背景:心内膜是心脏瓣膜发育和成熟的重要信号中心。基因分析已经确定了几种人类心内膜基因,这些基因的失活导致二叶主动脉瓣形成和钙化性主动脉瓣疾病。但关于非编码心内膜调节区域和上游因素在瓣膜发育和疾病中的作用的知识非常有限.
方法:我们通过与表达Notch配体的OP9基质细胞进行短期和长期共培养并抑制Notch活性来操纵小鼠胚胎心内膜细胞中的Notch信号传导。我们检查了每种情况的转录谱和染色质可及性景观,整合转录组,转录因子占用,染色质可及性,和蛋白质组数据集。我们用CRISPR-Cas9编辑的各种非编码调节元件的缺失生成了体外和体内模型,并验证了它们的调节潜力。
结果:我们确定了小鼠胚胎心内膜中对Notch配体的初级和次级转录反应,以及瓣膜发育和疾病中NOTCH依赖的转录特征。通过定义染色质可及性景观的变化,并与发育小鼠心内膜和成人瓣膜的景观相结合,我们确定了潜在的非编码调控元件,验证选定的候选人,提出相互作用的辅因子,并定义其监管活动的时间表。此外,我们发现,在心脏瓣膜发育过程中,通过抑制心内膜的核Yap(Yes相关蛋白)活性,与Hippo途径发生协同转录抑制.
结论:胚胎心内膜中Notch依赖性的序贯转录调控涉及多种因素。Notch通过这些因子激活某些非编码元素,同时抑制可能阻碍心脏瓣膜发育和稳态的元素。Biorxviv:https://www.biorxiv.org/content/10.1101/2023.03.23.53382v1.满。
The
endocardium is a crucial signaling center for cardiac valve development and maturation. Genetic analysis has identified several human endocardial genes whose inactivation leads to bicuspid aortic valve formation and calcific aortic valve disease, but knowledge is very limited about the role played in valve development and disease by noncoding endocardial regulatory regions and upstream factors.
We manipulated Notch signaling in mouse embryonic endocardial cells by short-term and long-term coculture with OP9 stromal cells expressing Notch ligands and inhibition of Notch activity. We examined the transcriptional profile and chromatin accessibility landscape for each condition, integrated transcriptomic, transcription factor occupancy, chromatin accessibility, and proteomic datasets. We generated in vitro and in vivo models with CRISPR-Cas9-edited deletions of various noncoding regulatory elements and validated their regulatory potential.
We identified primary and secondary transcriptional responses to Notch ligands in the mouse embryonic
endocardium, and a NOTCH-dependent transcriptional signature in valve development and disease. By defining the changes in the chromatin accessibility landscape and integrating with the landscape in developing mouse
endocardium and adult human valves, we identify potential noncoding regulatory elements, validated selected candidates, propose interacting cofactors, and define the timeframe of their regulatory activity. Additionally, we found cooperative transcriptional repression with Hippo pathway by inhibiting nuclear Yap (Yes-associated protein) activity in the
endocardium during cardiac valve development.
Sequential Notch-dependent transcriptional regulation in the embryonic
endocardium involves multiple factors. Notch activates certain noncoding elements through these factors and simultaneously suppresses elements that could hinder cardiac valve development and homeostasis. Biorxviv: https://www.biorxiv.org/content/10.1101/2023.03.23.533882v1.full.