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

项目概要/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。该过程由保守的心脏转录因子 (cTF) 调节，例如 NKX2.5、 GATA4、TBX5、MEF2C 和 Myocardin 相互配合并参与染色质重塑 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 GATA4 中的杂合甘氨酸到丝氨酸错义突变凸显了合作相互作用 (GATA4-G296S) 会破坏与 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 复合物相互作用（在项目 2 中进行研究），并且 这些因素共同促进胚胎中的心脏重编程。进一步添加 MEF2C 及其 co- 激活剂 Myocardin（在项目 3 中进行研究）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 与构成 核膜上的核孔复合体（NPC）。核孔蛋白可以控制核孔蛋白的激活或沉默 发育基因通过调节三维染色质结构，但具体的基因组如何 区域被招募到核膜仍不清楚。在这里，我们将检验 GATA4 的假设 TBX5 以谱系特异性方式与 NPC 蛋白相互作用，将基因组位点招募到核 膜在心脏分化过程中调节转录输出。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其相互作用 工程（由核心 C 支持），并在小鼠体内验证研究结果。我们建议有缺陷的 GATA4 和 TBX5 之间的相互作用破坏了蛋白质复合物与核孔蛋白的化学计量 （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 TBX5 或 GATA4 相互作用并共定位，以建立谱系特异性的三- 人类心肌细胞和重新编程的心肌细胞样细胞中的三维基因组结构；目标 2) 通过与 TBX5 或 GATA4 相互作用确定位于 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后果 重新编程；目标 3) 确定 GATA4 中引起人类疾病的突变的影响 破坏 TBX5 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，以及这些事件与之间的物理相互作用的相互依赖性 GATA4 和 TBX5。这些研究代表了对谱系特异性转录因子在 通过将特定基因组位点定位到 NPC 来调控基因转录，旨在开辟一个新领域 了解 CM 谱系规范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.