Gene regulatory networks for heart development

项目总结 先天性心脏病是最普遍和最严重的出生缺陷，发生在超过1%的 活产。先天性心脏病的主要亚型是房间隔或室间隔的缺陷，以及 传导系统缺陷，通常在个体内部共存。病毒的分子和细胞基础 先天性心脏缺陷仍然知之甚少，需要了解才能发展出新的 诊断和治疗方式。人类冠心病的遗传基础主要来自显性突变 转录因子(TF)和染色质修饰因子，导致它们的剂量减少。多么 转录调节剂剂量的减少会导致基因组功能的改变是未知的，也是未知的 知道这些改变的基因调控网络是如何扰乱心脏发育从而导致冠心病的。TBX5是 一种T-box转铁蛋白，其单倍体功能不全会导致与Holt-Oram综合征(HOS)相关的心脏缺陷。 我们开发了一种诱导多能干细胞(Ips)模型，同时，我们还研究了一种 新的TBX5依赖的CHD小鼠模型。基于强大的初步数据，包括单个细胞 RNAseq，我们提出了一个旨在阐明tbx5依赖基因的分子基础的项目。 在先天性心脏病形成过程中的监管网络。我们假设TBX5在特定的、易受攻击的细胞中的剂量 发育中的心脏中的群体，驱动基因调控网络，控制精细调控的细胞 行为，对心脏发育有影响。我们将通过阐明 在CHD的人iPSC模型中，由TBX5单倍体不足导致的基因组失调，以及 在小鼠模型中定义室间隔边界的细胞的遗传和细胞缺陷 TBX5单倍体缺乏症的体内研究。拟议中的实验，基于单细胞转录和 表观基因组学，将为导致常见CHD的分子事件提供令人兴奋的新见解。我们 提出三个具体目标。目标1是定义离散细胞中被破坏的基因调控网络 使用包括杂合子的TBX5等位基因系列的CHD人类细胞模型中的群体 和纯合子空iPS细胞系，以及单细胞RNAseq和ATACseq。目标2是识别表观基因组 通过在我们的TBX5等位基因系列中检查染色质占有率来研究TBX5单倍性不足的机制 Tbx5及其转录因子伙伴、染色质重构体和组蛋白修饰。目标3是描绘基因 单细胞在体室间隔祖细胞Tbx5单倍体功能不全的细胞学基础 RNAseq和ATACseq使用谱系标记的室间隔心肌细胞，结合空间 转录学。这些结果将揭示CHD体内基因调控网络的失调。少校 拟议工作的影响将是对CHD的重要机械性见解，并可广泛推广 转录因子剂量敏感基因调控机制。

PROJECT SUMMARY Congenital heart defects (CHDs) are the most prevalent and serious birth defects, occurring in over 1% of live births. Major subsets of congenital heart defects are defective septation of the atria or ventricles, and conduction system defects, which often co-exist within an individual. The molecular and cellular basis of congenital heart defects remains poorly understood, and an understanding is necessary to develop new diagnostic and therapeutic modalities. The genetic basis of human CHD is largely from dominant mutations in transcription factors (TFs) and chromatin-modifying factors, resulting in their reduced dosage. How reduced dosage of a transcriptional regulator translates to altered genomic function is not known, nor is it known how these altered gene regulatory networks then disrupt heart development to cause CHDs. TBX5 is a T-box TF, haploinsufficiency of which causes heart defects associated with Holt-Oram syndrome (HOS). We have developed an induced pluripotent stem (iPS) cell model of HOS, and in parallel, we have studied a new mouse model of TBX5-dependent CHD. Based on strong preliminary data including single cell RNAseq, we propose a project aimed at elucidating the molecular basis of TBX5-dependent gene regulatory networks in the formation of CHDs. We hypothesize that TBX5 dosage in specific, vulnerable cell populations in the developing heart, drives gene regulatory networks that control finely regulated cellular behaviors, with consequences for cardiac development. We will test this hypothesis by elucidating the genomic dysregulation that results from TBX5 haploinsufficiency in a human iPSC model of CHD, and the genetic and cellular defects in the cells that define the interventricular septum boundaries in a mouse model of TBX5 haploinsufficiency in vivo. The proposed experiments, based on single cell transcriptomics and epigenomics, will provide exciting new insights into the molecular events that lead to common CHD. We propose three specific aims. Aim 1 is todefine disrupted gene regulatory networks in discrete cell populations in a human cell model of CHD, using an allelic series of TBX5, which includes heterozygous and homozygous null iPS cell lines, and single cell RNAseq and ATACseq. Aim 2 is to identify epigenomic mechanisms for TBX5 haploinsufficiency by examining in our TBX5 allelic series chromatin occupancy of TBX5 and its TF partners, chromatin remodeler, and histone modifications. Aim 3 is to delineate the genetic and cellular basis for Tbx5 haploinsufficiency in interventricular septum progenitors in vivo by single cell RNAseq and ATACseq using lineage labeled interventricular septum cardiomyocytes, combined with spatial transcriptomics. These results will reveal in vivo gene regulatory networks dysregulated in CHD. The major impact of the proposed work will be important mechanistic insights into CHDs, and broadly generalizable mechanisms of transcription factor dosage-sensitive gene regulation.