Project 1: Regulation of gene networks through cardiac transcription factor interaction with the nuclear membrane

项目总结/摘要 项目1 心脏发育依赖于谱系特异性基因表达的逐步激活和抑制 程序.该过程由保守的心脏转录因子（cTF）如NKX2.5调节， GATA 4、TBX 5、MEF 2C和Myocardin相互作用与染色质重塑 复合物通过控制基因调控网络来建立细胞身份。的关键性质 GATA 4中的杂合甘氨酸-丝氨酸错义突变突出了协同相互作用 GATA 4-G296 S是一种能够破坏与TBX 5相互作用并导致先天性心脏病（CHD）的基因。tbx 5和 GATA 4与染色质重塑蛋白的BAF复合物相互作用（在项目2中研究）， 这些因素共同促进胚胎中的心脏重编程。进一步添加MEF 2C及其共- 激活剂Myocardin（在项目3中进行了研究）将成人心脏成纤维细胞重编程为心肌细胞样 细胞我们的初步数据显示，TBX 5和GATA 4与几种核孔蛋白相互作用， 核膜上的核孔复合体（NPC）。核孔蛋白可以控制激活或沉默 发育基因通过调控三维染色质结构，但如何具体的基因组 区域被募集到核膜仍然不清楚。在这里，我们将测试GATA 4 和TBX 5以谱系特异性方式与NPC蛋白相互作用，将基因组位点募集到细胞核中， 在心脏分化过程中，细胞膜调节转录输出。我们将使用hiPSC衍生的CM 其中使用基于CRISPR/Cas9的基因组可以破坏单个蛋白质或它们的相互作用 工程（由Core C支持），并在小鼠体内验证发现。我们建议有缺陷的 GATA 4和TBX 5之间的相互作用破坏了蛋白质与核孔蛋白复合物的化学计量 （由核心A支持），从而有助于改变心脏转录和表观遗传结果 与疾病相关（由核心B支持）。我们的具体目标如下：目标1）确定 核孔蛋白与TBX 5或GATA 4相互作用并共定位，以建立谱系特异性的三个- 人心肌细胞和重编程心肌细胞样细胞的三维基因组结构;目的 2)确定通过与TBX 5或GATA 4相互作用定位于NPC的增强子元件的性质， 以及心肌细胞分化过程中相关的表观遗传和转录结果， 目的3）确定GATA 4中的人类致病突变的影响， 破坏与TBX 5在3D基因组结构、基因座的转录和表观遗传状态上的相互作用 招募到核膜，以及这些事件对物理相互作用的相互依赖性， GATA 4和TBX 5。这些研究代表了对谱系特异性转录因子在肿瘤中作用的一项新研究。 通过特定基因组位点定位于NPC来调控基因转录，旨在开辟一个新的领域， 了解CM谱系特化过程中遗传信息的流动。

PROJECT SUMMARY/ABSTRACT PROJECT 1 Cardiac development relies on the stepwise activation and repression of lineage-specific gene expression programs. This process is regulated by conserved cardiac transcription factors (cTFs), such as NKX2.5, GATA4, TBX5, MEF2C and Myocardin, which cooperate with one another and chromatin-remodeling complexes to establish cellular identity by controlling gene regulatory networks. The critical nature of cooperative interactions is highlighted by a heterozygous glycine-to-serine missense mutation in GATA4 (GATA4-G296S) that disrupts interaction with TBX5 and causes congenital heart disease (CHD). TBX5 and GATA4 interact with the BAF complex of chromatin-remodeling proteins (investigated in Project 2), and together, these factors promote cardiac reprogramming in embryos. Further addition of MEF2C and its co- activator, Myocardin, (investigated in Project 3) reprograms adult cardiac fibroblasts into cardiomyocyte-like cells. Our Preliminary Data revealed that TBX5 and GATA4 interact with several nucleoporins that constitute the nuclear pore complex (NPC) at the nuclear membrane. Nucleoporins can control activation or silencing of developmental genes by regulating the three-dimensional chromatin architecture, but how specific genomic regions are recruited to the nuclear membrane remains unclear. Here, we will test the hypothesis that GATA4 and TBX5 interact in a lineage-specific fashion with NPC proteins to recruit genomic loci to the nuclear membrane to regulate the transcriptional output during cardiac differentiation. We will use hiPSC-derived CMs in which disruption of individual proteins or their interaction is possible using CRISPR/Cas9-based genome engineering (supported by Core C), and validate the findings in vivo in mice. We propose that the defective interaction between GATA4 and TBX5 disrupts the stoichiometry of the protein complex with nucleoporins (supported by Core A) and, thereby, contributes to the altered cardiac transcriptional and epigenetic outcome associated with disease (supported by Core B). Our specific aims are as follows: Aim 1) determine which nuclear pore proteins interact and co-localize with TBX5 or GATA4 to establish the lineage-specific three- dimensional genomic architecture in human cardiomyocytes and reprogrammed cardiomyocyte-like cells; Aim 2) determine the nature of enhancer elements localized to the NPC through interaction with TBX5 or GATA4, and the related epigenetic and transcriptional consequences during cardiomyocyte differentiation and reprogramming; and Aim 3) determine the effects of the human disease–causing mutation in GATA4 that disrupts interaction with TBX5 on the 3D genomic architecture, the transcriptional and epigenetic states of loci recruited to the nuclear membrane, and the interdependence of these events on physical interaction between GATA4 and TBX5. These studies represent a novel investigation into the role for lineage-specific TFs in regulating gene transcription by localization of specific genomic loci to the NPC and aim to open a new field in understanding the flow of genetic information during CM lineage specification.