequently, VitaminA/RA deficiency and excess in this developmental window lead to structural abnormalities in the heart. Thus, understanding the mechanisms that regulate RA signaling homeostasis is a major goal in the heart field. Here, we adopted a functional genomic approach at single-cell resolution to investigate the Retinoic Acid- regulatory network in human embryonic stem cell-derived CPCs (hESC-CPCs). Our preliminary data suggest that the Hippo effectors YAP1 and TEAD4 are non-canonical effectors of RA signaling critical for atrial cardiomyocyte differentiation. Our studies show that YAP deletion affects the expression of RA-target genes and phenotypic properties of atrial cardiomyocytes (CMs). Furthermore, our single-cell ATAC-seq analysis in WT versus YAP KO CPCs suggest that YAP facilitates accessibility of key atrial transcription factors (TFs) to the chromatin, including MEIS2, HOXA1 and GATA6, supporting a pivotal role of YAP in the TF composition of atrial enhancers (Aim1). Interestingly, our ongoing studies show that RA signaling induces genome-wide de-novo recruitment of YAP:TEAD4 to atrial enhancers in CPCs, independently of the activity of the Hippo-kinases. Our findings suggest that RA recruits YAP to the chromatin through the RA-activated steroid nuclear receptor NR2F2. NR2F2 is a key RA-effector, that regulates the acquisition of atrial lineages in hESC-CPCs and pSHF progenitors. Moreover human NR2F2 mutations lead to CHD. Therefore, we will investigate a novel non-canonical function of YAP and TEAD4 as NR2F2 cofactors in regulating atrial specification, highlighting the potential of YAP/TEAD as possible CHD modifiers (Aim2). Accordingly, our ongoing studies in mouse models and iPSCs revealed that the 22q11.2 genetic deletion or DiGeorge Syndrome (DGS) lead to the inactivation of YAP signaling. DGS is one of the main causes of human CHD, which is largely attributed to the haploinsufficiency in TBX1 in concert with downstream pathways, such as Vitamin A/RA. It has been shown that TBX1 loss in the SHF lead to unbalanced Vitamin A signaling, which evokes in erratic specification of these cells and CHD. Therefore, YAP inactivation in DGS may contribute to abnormal RA signaling in SHF cells and yet, CHD. Thus, we will examine whether restoring the activity of YAP in a mouse model of DGS improves CHD phenotypes. Finally, in collaboration with the UCLA CHD Biocore, we will validate the impact of the Retinoic Acid and YAP pathways in the phenotypes of biopsies of infants with CHD and DGS (Aim3). Findings from these aims will provide novel mechanist insights of normal CPC differentiation and into alterations that cause defective CPC differentiation in CHD.