Defining the downstream genetic networks regulated by GATA6 during human cardiogenesis using iPSC and hESC models

摘要 在模式生物中的遗传功能丧失研究已经阐明了GATA转录因子家族 调节心脏形态发生的关键方面，包括早期生殖层构型和心脏祖细胞 规范和差异化。重要的是，人类致心因子GATA6的杂合子突变 与各种形式的先天性心脏病(CHD)有关，如流出道和间隔缺陷。 含有这些GATA6突变的冠心病患者的表型多样性可能是由于一种未知的原因 修饰或相互作用基因的变异组合，这些基因汇聚在一起影响心脏表型。一个 因此，对控制人类心脏发育的GATA6基因调控网络有更好的理解 对于推进治疗是必不可少的。在这里，我建议利用心脏定向分化人类的多能性 干细胞(HPSCs)作为研究GATA6功能的系统。初步证据表明GATA6-/-hPSCs 不能产生心肌细胞或表达心脏祖细胞的标志，并已减少心脏 与野生型(WT)对照相比，心脏定向分化过程中中胚层标志物的表达。 此外，GATA6/-hPSCs生成心肌细胞的效率较低，心脏祖细胞减少 标记基因表达与WT细胞的比较，揭示了GATA6杂合子中没有 在之前的动物研究中已有报道。基于这些数据，我打算研究GATA6在心脏疾病中的功能。 心脏发生过程中的中胚层图案化和GATA6单倍体功能不全通过两个特定的目标。在目标1中， 我计划在人类心脏形成的最早阶段发现GATA6的功能。两名候选人 首先将研究从RNA-seq数据中确定的GATA6活性的下游目标LGR5和RIPPLY1 通过基因获得和功能丧失的研究。GATA6 CHIP-SEQ和ATAC-SEQ也将在#年执行。 GATA6-/-和WT细胞在心脏分化早期识别GATA6和WT的直接靶点 分别受染色质不可及性影响的调控基因。在目标2中，我建议定义 心脏发生过程中人类GATA6单倍体功能不全的后果。诱导多能干细胞(IPSC) 从一名CHD患者中创建了一株含有GATA6(c.1071delG)杂合突变的株 分化为心肌细胞的效率低于WT iPSC系。在这里，工程GATA6/-hESCs和 患者来源的GATA61071delG/IPSCs将被分化为心肌细胞，并在不同的阶段进行检测 心脏发生，以仔细定义心脏表型。GATA6/-细胞维甲酸表达增加 酸(RA)信号相关基因，这可能是心脏表型的基础。因此RA信号将被抑制 目的是挽救GATA6单倍体功能不全的表型。最后，辨证的成绩单 GATA6/-hESC和患者来源的IPSC株将使用RNA-SEQ进行分析，以发现新的修饰 可能与冠心病相关的基因。总体而言，这些研究将扩大我们对糖尿病遗传学的理解。 人类心脏发育，并有可能为未来的CHD治疗奠定基础。

ABSTRACT Genetic loss-of-function studies in model organisms have elucidated that the GATA family of transcription factors regulate crucial aspects of heart morphogenesis including early germ layer patterning and cardiac progenitor cell specification and differentiation. Importantly, heterozygotic mutations of the cardiogenic factor GATA6 in humans are associated with various forms of congenital heart disease (CHD) such as outflow tract and septal defects. The phenotypic diversity of CHD in patients containing these GATA6 mutations is likely due an unknown combination of variants in modifying or interacting genes that converge to influence the cardiac phenotype. A greater understanding of the GATA6 genetic regulatory network that controls human heart development is thus essential for advancing therapies. Here, I propose utilizing cardiac-directed differentiating human pluripotent stem cells (hPSCs) as a system to study GATA6 function. Preliminary evidence showed that GATA6-/- hPSCs failed to generate cardiomyocytes or express markers of cardiac progenitors, and had reduced cardiac mesoderm marker expression during cardiac-directed differentiation compared to wild-type (WT) controls. Furthermore, GATA6+/- hPSCs generated cardiomyocytes less efficiently and had reduced cardiac progenitor marker gene expression compared to WT cells, revealing a phenotype in GATA6 heterozygotes that has not been reported in previous animal studies. Based on these data, I aim to study GATA6 function during cardiac mesoderm patterning and GATA6 haploinsufficiency during cardiogenesis through two Specific Aims. In Aim 1, I plan to discover the function of GATA6 during the earliest stages of human cardiogenesis. Two candidate downstream targets of GATA6 activity identified from RNA-seq data, LGR5 and RIPPLY1, will first be studied through genetic gain- and loss-of-function studies. GATA6 ChIP-seq and ATAC-seq will also be performed in GATA6-/- and WT cells during early stages of cardiac differentiation to identify direct targets of GATA6 and regulatory genes affected by chromatin inaccessibility, respectively. In Aim 2, I propose to define the consequence of human GATA6 haploinsufficiency during cardiogenesis. An induced pluripotent stem cell (iPSC) line was created from a CHD patient containing a heterozygous mutation in GATA6 (c.1071delG) that differentiates to cardiomyocytes less efficiently than a WT iPSC line. Here, engineered GATA6+/- hESCs and patient-derived GATA61071delG/+ iPSCs will be differentiated to cardiomyocytes and examined at distinct stages of cardiogenesis to carefully define the cardiac phenotype. GATA6+/- cells have increased expression of retinoic acid (RA) signaling related genes, which may underlie the cardiac phenotype. RA signaling will thus be inhibited with the goal of rescuing the phenotype of GATA6 haploinsufficiency. Finally, the transcriptomes of differentiating GATA6+/- hESC and patient-derived iPSC lines will be analyzed using RNA-seq to discover novel modifying genes that may be relevant to CHD. Overall, these studies will expand our understanding of the genetics of human heart development and potentially lay a foundation for future CHD therapies.