GATA4 Is a Direct Transcriptional Activator of Cyclin D2 and Cdk4 and Is Required for Cardiomyocyte Proliferation in Anterior Heart Field-Derived Myocardium

Gata4 AHF knockout embryos have profound myocardial proliferation defects. (A to H) Immunohistochemical analyses of proliferation markers on transverse sections show that Gata4 AHF knockout embryos (B, D, F, and H) have reduced proliferation compared to control embryos (A, C, E, and G) at E10.5. (A and B) Gata4 AHF knockout embryos display decreased staining of the nuclear antigen Ki67 (brown) in the right ventricular myocardium and interventricular septum compared to control embryos (asterisks). (C and D) Closer view of the right ventricle (RV) shows that Ki67 staining in Gata4 AHF knockout hearts is reduced in the myocardium (myo) but not in other regions where Gata4 was not inactivated, such as the epicardium (epi). (E and F) BrdU incorporation is diminished in the myocardium of the right ventricle of Gata4 AHF knockout embryos compared to control embryos (asterisks). (G and H) Expression of the mitotic marker phospho-histone H3 (pHH3) is reduced in the right ventricle in Gata4 AHF knockout embryos compared to control littermates (arrowheads). No differences in the staining of any of these proliferation markers between Gata4 AHF knockout and control embryos was observed in the left ventricle (LV). (I and J) TUNEL staining on transverse sections of embryonic hearts shows no difference in apoptosis between Gata4 AHF knockout and control embryos at E10.5. Genotypes for control (Gata4^flox/flox) and Gata4 AHF knockout (Gata4^flox/flox; Mef2c-AHF-Cre^Tg/0) embryos are indicated. (K and L) Quantification of BrdU (K)- and pHH3 (L)-labeled cells shows a significant decrease in proliferation in the right ventricle of conditional knockout (CKO) embryos compared to littermate controls. The total number of DAPI-labeled cells and the number of BrdU- or pHH3-labeled cells were determined by counting cells in a series of sections from three CKO and three control hearts. Data are presented as the mean percentages of cells labeled with BrdU or pHH3 plus standard errors of the means from three hearts of each genotype. P values were calculated using a two-tailed, unpaired t test.

GATA4 regulates multiple cell cycle control genes. Affymetrix gene expression data were analyzed by gene set enrichment (72). Several sets of genes with known roles in cell cycle regulation showed statistically significant, concordant differences between control (Gata4^flox/+) and Gata4 CKO^Nkx (Gata4^flox/flox; Nkx2-5^Cre/+) hearts. The heat map of genes comprising the cell cycle gene set with the most significant statistical score (Brentani cell cycle gene set) is shown (10). Color indicates degree of upregulation (red) or downregulation (blue) relative to the mean expression across all samples (see the color scale at the bottom). Numerous cell cycle control genes were significantly downregulated in Gata4 CKO^Nkx hearts compared to controls, including Cyclin D2 (CCND2), Cyclin A2 (CCNA2), and Cdk4, which are denoted by arrows.

Gata4 inactivation leads to decreased expression of cell cycle proteins. Immunohistochemical staining of transverse sections with anti-cyclin D2 (A and B), anti-Cdk4 (C and D), and anti-cyclin A2 (E and F) antibodies shows that the expression of all three cell cycle proteins is dramatically reduced in the right ventricular myocardium in Gata4 AHF knockout embryos (B, D, and F) compared to that in littermate control embryos (A, C, and E) at E10.5 (asterisks). In panels A to D, staining for cyclin D2 and Cdk4 is red and nuclei have been counterstained with DAPI (blue). In panels E and F, cyclin A2 is stained in brown. No differences in cyclin D2, Cdk4, or cyclin A2 protein expression between knockout and control embryos were observed in regions outside the Mef2c-AHF-Cre domain, such as the left ventricle (LV). Genotypes for control (Gata4^flox/flox) and Gata4 AHF knockout (Cre^Tg/0;Gata4^flox/flox) embryos are indicated. RV, right ventricle.

GATA4 binds directly to the Cyclin D2 and Cdk4 promoters in vivo and in vitro. (A and B) Schematic representations of the mouse Cyclin D2 and Cdk4 promoters. The Cyclin D2 construct encompasses nucleotides −671 to +260 relative to the transcriptional start site (bent arrow). The Cdk4 construct encompasses nucleotides −771 to +56 relative to the transcriptional start site (bent arrow). Boxes denote consensus GATA binding sites in the Cyclin D2 (Gata I [GI], Gata II, and Gata III) and Cdk4 (Gata I/II and Gata III) promoters. Arrowheads indicate the locations of primers used to amplify regions of the Cyclin D2 and Cdk4 promoters, containing consensus GATA sites, in ChIP assays. (C and D) Recombinant GATA4 proteins were transcribed and translated in vitro and used in EMSA with radiolabeled double-stranded oligonucleotides encompassing the CyclinD2 Gata I (C, lanes 1 to 4), Gata II (C, lanes 5 to 8), and Gata III (C, lanes 9 to 12) sites and the Cdk4 Gata I/II (D, lanes 1 to 4) and Gata III (D, lanes 5 to 8) sites. Lanes 1, 5, and 9 (C) and lanes 1 and 5 (D) contain reticulocyte lysate without recombinant GATA4 (−). GATA4 efficiently bound to all GATA sites in the Cyclin D2 and Cdk4 promoters in vitro. mI, mutant version of the Gata I site. (E) GATA4 binds to the endogenous Cyclin D2 and Cdk4 promoters in vivo. Differentiated P19CL6 cardiomyocytes were subjected to ChIP to detect endogenous GATA4 bound to the Cyclin D2 and Cdk4 promoters using anti-GATA4 antibody. Following ChIP, the Cyclin D2 promoter was detected using primers P1 and P2 (lanes 1 to 3) and the Cdk4 promoter was detected using primers P1 and P2 (lanes 4 to 6) and primers P3 and P4 (lanes 7 to 9). In addition, primers were used to detect the second exon of Cyclin D2 as a nonspecific control (lanes 10 to 12). PCR products were analyzed by agarose gel electrophoresis. Lanes 3, 6, 9, and 12 contain PCR products obtained following ChIP using anti-GATA4 antibody (α-G4). Lanes 1, 4, 7, and 10 contain PCR products obtained following ChIP using a nonspecific anti-IgG (α-IgG). Lanes 2, 5, 8, and 11 contain PCR products from input DNA (Inp) amplified prior to immunoprecipitation. ChIP products were detected only from promoter regions in samples where anti-GATA4 antibody was used. Sizes in bp are shown at the left.