Gene regulatory networks for heart development

项目摘要 先天性心脏缺陷（CHD）是最普遍和最严重的出生缺陷，发生在1%以上的 活产先天性心脏病的主要亚类是心房或心室的间隔缺损， 传导系统的缺陷，这往往同时存在于一个人。的分子和细胞基 先天性心脏病仍然知之甚少，了解是必要的，以开发新的 诊断和治疗方式。人类CHD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，导致其剂量减少。如何 减少剂量的转录调节因子翻译为改变的基因组功能是未知的，也不是 已知这些改变的基因调控网络如何破坏心脏发育，从而导致CHD。TBX 5是 T盒TF，其单倍不足导致与Holt-Oram综合征（HOS）相关的心脏缺陷。 我们已经建立了一个诱导多能干细胞（iPS）模型的HOS，并在平行，我们已经研究了一个 TBX 5依赖性CHD的新小鼠模型。基于强有力的初步数据，包括单细胞 RNAseq，我们提出了一个旨在阐明TBX 5依赖基因的分子基础的项目 监管网络在冠心病形成中的作用。我们假设TBX 5在特定的脆弱细胞中的剂量 在发育中的心脏，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TBX 5单倍不足引起的基因组失调，以及 在小鼠模型中定义室间隔边界的细胞中的遗传和细胞缺陷 TBX 5单倍型不足。基于单细胞转录组学和 表观基因组学，将提供令人兴奋的新见解的分子事件，导致常见的冠心病。我们 提出三个具体目标。目的1是确定离散细胞中被破坏的基因调控网络 在CHD的人细胞模型中，使用TBX 5的等位基因系列，其包括杂合的 和纯合无效iPS细胞系，以及单细胞RNAseq和ATACseq。目的二是鉴定表观基因组 通过检测我们的TBX 5等位基因系列染色质占有率， TBX 5及其TF伙伴，染色质重塑和组蛋白修饰。目的3是描述遗传 Tbx 5单倍性不足的细胞学基础 使用谱系标记的室间隔心肌细胞的RNAseq和ATACseq，结合空间 转录组学这些结果将揭示在CHD体内基因调控网络失调。主要 所提出的工作的影响将是对冠心病的重要的机理性见解，并具有广泛的普遍性 转录因子剂量敏感性基因调控的机制。

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.