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

项目1：通过心脏转录因子与核膜相互作用调节基因网络

• 批准号: 10245029
• 负责人: DEEPAK SRIVASTAVA
• 金额: $ 57.97万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10245029
• 关键词: 3-Dimensional; Adult; Affect; Architecture; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiac development; Cardiomyopathies; Cells; Chromatin; Chromatin Remodeling Factor; Collaborations; Complex; Data; Developmental Gene; Disease; Embryo; Epigenetic Process; Event; Failure; Fibroblasts; GATA4 gene; Gene Expression; Gene Expression Regulation; Genes; Genetic Enhancer Element; Genetic Transcription; Genome engineering; Genomic Segment; Genomics; Glycine; Heart Diseases; Human; Impairment; Individual; Intervention; Investigation; Maps; Missense Mutation; Mus; Mutation; Nature; Nuclear Envelope; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Outcome; Output; Peripheral; Pore Proteins; Process; Proteins; Regulator Genes; Repression; Role; Serine; Specificity; TBX5 protein; Testing; Tissues; base; combinatorial; congenital heart disorder; disease-causing mutation; gene repression; genetic information; genetic regulatory protein; genomic locus; human disease; in vivo; induced pluripotent stem cell; member; myocardin; novel; nucleocytoplasmic transport; programs; protein complex; recruit; scaffold; stoichiometry; transcription factor

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.

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表达 程序。此过程由配置的心脏转录因子（CTF）（例如NKX2.5， GATA4，TBX5，MEF2C和Mycardin，彼此辅导和染色质复制 复合物通过控制基因调节网络来建立细胞身份。批判性质 GATA4中的杂合甘氨酸对丝氨酸的错义突出突出了合作互动 （GATA4-G296）破坏了与TBX5的相互作用并引起先天性心脏病（CHD）。 tbx5和 GATA4与染色质复制蛋白的BAF复合物相互作用（在项目2中进行了研究），并且 这些因素共同促进了胚胎中的心脏重编程。进一步添加了MEF2C及其共同 激活剂，心肌（在项目3中进行了研究）将成人心脏成纤维细胞重新编程为类似心肌细胞样 细胞。我们的初步数据表明，TBX5和GATA4与构成几种核蛋白相互作用 核膜上的核孔复合物（NPC）。核苷可以控制激活或沉默 发育基因通过调节三维染色质结构，但特定基因组如何 招募到核膜的区域尚不清楚。在这里，我们将测试GATA4的假设 和TBX5以特异性方式与NPC蛋白相互作用，以募集基因组局部为核 膜调节心脏分化过程中的转录输出。我们将使用HIPSC衍生的CMS 使用基于CRISPR/CAS9的基因组可能会破坏单个蛋白质或它们的相互作用 工程（由Core C支持），并验证小鼠体内的发现。我们建议有缺陷 GATA4和TBX5之间的相互作用破坏了蛋白质复合物与核苷的化学计量法 （由核心A支持），从而有助于改变心脏转录和表观遗传结果 与疾病有关（由核心B支持）。我们的具体目的如下：目标1）确定哪个 核孔蛋白与TBX5或GATA4相互作用并共定位，以建立谱系特定的三 人类心肌细胞和重编程的心肌细胞样细胞中的尺寸基因组结构；目的 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 重编程；目标3）确定人类疾病引起的突变在GATA4中的影响 在3D基因组架构，基因座的转录和表观态上破坏与TBX5的相互作用 招募到核膜，以及这些事件在物理相互作用之间的相互依存关系 GATA4和TBX5。这些研究代表了对谱系特异性TF作用的新研究 通过将特定基因组局部定位到NPC来调节基因转录，并​​旨在打开一个新领域 了解CM谱系规范期间遗传信息的流动。