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Fig. 5 | BMC Developmental Biology

Fig. 5

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

Fig. 5

Overexpression of GLI2 recruits BRG1 to a GLI2-specific Mef2c gene element. a Anti-Flag immunoprecipitation in nuclear extracts from day 3 mES[GLI2] or mES[Ctrl] cells. Immunoprecipitated proteins were probed by western blot using anti-BRG1 or anti-GLI2 antibodies. b A schematic representation of the Mef2c gene and its major sites of interest. The scale bar represents coordinates of the Mef2c gene in the mouse genome (mm10 genome assembly). This schematic was constructed using the UCSC Genome Browser (http://genome.ucsc.edu), TRANSFAC, and data from previous publications [33, 8284, 94, 96101]. GLI-specific Mef2c sites A-I are marked with beige circles. GLI2 has been shown to bind only sites B-I (circles outlined in grey). Other associating proteins, including BRG1 and MyoD, are depicted with coloured circles as outlined in the legend. A detailed description of the associating protein sites can be found in Table 3. Chr: Chromosome. SHF I: ISL-1-dependent SHF enhancer. SHF II: NKX2-5/FOXH1-dependent SHF enhancer. c Anti-BRG1 ChIP was performed on day 4 differentiating P19[GLI2] and P19[Ctrl] cultures with sequential qPCR analyses of GLI-specific Mef2c sites A-I, depicted in (B). One-tailed Student’s T-tests were used for the ChIP statistical analyses; n = 3. All error bars represent +/- SEM. All grey lines represent paired T-tests; all black lines represent unpaired T-tests; ( ) p < 0.05. d Gli1 and Brg1 mRNA expression levels were assessed by qPCR in differentiating P19 EC cultures, treated with MeOH vehicle or KAAD-cyclopamine. These levels were normalized to β-actin and presented as a fold-change over MeOH-treated culture expression levels from the same day. e The effect of KAAD-cyclopamine (black bars) or vehicle (grey bars) treatment on the expression level of Gli1 and indicated cardiomyogenesis-specific genes was assessed using qPCR analysis. Expression levels were normalized to β-actin, calibrated to day 0 untreated culture expression levels, and presented as a percentage of the highest expression level recorded, per gene. f Anti-BRG1 ChIP was performed on day 4 differentiating P19 EC cultures that were treated with either MeOH vehicle (grey bars) or KAAD-cyclopamine (black bars). Each ChIP was followed by qPCR analyses on a gene desert region (negative control), β-actin (positive control), and Mef2C site C from panel b. All error bars represent +/- SEM; n = 3. Two-tailed Student’s T-tests were used for statistical analyses. All grey lines represent paired T-tests; all black lines represent unpaired T-tests; ( ) p < 0.05

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