Abstract
Introduction: Recent whole exome sequencing carried out in patients with congenital heart disease revealed an over-representation of mutations in members of the Histone 3 Lysine 4 Methyltransferase Complex, indicating that the methylation of Histone 3 Lysine 4 (H3K4) is of particular importance during heart development. In the tri-methylated state (H3K4me3) this signature actively promotes gene transcription through the recruitment of the Pre-Initiation Complex to the promoter. There are currently no data regarding the role of the H3K4me3 modification in cardiogenesis. We investigated the hypothesis that the acquisition of H3K4me3 is necessary for Epicardial Epithelial-to-Mesenchymal Transformation (EMT). Methods: Global patterns of H3K4 tri-methylation were investigated in epicardial cells undergoing EMT in the embryonic quail heart in-vivo, via co-staining of H3K4me3 with the epicardial marker pan-cytokeratin, and the mesenchymal marker alpha-Smooth Muscle Actin. For the in-vitro investigation of H3K4me3 during Epicardial EMT, quail epicardial explants were induced to undergo mesenchymal transformation via treatment with Fetal Bovine Serum alone, and in the presence of an H3K4 methyltransferase inhibitor. Epicardial EMT was characterized by the induction of Snail expression, a loss of E-cadherin, the expression of alpha-Smooth Muscle Actin, and an increase in nuclear area. The acquisition of new H3K4me3 was investigated via Immunohistochemistry. Results: There is a global acquisiton of H3K4me3 in quail epicardial cells undergoing EMT in-vivo and in-vitro. Inhibition of H3K4 methyltransferase activity impaired Snail gene expression, rescued E-cadherin, prevented the expression of alpha-Smooth Muscle Actin, and prevented nuclear enlargement in epicardial cells in-vitro, while having no effect on cell proliferation. Conclusions: We conclude that the acquisition of tri-methylated H3K4 is featured in Epicardial EMT both in-vivo and in-vitro in embryonic quail epicardium. This acquisition is necessary for Epicardial Epithelial-to-Mesenchymal Transformation in-vitro. These data represent the first potential explanation as to how mutations in the H3K4 Methyltransferase Complex impair heart development.
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