The role of Smyd4 in regulating cardioprogenitor specification.

项目摘要 SET和MyND结构域包含蛋白(SMYDS)家族成员是独特的赖氨酸甲基转移酶 通过调节组蛋白H3赖氨酸残基在胚胎发育过程中发挥重要作用 转录激活和抑制功能。SMYD1以其调节心肌细胞的功能而闻名 分化和成熟。我们先前对SMYD4在斑马鱼中的作用的研究表明，SMYD4是 对早期胚胎发育和心脏发生至关重要。我们最近对smyd4消融基因的转录分析 斑马鱼囊胚和早期原肠胚胎揭示了基因调控网络的重大变化 参与中胚层的分化和心脏祖细胞的分化。这些 有数据表明，smyd4可能在显性表达之前参与了心源性谱系的指定和分化。 心脏编队。重要的是，我们证明了新的SMYD4基因变异与 先天性心脏病(CHDS)。建立SMYD4基因关联的机制基础 CHDS，我们分析了SMYD4在人胚胎干细胞中的时间表达模式 (HESCs)及其诱导分化为心肌细胞。我们发现SMYD4在整个 人胚胎干细胞向心肌细胞分化的过程。人胚胎干细胞SMYD4的基因消融(hESC-SMYD4KO) 导致中胚层规格和CPC分化显著受损，从而表型复制 在smyd4突变斑马鱼中观察到的早期发育和心脏缺陷。附加转录本分析 进一步支持hESCs-SMYD4KO诱导的心肌分化被严重改变，其中 HESCs-SMYD4KO采用更多的神经外胚层血统。定量质谱分析显示 BACH1，干细胞多能性和谱系规范的关键转录调节因子，作为一种强大的 SMYD4结合伙伴。基于这些初步数据，我们假设SMYD4作为一个关键 通过与BACH1的相互作用，表观遗传学调节中胚层的规范和心脏发育。在……里面 本项目针对SMYD4的生物学和分子功能提出了3个具体的研究目标 中胚层规格与心脏发育。AIM 1检验了SMYD4对 通过进行遗传挽救实验和一系列实验来确定中胚层的规格和心脏分化 全基因组综合分析，包括单细胞RNA-SEQ和DNA占有率分析，以确定 突变的hESCs在心肌细胞分化过程中可能改变的组蛋白表观遗传格局。目标 2验证了BACH1-SMYD4复合体对中胚层的表观遗传调控至关重要的假设 通过确定蛋白质-蛋白质相互作用结构域和BACH1- SMYD4特异性靶向基因的切割和运行分析。AIM 3测试了SMYD4是必需的假设 SMYD4突变小鼠品系的产生和特性对哺乳动物心脏发育的影响。

PROJECT ABSTRACT Members of SET and MYND domain-containing protein (SMYDs) family are unique lysine methyltransferases that play important roles during embryogenesis via the regulation of histone H3 lysine residues that impart both transcriptional activation and repression functions. SMYD1 is known for its function in regulating cardiomyocyte differentiation and maturation. Our previous study on the role of SMYD4 in Zebrafish suggesting that smyd4 is critical to early embryonic development and cardiogenesis. Our recent transcriptomic analysis of smyd4-ablated zebrafish blastula and early gastrula embryos has revealed significant changes in gene regulatory networks involved in the specification of mesendoderm and the differentiation of cardiac progenitor cells (CPCs). These data suggest that smyd4 may be involved in cardiogenic-lineage specification and differentiation prior to overt heart formation. Importantly, we demonstrate that de novo SMYD4 genetic variants are associated with congenital heart defects (CHDs). To establish the mechanistic basis for the genetic association of SMYD4 to CHDs, we have analyzed the temporal expression pattern of SMYD4 within human embryonic stem cells (hESCs) and their induced differentiation into cardiomyocytes. We find that SMYD4 is expressed throughout the differentiation process from hESCs to cardiomyocytes. Genetic ablation of SMYD4 in hESCs (hESC-SMYD4KO) resulted in dramatically compromised mesodermal specification and CPC differentiation, thus phenocopying the early developmental and cardiac defects observed in smyd4 mutant zebrafish. Additional transcriptomic analysis further supports that induced cardiomyogenic differentiation of hESCs-SMYD4KO is severely altered, where hESCs-SMYD4KO adopt a more neuroectodermal lineage fate. Quantitative mass spectrometry analysis reveals that BACH1, a critical transcriptional regulator of stem cell pluripotency and lineage specification, as a strong SMYD4 binding partner. Based on these preliminary data, we hypothesize that SMYD4 functions as a critical epigenetic regulator for mesendoderm specification and cardiac development via its interaction with BACH1. In this project, we propose 3 specific aims to define the biological and molecular function of SMYD4 in mesendoderm specification and cardiac development. Aim 1 tests the hypothesis that SMYD4 is critical to mesoderm specification and cardiogenic differentiation by performing genetic rescue experiment and a series of genome-wide integrated analyses, including the single-cell RNA-seq and DNA occupancy analyses, to define the potentially altered histone epigenetic landscape during cardiomyocyte differentiation in mutant hESCs. Aim 2 tests the hypothesis that BACH1-SMYD4 complexes are critical to the epigenetic regulation of mesoderm specification and cardiogenic differentiation by determining the protein-protein interaction domains and BACH1- SMYD4 specific targeted genes using CUT&RUN analysis. Aim 3 tests the hypothesis that SMYD4 is essential to mammalian cardiac development by the generation and characterization of SMYD4 mutant mouse lines.