GATA-dependent regulatory switches establish atrioventricular canal specificity during heart development

Abstract

The embryonic vertebrate heart tube develops an atrioventricular canal that divides the atrial and ventricular chambers, forms atrioventricular conduction tissue and organizes valve development. Here we assess the transcriptional mechanism underlying this localized differentiation process. We show that atrioventricular canal-specific enhancers are GATA-binding site-dependent and act as switches that repress gene activity in the chambers. We find that atrioventricular canal-specific gene loci are enriched in H3K27ac, a marker of active enhancers, in atrioventricular canal tissue and depleted in H3K27ac in chamber tissue. In the atrioventricular canal, Gata4 activates the enhancers in synergy with Bmp2/Smad signalling, leading to H3K27 acetylation. In contrast, in chambers, Gata4 cooperates with pan-cardiac Hdac1 and Hdac2 and chamber-specific Hey1 and Hey2, leading to H3K27 deacetylation and repression. We conclude that atrioventricular canal-specific enhancers are platforms integrating cardiac transcription factors, broadly active histone modification enzymes and localized co-factors to drive atrioventricular canal-specific gene activity.

Figure 1. GATA sites are required to mediate repression of AV canal genes in chamber myocardium.

Localization of transgene expression in E11.5 mouse hearts. (a,b) The cTnT transgene is expressed both in the AV canal myocardium and the chamber myocardium. (c,d) cGata6-cTnT transgenics show predominant expression in the AV canal, similar to the pattern of the Tbx2-cTnT transgenes (e,f). (g,h) Mutation of the two GATA sites in the Tbx2 regulatory region removes the repression in the chambers. Ratios denote proportion of embryos showing chamber repression. Scale bar represents 100 μm. (la) left atrium; (ra) right atrium; (avc) AV canal; (lv) left ventricle; (rv) right ventricle; (oft) outflow tract. (i) Browser view of Tbx2 gene locus with ChIP-seq profiles of Gata4 (ref. 18) (black) and p300 (ref. 24) (black) in heart, Smad4 (grey) in human ESC and Hey2 (blue) in mouse ESC. The 380 bp Tbx2 (−2700/−2300) enhancer region is depicted as a black box.

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 hearts (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).

Figure 3. The Gata4-Smad-Hat complex induces H3K27 acetylation and AV canal gene activation.

(a) Luciferase reporter assays on the 102 bp cGata6 enhancer. Plasmids encoding full-length Gata4 protein, the luciferase reporter construct with a minimal promoter (minP) or containing the cGata6 enhancer with the same minimal promoter (cGata6_minP) were co-transfected in C2C12 cells. The cells were incubated with or without the recombinant human BMP2. Statistical test was conducted using two- and three-way analysis of variance (ANOVA) (mean±s.e.m., n=3, ^#P<0.05 for Gata4 or BMP2 treatment versus control, *P<0.05 for Gata4 and BMP2 treatment versus Gata4 or BMP2 treatment). (b) Luciferase reporter assays on the 102 bp cGata6, the 380 bp Tbx2, 660 bp Cx30.2 and the 356 bp cTnI genomic fragments. Constructs were co-transfected with Gata4, Alk3CA, Smad1 and Smad4 expression vectors into Cos-7 cells. Luciferase activity was determined and normalized as fold over the reporter alone (mean±s.e.m., n=6, ^#P<0.05 for Gata4 or BMP2 effectors versus control, *P<0.05 for Gata4 and BMP2 effectors versus Gata4 or BMP2 effectors, using two- and three-way ANOVA). (c) E9.5 chambers–Katushka/AV canal-EGFP transgenic hearts were cultured in the presence or absence of 200 ng ml⁻¹ Bmp2 for 48 h, and expression of AV canal (Tbx2, Tbx3, EGFP) and chamber markers (Nppa, Nppb, Kat) was analysed with real time RT–PCR. Data were normalized to HPRT and expressed as folds of increase over untreated samples. (d) Untreated and Bmp2-treated E9.5 hearts were subjected to ChIP assay analyses with anti-H3K27ac antibody. Histograms represent enrichment for Tbx2, Cx30.2 and cTnI regulatory enhancers (mean±s.e.m., n=3, *P<0.05 using the Student’s two-tailed t-test). (e) ChIP–qPCR analysis with Smad4 antibody on E10.5 embryo extracts from AV canal/outflow tract and chamber myocardium. Primers amplifying the BRE of the Id2 promoter serve as a positive ChIP control. Pulled-down DNA fragments were analysed with real-time PCR and enrichment indicates the ratio of ChIPed DNA to negative control regions, normalized for input DNA (mean±s.e.m., n=3, *P<0.05 using the Student’s two-tailed t-test). (f) Cartoon depicting the overlapping expression patterns of Bmp2/Smad4 and Gata4 at stage E9.5–E10.5.

Figure 4. The Gata4/Hey/Hdac complex induces H3K27 deacetylation and AV canal gene repression.

(a) Luciferase reporter assays on 102 bp cGata6, the 380 bp Tbx2, 660 bp Cx30.2 and the 356 bp cTnI genomic fragments. Constructs were co-transfected with Gata4, Alk3CA, Smad1 and Smad4, HDAC1 and 2 expression vectors into Cos-7 cells (mean±s.e.m., n=6, *P<0.05 for Gata4, BMP2 effectors and HDAC1, 2 vs Gata4 and BMP2 co-transfection using two- and three-way analysis of variance (ANOVA)). (b) qPCR analysis of mRNA isolated from AV canal-EGFP and chambers–Katushka sorted cells. Values are normalized against EGFP population. Immunohistochemistry for Hdac1 (c) and 2 (d) proteins on sections of E10.5 hearts. Scale bars represent 100 μm. (e) Luciferase reporter assays on AV canal enhancers with expressing vectors encoding for Gata4, Bmp2 effectors and the notch target genes Hey1 and 2 (mean±s.e.m., n=6, *P<0.05 for Gata4, BMP2 effectors and Hey1, 2 versus Gata4 and BMP2 effectors co-transfection, using two- and three-way ANOVA). (f) E9.5 chambers–Katushka/AV canal-EGFP transgenic hearts were cultured in the presence or absence of 50 μmol l⁻¹ γ-secretase inhibitor (DAPT) for 48 h, and expression of AV canal and chamber markers was analysed with real time RT–PCR. Data were normalized to HPRT and expressed as folds of increase over untreated samples. (g) Untreated and DAPT-treated E9.5 hearts were subjected to ChIP assay analyses with anti-H3K27ac antibody. Pulled-down DNA fragments were analysed with RT–PCR and enrichment indicates the ratio of ChIPed DNA to negative control regions, normalized for input DNA. (h) Cartoon depicting the overlapping expressions of Hey1, 2 and Gata4 at stage E9.5–E10.5.

Figure 5. GATA sites serve as a scaffold to recruit functionally distinct complexes.

(a,b) In vitro reporter assays with the cGata6 (102 bp) and Tbx2 (380 bp) luciferase constructs containing mutations for GATA sites (cGata6muGATA, Tbx2muGATA). Cos-7 cells co-transfected with Gata4, Alk3CA, Smad1 and 4, Hey1, 2, HDAC1 and 2 expressing vectors or not were treated in the absence or presence of 30 nmol l⁻¹ TSA (mean±s.e.m., n=3, ^#P<0.05 for Gata4 or TSA/BMP2 treatment versus Gata, TSA or BMP2 alone, *P<0.05 for mutated versus wild-type enhancers using two- and three-way analysis of variance (ANOVA)). (c) In vitro reporter assays on 102 bp cGata6 and mutated 47 bp cGata6 enhancer (cGata6muSP). Cos-7 cells were co-transfected with Gata4 and Gata4 co-activators and co-repressors (mean±s.e.m.; n=3; ^#P<0.05 for HDAC1, 2 or Hey1, 2 versus Gata4 and BMP2 effectors; *P<0.05 for HDAC1, 2 and Hey1, 2 versus HDAC1, 2. ^$P<0.05 for HDAC1, 2 and Hey1, 2 versus Hey1, 2 using three-way ANOVA. (d) GATA-binding site-enhancers recruit a Gata4/Smad4/Hat transcriptional activation complex in the AV canal and a Gata4/Hey1,2/Hdac transcriptional repression complex in the chambers, which coordinately establish AV canal gene specificity.