Abstract
Expression of the MADS domain transcription factor Myocyte Enhancer Factor 2 (MEF2) is regulated by numerous and overlapping enhancers which tightly control its transcription in the mesoderm. To understand how Mef2 expression is controlled in the heart, we identified a late stage Mef2 cardiac enhancer that is active in all heart cells beginning at stage 14 of embryonic development. This enhancer is regulated by the NK-homeodomain transcription factor Tinman, and the GATA transcription factor Pannier through both direct and indirect interactions with the enhancer. Since Tinman, Pannier and MEF2 are evolutionarily conserved from Drosophila to vertebrates, and since their vertebrate homologs can convert mouse fibroblast cells to cardiomyocytes in different activator cocktails, we tested whether over-expression of these three factors in vivo could ectopically activate known cardiac marker genes. We found that mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic Hand and Sulphonylurea receptor (Sur) expression. By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of Hand and Sur was observed in almost all embryos analyzed. Two additional cardiac markers were also expanded in their expression. Our results demonstrate the ability to initiate ectopic cardiac fate in vivo by the combination of only three members of the conserved Drosophila cardiac transcription network, and provide an opportunity for this genetic model system to be used to dissect the mechanisms of cardiac specification.
Highlights
Human heart defects are the most common form of congenital birth defects, and a leading contributor to morbidity and mortality in later life [1,2]
Such network components include the NK homeodomain factor NKX2.5, with mutations leading to malformed cardiac structures, cardiomyopathy and irregular conduction [4,5,6,7,8,9]; the GATA factor GATA4, with mutations leading to septal defects [10]; and the MADS domain transcription factor Myocyte enhancer factor-2 (MEF2), with mutations leading to inherited coronary artery disease [11]
We show that its activity is dependent upon a single Tin consensus binding site which is capable of binding to the Tin protein in vivo, confirming the ChIP-sequencing observations of Jin et al indicating that this region of Mef2 is responsive to Tin [35]
Summary
Human heart defects are the most common form of congenital birth defects, and a leading contributor to morbidity and mortality in later life [1,2]. Key factors in the regulatory network are known to be encoded by genes for which mutations cause heart defects in humans. Such network components include the NK homeodomain factor NKX2.5, with mutations leading to malformed cardiac structures, cardiomyopathy and irregular conduction [4,5,6,7,8,9]; the GATA factor GATA4, with mutations leading to septal defects [10]; and the MADS domain transcription factor Myocyte enhancer factor-2 (MEF2), with mutations leading to inherited coronary artery disease [11]. Defining transcriptional networks for complex biological processes provides important insight into the mechanisms of diseases affecting that process
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