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

项目摘要/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。这一过程受保守的心脏转录因子(CTF)调控，如NKX2.5， 相互协同的GATA4、TBX5、MEF2C和Myocardin与染色质重塑 通过控制基因调控网络来建立细胞身份的复合体。的批判性 GATA4中甘氨酸到丝氨酸的杂合错义突变突出了合作性相互作用 (GATA4-G296S)，干扰与TBX5的相互作用并导致先天性心脏病(CHD)。TBX5和 GATA4与染色质重塑蛋白的BAF复合体相互作用(在项目2中进行了研究)，以及 这些因素共同促进了胚胎的心脏重编程。进一步增加MEF2C及其联名- 激活剂Myocardin(在项目3中研究)将成人心脏成纤维细胞重新编程为心肌细胞样细胞 细胞。我们的初步数据显示，TBX5和GATA4与几种核孔素相互作用，这些核孔素构成 核膜上的核孔复合体(NPC)。核孔素可以控制细胞的激活或沉默 发育基因通过调节三维染色质结构，但如何特异的基因组 核膜上是否有区域被征募仍不清楚。在这里，我们将检验GATA4的假设 和Tbx5以谱系特异性的方式与NPC蛋白相互作用，将基因组基因座招募到核中 在心脏分化过程中调节转录输出的膜。我们将使用HiPSC衍生的CMS 其中使用基于CRISPR/Cas9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。我们建议有缺陷的人 GATA4和TBX5之间的相互作用破坏核孔蛋白与蛋白质复合体的化学计量 (由核心A支持)，因此有助于心脏转录和表观遗传结果的改变 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TBX5或GATA4相互作用和共定位，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空间基因组结构；目的 2)确定通过与TBX5或GATA4相互作用定位于NPC的增强子元件的性质， 以及心肌细胞分化和转录过程中相关的表观遗传和转录后果 重新编程；以及目标3)确定GATA4中人类致病突变的影响 干扰与TBX5在3D基因组结构、基因座转录和表观遗传状态上的相互作用 募集到核膜上，以及这些事件之间的相互依赖物理相互作用 GATA4和TBX5。这些研究代表了一项新的研究，即特定血统的TF在 通过定位鼻咽癌特异基因组位点来调控基因转录，旨在开辟鼻咽癌基因转录调控的新领域 了解CM血统规范过程中的遗传信息流。

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.