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Abstract

The controls of early lineage cells and cardiac progenitor cells specification, migration, and differentiation by a regulatory network consisting of multiple transcription factors and signaling molecules are important during early lineages formation and heart development. Beyond the genetic level, regulation of those transcription factors and signaling molecules on the epigenetic level is also essential. Histone modification is one of the most important and well-studied epigenetic regulations. Modification such as methylation at different amino acid residues on histone allows modified histone to regulate gene expression repressively or actively. Bivalent domain consisting of both repressive mark trimethylation at lysine 27 residue of histone protein 3 (H3K27me3) and active trimethylation at lysine 4 residue of histone protein 3 (H3K4me3) on key developmental gene allows tight regulation of gene expression and quick response of a cell to extracellular stimulation. Many key developmental genes are marked by bivalent domain and thus are poised for later lineage-specific activation or repression. Jumonji domain-containing protein 6 (Jmjd6) plays an essential role in mouse embryogenesis; however, the detailed mechanism is poorly understood. Here I found that Jmjd6 ablation results in imbalanced germ layer formation in which mesoderm and endoderm lineages are impaired, and ectoderm is favored. Similarly, at later stages, during the commitment and differentiation of progenitor cells to different populations, Jmjd6 plays a key role by regulating chromatin state at lineage-specific regulators. Mechanically, I found less active histone marker H3K4me3 and more repressive histone marker H3K27me3 accumulation, leading to the downregulation of many essential genes for lineage specification and cardiac morphogenesis genes. Using proteomic analysis, I found that JMJD6 interacts with many spliceosome components. Consistent with the protein studies, alternative splicing analysis revealed that Jmjd6 regulates splicing variety on chromatin modification and RNA splicing genes. I focused that the Prmt6 methyltransferase is alternatively spliced to significantly increased PRMT6 protein level. As a methyltransferase for asymmetric dimethylation at arginine 2 residue of histone protein 3 (H3R2me2a), upregulated PRMT6 protein results in increased H3R2me2a and decreased H3K4me3 modification on early lineage II specification and heart morphogenesis genes. Polycomb repressive complex 1 (PRC1) and Polycomb repressive complex 2 (PRC2) are the critical players for depositing repressive histone marks Ubiquitination at lysine 119 residue of histone protein 2A (H2AK119ub) and H3K27me3, respectively. Cbx7, a member of the PRC1 complex, is increased in mesodermal and cardiac progenitor cells differentiated from Jmjd6 knockout mouse embryonic stem cells (mESCs). Increased CBX7 protein leads to higher H2AK119ub, which recruits the PRC2 complex. Ezh2 from the PRC2 complex methylates lysine residue at histone 3 to form H3K27me3. The imbalance of bivalent chromatin marks causes the downregulation of early lineage specification and cardiac morphogenesis gene expression. This study highlights the essential roles of Jmjd6 in early cell lineage choice and heart development by maintaining bivalent chromatin marks balance at the promoter. The transcription factor Isl1 marks the second heart field (SHF) cardiac progenitor cell population and is crucial for cardiac lineage differentiation and heart development. In addition, the expression of Isl1 is tightly regulated to express in second heart field progenitor cells during cardiogenesis. Post-translational modifications of proteins significantly alter their physical and chemical properties, including their folding, stability, activity and function. This study shows that p38α could phosphate Isl1 at S269 and protect it from degradation. Manipulating p38 activity can largely affect the Isl1 protein level in cardiac progenitor cells. Inhibiting p38 activity with an inhibitor or knocking down p38α impairs cardiac progenitor cells and cardiomyocyte marker gene expression, which leads to impaired cardiogenesis. Moreover, Inhibition of p38 in zebrafish or mouse embryos affects heart morphogenesis and function in vivo. In summary, the phosphorylation of Isl1 at S269 by p38α plays a key role in its protein stability and cardiogenesis.