The role of Smyd4 in regulating cardioprogenitor specification.

项目摘要 SET和MYND结构域蛋白（SMYDs）家族成员是一类独特的赖氨酸甲基转移酶 在胚胎发生过程中通过调节组蛋白H3赖氨酸残基发挥重要作用， 转录激活和抑制功能。SMYD 1以其调节心肌细胞的功能而闻名 分化和成熟。我们之前关于SMYD 4在斑马鱼中的作用的研究表明，SMYD 4是一种重要的蛋白质， 对早期胚胎发育和心脏发生至关重要。我们最近的转录组学分析， 斑马鱼囊胚和早期原肠胚的基因调控网络发生了重大变化 参与中内胚层的特化和心脏祖细胞（CPC）的分化。这些 数据表明，smart 4可能参与了心源性谱系的特化和分化， 心脏形成。重要的是，我们证明了新生SMYD 4遗传变异与以下相关： 先天性心脏病（CHD）。建立SMYD 4基因与人乳腺癌细胞株之间的遗传关联的机制基础， CHDs，我们分析了SMYD 4在人胚胎干细胞中的时间表达模式 （hESC）及其诱导分化为心肌细胞。我们发现SMYD 4在整个细胞中表达， 从hESC向心肌细胞的分化过程。hESC中SMYD 4的遗传消除（hESC-SMYD 4KO） 导致中胚层特化和CPC分化严重受损，从而表型模仿了 在smart 4突变斑马鱼中观察到的早期发育和心脏缺陷。其他转录组学分析 进一步支持hESCs-SMYD 4KO诱导的心肌分化被严重改变， hESC-SMYD 4KO采用更多神经外胚层谱系命运。定量质谱分析显示 BACH 1是干细胞多能性和谱系特化的关键转录调节因子， SMYD 4结合伴侣。基于这些初步数据，我们假设SMYD 4作为一个关键的功能， 通过与BACH 1的相互作用对中内胚层特化和心脏发育的表观遗传调节剂。在 在本项目中，我们提出了3个具体目标来定义SMYD 4的生物学和分子功能， 中内胚层特化和心脏发育。目的1检验SMYD 4对以下假设至关重要： 中胚层特化和心源性分化，通过进行遗传拯救实验和一系列 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 突变型hESC心肌细胞分化过程中可能改变的组蛋白表观遗传景观。目的 2检验了BACH 1-SMYD 4复合物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BACH 1- 使用CUT&RUN分析的SMYD 4特异性靶向基因。目的3检验SMYD 4是必不可少的假设 通过SMYD 4突变小鼠系的产生和表征，将其应用于哺乳动物心脏发育。

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.