Tbx1 represses Mef2c gene expression and is correlated with histone 3 deacetylation of the anterior heart field enhancer

Figures

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  Fig. 1.. Tbx1 negatively regulates Mef2c expression in C2C12 at day 3. (A,B) Time course of Tbx1 (A) and Mef2c (B) expression during in vitro C2C12 myoblast differentiation, with or without transient transfection of Tbx1. Note that Tbx1 negatively regulates Mef2c expression at day 3 of differentiation (B). (C) Mef2c mRNA levels are affected by Tbx1 overexpression 24 h after transfection (performed at day 2). Data are from three experiments (mean±s.d.); *P<0.05. The arrows in A and B indicate the time of transfection (24 h before induction of differentiation, which is on day 0).

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  Fig. 2.. Acetylation of histone 3 in the AHF enhancer region of Mef2c is reduced by Tbx1 overexpression. (A) Examples of ChIP-seq data profiles as shown using the UCSC genome browser (https://genome.ucsc.edu) in the genomic region containing the AHF enhancer of Mef2c (GenBank AY324098): H3K27Ac profiles at four stages of cardiomyocyte differentiation of mouse ESCs: undifferentiated ESCs (ESC), cells expressing mesodermal markers (MES), cardiac precursors (CP), cardiomyocytes (CM); H3K27Ac ChIP-seq data profiles in E14.5 heart tissue; Tbx1 ChIP-seq data profiles. Arrows indicate the primers used for real-time PCR amplification. (B) q-ChIP assay of AcH3 on differentiating C2C12 cells (day 3) transfected with an empty vector (EV) or with a vector overexpressing Tbx1 (Tbx1), followed by quantitative real-time PCR. Data are from three experiments (mean±s.d.); *P<0.05.

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  Fig. 3.. Tbx1 regulates the Mef2c AHF enhancer in vivo. (A-E) Immunofluorescence of transverse sections of E9.5 (20 somites) embryos with the genotype indicated, using anti-Cre (green) and anti-Isl1 (red) antibodies. The first row of sections (A) corresponds to a level immediately below the outflow tract. The second row (B) is just anteriorly to the inflow tract, while the third fourth and fifth rows (C-E) correspond to the inflow tract. Note that the Cre signals in A and B are comparable in the three genotypes, while in C-E, reduced dosage of Tbx1 is associated with expansion of Cre expression (dashed line arrows). We examined three embryos per genotype. Fig. S3 shows Tbx1 gene expression in similar sections. A, atrium; DPW, dorsal pericardial wall (line arrowhead); IFT, inflow tract; NT, neural tube; OFT, outflow tract; Ph, pharynx. Scale bar: 100 μm.

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  Fig. 4.. Transverse sections of a Tbx1+/lacZ embryo at E9.5 (22 somites) after staining with the β-gal chromogenic substrate salmon-gal. (A-E) Sections are at similar levels as in Fig. 3A-E. Scale bars: 100 µm. (F,G) Left and right sides of the whole-mount stained embryo from which sections in A-E were derived. (H) Wild-type littermate with negative staining. DPW, dorsal pericardial wall; IFT, inflow tract; Ph, pharynx.

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  Fig. 5.. Experimental design for the generation and cardiac differentiation of control and 22q11.2DS hiPSCs. hiPSCs were generated by overexpression of retroviral transgenes for OCT3/4, SOX2 and KLF4 in primary skin fibroblasts from a 22q11.2DS (DGS) patient and from an unrelated healthy donor. Two hiPSCs clones from each individual were differentiated in cardiac progenitors and analyzed by qRT-PCR.

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  Fig. 6.. Analysis of MEF2C and GATA4 expression in control and 22q11.2DS hiPSC-derived cardiac cells. (A,B) qRT-PCR-based MEF2C (A) and GATA4 (B) expression in two hiPSC clones from a control (CTR) individual and two from a 22q11.2DS (DGS) patient at days 4, 8 and 12 of cardiac differentiation. n=3, *P<0.05 vs day 0.

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  Fig. 7.. Acetylation of histone 3 in MEF2C and GATA4 loci is enhanced in differentiating 22q11.2DS hiPSCs. (A,B) Localization of primer pairs (arrows) used for ChIP experiments with human cells on MEF2C and GATA4 genes. ‘AHF Enhancer’ indicates a region of high similarity with the murine AHF enhancer. For reference, we report ENCODE ChIP-seq data for H3K27Ac for two cell types, undifferentiated human embryonic stem cells (H1-hESC) and human skeletal muscle myoblasts (HSMM). The GATA4 promoter region is not acetylated in these two cell types. (C) ChIP assays with an anti-AcH3 antibody in differentiating control and 22q11.2DS hiPSCs, followed by quantitative real-time PCR.