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Figure 1 | BMC Biology

Figure 1

From: Remodeling of the chromatin structure of the facioscapulohumeral muscular dystrophy (FSHD) locus and upregulation of FSHD-related gene 1 (FRG1) expression during human myogenic differentiation

Figure 1

Facioscapulohumeral muscular dystrophy-related gene 1 ( FRG1 ) upregulation during myogenic differentiation is marked by a switch between H3K27me3 and Polycomb factors with H3K4me3 on its promoter. FRG1 mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) (a) and western blotting (b) in myoblasts and myotubes (8 days of differentiation); 18S rRNA and b tubulin were used as controls. (c) Total RNA from adult tissues was tested for FRG1 expression by means of RT-PCR; 18S rRNA was used as a control. Histograms in A and C represent FRG1 expression over 18S rRNA. (d) Chromatin immunoprecipitation (ChIP) assays of (i) myoblasts and myotubes, and (ii) HeLa and lymphoblasts, using antibodies against H3K4me3 (K4), H3K9me3 (K9) and H3K27me3 (K27). Input DNA (+) represents total chromatin, and IgG the immunoprecipitation by normal rabbit IgG. The amplified FRG1 promoter subregion corresponds to FRG1 A in (f)(i). The G6PD promoter was amplified as a negative H3K27me3 control. (e) ChIP analyses of control (CN) and facioscapulohumeral muscular dystrophy (FSHD) myoblasts and myotubes, indicating the standard error of the mean. A two-tailed t test was used for statistical analysis; the asterisks indicate the statistically significant differences at α = 0.05. CN-K27me3/CN-K4me3 in myoblasts: P = 0.0167, n = 3; FSHD-K27me3/FSHD-K4me3 in myoblasts: P = 0.0157, n = 4; CN-K27me3/CN-K4me3 in myotubes: P = 0.0006, n = 3; FSHD-K27me3/FSHD-K4me3 in myotubes: P < 0.0001, n = 4. The RT-PCR primer pairs were 4q specific [11], and are shown in Additional file 3; the anti-FRG1P antibody is specific for a 4q FRG1 peptide [21]. (f)(i) A schema of the FRG1 promoter showing the position of one CarG box responsive element (in red) and two E-boxes (in green) in relation to the ATG and transcription start site (+1), and the PvuII site. The arrowheads indicate the primer positions for the FRG1 A and FRG1 B PCRs. (ii) ChIP and methylated DNA immunoprecipitation (MeDIP) experiments on myoblasts and myotubes using the anti-H3K27me3 (K27me3), anti-Ezh2, anti-YY1, and anti-5-methyl cytidine (5meCy) antibodies. All PCR experiments were performed in a linear range of amplification, and band intensities were measured using a Typhoon 9200 phosphoscanner and Image Quant analysis software; after subtracting the signals derived from IgG immunoprecipitation, the results were expressed as percentages of input DNA.

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