Cardiogenesis: Molecular Mechanisms

 描述(由申请人提供)：我的实验室利用控制心脏发生的信号、转录和翻译调节因子的知识，试图将心脏成纤维细胞重新编程为心肌细胞状态。虽然没有单一的转录因子(Tf)或microRNA产生心肌样细胞，但Gata4、MEF2C和Tbx5(GMT)的组合诱导了表型的根本转变，小鼠成纤维细胞形成了组织良好的肌节，基因表达发生了广泛和表观稳定的转变。在体外，只有一小部分人完全重新编程到收缩活动点。然而，在心脏损伤后将GMT病毒导入体内后，小鼠心脏成纤维细胞重新编程为收缩细胞的比率要高得多，并改善了心功能。其他人也报道了添加其他因素后可能改善重编程的类似结果，包括小分子SB431542抑制转化生长因子-？信号转导，这导致了GMT重编程的质量和效率更高。虽然将内源性心脏成纤维细胞重新编程为心肌细胞样细胞是一种有希望的心脏再生方法，但努力提高重新编程的程度是重要的。为此，我们启动了研究，以揭示GMT从根本上改变细胞状态的机制。全基因组时间转录组研究和染色质免疫沉淀(CHIP)显示，每个重编程因子的早期变化逐渐将成纤维细胞重新编程为心肌细胞状态。深入了解重编程因子通过广泛的转录和表观遗传学改变诱导心肌细胞表型的机制，将有助于识别可以提高效率的因素，并揭示需要激活以建立和维持心肌细胞的网络。在这里，我们将测试以下假设：随着时间的推移，GMT以组合方式在增强剂上动态发挥作用，以表观遗传方式改变基因组格局，以及体内环境和SB431542在体外增强表观遗传转移。我们将从三个目标检验这些假说：(1)结合单细胞RNA测序技术，确定在体外和体内心脏重编程的进展阶段表达GMT的转录后果；(2)确定体外和体内细胞转换过程中心脏重编程因子在全基因组中的占有率，并将它们与临时基因表达变化相关联；(3)确定在心脏重编程过程中如何建立全基因组的表观遗传学变化，包括组蛋白甲基化、乙酰化和DNA甲基化，以及如何在重编程因子占据的座位上特定地改变表观遗传格局，导致转录变化。这些目标的完成将揭示从一个体细胞到另一个体细胞的细胞重编程的复杂机制，并将阐明心脏重编程的潜在障碍，这些障碍将成为提高效率的目标。

 DESCRIPTION (provided by applicant): My laboratory leveraged knowledge of signaling, transcriptional and translational regulators that control cardiogenesis to attempt to reprogram cardiac fibroblasts toward the cardiomyocyte state. While no single transcription factor (TF) or microRNA generates cardiomyocyte-like cells, a combination of Gata4, Mef2c and Tbx5 (GMT) induced a fundamental switch in phenotype in which mouse fibroblasts developed well-organized sarcomeres and had a broad and epigenetically stable shift in gene expression. In vitro, only a small percentage fully reprogrammed to the point of contractile activity. However, viral introduction of GMT in vivo after cardiac injury resulted in a much higher rate of cardiac fibroblasts reprogramming to contractile cells and improved cardiac function in mice. Similar results with potentially improved reprogramming upon addition of other factors were reported by others, including inhibition of Tgf-ß signaling by the small molecule SB431542, which results in greater quality and efficiency with GMT reprogramming. While reprogramming endogenous cardiac fibroblasts into cardiomyocyte-like cells is a promising approach for cardiac regeneration, efforts to improve the degree of reprogramming are important. To this end, we initiated studies to reveal the mechanism by which GMT fundamentally alters the cell state. Genome-wide temporal transcriptome studies and chromatin immunoprecipitation (ChIP) with each reprogramming factor demonstrate early changes that progressively reprogram fibroblasts toward the cardiomyocyte state. A deep understanding of the mechanism by which reprogramming factors can induce the cardiomyocyte phenotype through broad transcriptional and epigenetic changes will help identify factors that could improve efficiency and reveal networks that need to be activated to establish and maintain cardiomyocytes. Here, we will test the hypotheses that GMT functions dynamically in a combinatorial fashion on enhancers over time to epigenetically alter the genomic landscape, and that the epigenetic shift is enhanced by the in vivo environment and by SB431542 in vitro. We will test these hypotheses in three aims: (1) To determine the transcriptional consequences of expressing GMT during the progressive stages of in vitro and in vivo cardiac reprogramming, incorporating single cell RNA sequencing technology; (2) To determine the genome-wide occupancy of cardiac reprogramming factors during cell conversion in vitro and in vivo and to correlate them with temporal gene expression changes; (3) To define how genome-wide epigenetic changes, including histone methylation, acetylation, and DNA methylation are established during the cardiac reprogramming process and how the epigenetic landscape is altered specifically at loci occupied by reprogramming factors, resulting in transcriptional changes. Completion of these aims will reveal the complex mechanisms underlying cellular reprogramming from one somatic cell to another and will illustrate potential barriers to cardiac reprogramming that will serve as targets to improve efficiency.