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The myocyte enhancer factor 2 (MEF2) transcription factor requires interactions with co-factors for precise regulation of its target genes. Our lab previously reported that the mammalian MEF2A isoform regulates the cardiomyocyte costamere, a critical muscle-specific focal adhesion complex involved in contractility, through its transcriptional control of genes encoding proteins localized to this cytoskeletal structure. To further dissect the transcriptional mechanisms of costamere gene regulation and identify potential co-regulators of MEF2A, a bioinformatics analysis of transcription factor binding sites was performed using the proximal promoter regions of selected costamere genes. One of these predicted sites belongs to the early growth response (EGR) transcription factor family. The EGR1 isoform has been shown to be involved in a number of pathways in cardiovascular homeostasis and disease, making it an intriguing candidate MEF2 coregulator to further characterize. Here, we demonstrate that EGR1 interacts with MEF2A and is a potent and specific repressor of MEF2 transcriptional activity. Furthermore, we show that costamere gene expression in cardiomyocytes is dependent on EGR1 transcriptional activity. This study identifies a mechanism by which MEF2 activity can be modulated to ensure that costamere gene expression is maintained at levels commensurate with cardiomyocyte contractile activity.

(B) Self-immunoprecipitation of the myc-EGR1 protein shows efficient expression and purification. https://doi.org/10.1371/journal.pone.0131619.g002 thumbnail Download: * PPT PowerPoint slide * PNG larger image * TIFF original image Fig 4. (E) qRT-PCR analysis of 13 MEF2-dependent costamere genes shows 11 of these genes are down-regulated when EGR1 is overexpressed in NRVMs; fold change is in comparison to expression levels in the Adβgal control. (C) EGR1-depletion results in upregulated costamere gene expression in NRVMs. qRT-PCR analysis of 13 MEF2-dependent costamere genes in EGR1 siRNA knockdown NRVMs shows that eight of the genes are significantly upregulated, and the majority of the remaining genes show a nonsignificant trend towards upregulation when EGR1 is knocked down; fold change is in comparison to expression levels in the negative siRNA knockdown controls, results were normalized to 18s.