Figure 2. Repression of GATA-dependent AV canal enhancers in chamber myocardium is mediated by Hdacs and deacetylation of histone H3K27.

Whole-mount fluorescence microscopy of double transgenic E10.5 embryo (a) and E10.5 heart (b). (c) Fluorescence-activated cell sorting profile of double AV canal-EGFP and chambers–Katushka transgenic E10.5 hearts. Scale bars represent 100 μm. (d) Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP–qPCR) assay with H3K27ac antibody using EGFP and Kat sorted cells. Bars represent mean±s.d. (n=3). (e) Alignment of H3K27a ChIP-seq profiles from EGFP (green), Kat (red) sorted cells and E14.5 whole hearts24 (black). (f) ChIP–qPCR assay with H3K27ac antibody using dissected AV canal/outflow tract and chamber myocardium extracts. Bars represent mean±s.e.m. (n=3). Statistical test was conducted using the Student’s two-tailed t-test (*P<0.05). (g) E10.5 cGata6-hsp68 transgenic hearts were cultured in the presence or absence of 50 nmol l⁻¹ TSA for 48 h. TSA-treated littermate hearts displayed pan-myocardial lacZ expression. In control medium, the reporter gene remained confined to the AV canal. Scale bar represents 100 μm. (h) E9.5 chambers–Katushka/AV canal-EGFP transgenic hearts were cultured in the presence or absence of 50 nmol l⁻¹ TSA for 48 h, and expression of AV (Tbx2, Tbx3, EGFP) and chamber markers (Nppa, Nppb, Katushka) was analysed with real time RT–PCR. Data were normalized to HPRT and expressed as folds of increase over untreated samples. (i) Luciferase reporter assays on 102 bp cGata6, the 380 bp Tbx2, 660 bp Cx30.2 and the 356 bp cTnI genomic fragments. Cos-7 cells co-transfected with Gata4 expressing vector or not were treated in the absence or presence of 30 nmol l⁻¹ TSA. Bars represent mean±s.e.m. (n=6). Statistical test was conducted using the two- and three-way analysis of variance (^#P<0.05 for Gata4 or TSA treatment versus control, *P<0.05 for Gata4 and TSA treatment versus Gata4 or TSA treatment).