BRG1 interacts with GLI2 and binds Mef2c gene in a hedgehog signalling dependent manner during in vitro cardiomyogenesis

Abstract Background The Hedgehog (HH) signalling pathway regulates cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (mES) cell model. To further assess the transcriptional role of HH signalling during cardiomyogenesis in stem cells, we studied the effects of overexpressing GLI2, a primary transducer of the HH signalling pathway, in mES cells. Results Stable GLI2 overexpression resulted in an enhancement of cardiac progenitor-enriched genes, Mef2c, Nkx2-5, and Tbx5 during mES cell differentiation. In contrast, pharmacological blockade of the HH pathway in mES cells resulted in lower expression of these genes. Mass spectrometric analysis identified the chromatin remodelling factor BRG1 as a protein which co-immunoprecipitates with GLI2 in differentiating mES cells. We then determined that BRG1 is recruited to a GLI2-specific Mef2c gene element in a HH signalling-dependent manner during cardiomyogenesis in P19 EC cells, a mES cell model. Conclusions Thus, we propose a mechanism where HH/GLI2 regulates the expression of Mef2c by recruiting BRG1 to the Mef2c gene, most probably via chromatin remodelling, to ultimately regulate in vitro cardiomyogenesis

Additional file 5: Table S1. of BRG1 interacts with GLI2 and binds Mef2c gene in a hedgehog signalling dependent manner during in vitro cardiomyogenesis

A complete list of gene ontology biological processes significantly enriched among genes within 50 kb of a BRG1-associating site and GLI consensus binding motif. (XLSX 490 kb

Additional file 4: Figure S4. of BRG1 interacts with GLI2 and binds Mef2c gene in a hedgehog signalling dependent manner during in vitro cardiomyogenesis

Expression of Brg1 in mES and P19 cells overexpressing Gli2. qPCR analysis of Brg1 mRNA expression levels in (A) mES[Ctrl] (white bars) and mES[GLI2] (grey bars) cells, or (B) P19[Ctrl] (white bars) and P19[GLI2] (grey bars) cells. For (A-B) expression levels were normalized to β-actin, calibrated to day 0 mES[Ctrl] or P19[Ctrl] culture expression levels, and presented as a percentage of the highest expression level recorded, per gene. Two-tailed Student’s T-tests were used for the mRNA statistical analyses; n = 3. (PDF 341 kb