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/维甲酸(RA)信号转导是主宰 心脏细胞规格的调节器。RA诱导心脏祖细胞的后部特异化 第二心场(PSHF)，即位于心房和静脉窦结构的心池。 因此，维生素A/RA在这一发育窗口中的缺乏和过剩导致结构性 心脏异常。因此，了解调节RA信号动态平衡的机制是 在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来 研究人胚胎干细胞来源的CPC(hESC-CPC)中的维甲酸调节网络。 我们的初步数据表明，河马效应器YAP1和TEAD4是RA的非典范效应器 信号转导对心房肌细胞分化至关重要。我们的研究表明，YAP缺失会影响 心房肌细胞(CMS)RA靶基因表达及表型特征此外，我们的 在WT和YAP KO CPC中的单细胞ATAC-SEQ分析表明，YAP促进了密钥的可获得性 心房转录因子(TF)到染色质，包括MEIS2、HOXA1和GATA6，支持一个枢轴 YAP在心房增强剂(Aim1)转铁蛋白组成中的作用有趣的是，我们正在进行的研究表明，RA 信号诱导CPC中YAP：TEAD4对心房增强剂的全基因组从头募集 不依赖于河马激动酶的活性。我们的发现表明，RA招募YAP进入染色质 通过RA激活的类固醇核受体NR2F2。NR2F2是一个关键的RA效应因子，它调节 在hESC-CPC和pSHF祖细胞中获得心房谱系。此外，人类NR2F2突变导致 先心病。因此，我们将研究YAP和TEAD4作为NR2F2辅助因子的一个新的非正则函数 调节心房规范，突出YAP/TEAD作为可能的CHD修饰剂的潜力(AIM2)。 因此，我们在小鼠模型和ipscs中正在进行的研究表明，22q11.2基因缺失或 DiGeorge综合征(DGS)导致YAP信号失活。DGS是人类致病的主要原因之一 CHD，这在很大程度上归因于TBX1单倍体不足与下游通路的协同，如 维生素A/RA。已有研究表明，SHF中TBX1的缺失导致维生素A信号的失衡，这是 引起这些细胞的不稳定规格和CHD。因此，DGS中的YAP失活可能有助于 SHF细胞中RA信号异常，但仍有CHD。因此，我们将研究是否恢复YAP的活动 在DGS小鼠模型中，DGS改善了CHD的表型。最后，与加州大学洛杉矶分校的CHD BioCore合作，我们 将验证维甲酸和YAP信号通路在婴儿的活体组织表型中的影响 CHD和DGS(Aim3)。这些目标的发现将为正常的CPC提供新的机械学家的见解 分化并转化为导致冠心病患者CPC分化缺陷的改变。

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.