Yamak A , Latinkic BV , Dali R , Temsah R , Nemer M .

Canadian Institutes of Health Research , PG/11/73/29095 British Heart Foundation , BHF_PG/11/73/29095 British Heart Foundation ,  CIHR

	Fig. 1. GATA4 and Cyclin D interaction. (A and B) Transient transactivation of CycD2 (ccnd2), Nppa, and the minimal GATA promoter by GATA4 and CycD2 in NIH 3T3 cells (A) and in HL-1 atrial cardiomyocytes (B); 250 and 500 ng of GATA4 expression vector and 3 and 4 μg of CycD2 expression vector were used in the case of NIH cells and 25 ng of GATA4 and 0.5, 1, and 3 μg of CycD2 expression vectors were used in the case of HL-1 cells. (C) Transcript levels of luciferase, ccnd2, and Gata4 in the heart and liver of GATA-Luc transgenic mice crossed with HA-ccnd2 mice. Note the increase of luciferase mRNA levels of CycD2.GATA-Luc mice relative to those of GATA-Luc mice specifically in the heart (Left). (Center) Control to show the overexpression of CycD2 in the hearts of CycD2.GATA-Luc mice but not in the liver. (Right) No effect of CycD2 overexpression on Gata4 mRNA levels. The results are shown as means ± SEM (n = 3). *P < 0.05; **P < 0.01. (D) Enrichment of CycD2 and GATA4 on CycD2 promoter, as revealed by ChIP. Dessert is used as a negative gene. IgG is a negative control. Flag-GATA4 and Flag-CycD2 C2C12 stable cell lines were used. The results are means ± SEM (n = 3). *P < 0.05 vs. IgG. (E) CycD2 coimmunoprecipitates with GATA4 in vivo. Nuclear extracts from 293T cells transfected with Flag-GATA4 and/or HA-CycD2 expression vectors were immunoprecipitated using an anti-Flag antibody, separated on 8% (vol/vol) SDS/PAGE, transferred to poly(vinylidene difluoride) (PVDF) membranes, and subjected to immunoblotting using anti-HA, anti-CycD2, or anti-GATA4 antibodies. The results here are from one representative experiment of three. For consistency, the relevant lanes were spliced from the same blot image and assembled in the order shown. (F) Immunocytochemical analysis of 293T cells transiently transfected with 100 ng of Flag-GATA4 with or without 1 μg of HA-CycD2. Red is GATA4 staining; green is CycD2; blue marks nuclei (Hoechst). Note cotransfection of GATA4 with CycD2 does not affect GATA4 nuclear levels.
	Fig. 2. Specificity of GATA4-CycD2 interaction. (A, Left) Transient transactivation of GATA-dependent promoter by GATA4 and CycD1 or CycD2 in NIH 3T3 cells; 500 ng of GATA4 and 4 μg of CycD1 or CycD2 expression vectors were used. Note that CycD1 does not synergize but rather inhibits GATA4 activity. #P < 0.005 vs. GATA4. (Right) Transient transactivation of GATA-dependent promoter by GATA4 and CycD1 in NIH 3T3 cells; 10 ng of GATA4 and 100, 200, 500, and 3,000 ng of CycD1 were used. Note that CycD1 inhibits GATA4 activity in a dose-dependent manner. (B) Transient transactivation of GATA-dependent promoter by GATA1, -2, -3, -4, -5, or -6 and CycD2 in NIH 3T3 cells; 250 and 500 ng of GATA and 4 μg of CycD2 expression vectors were used in the cases of GATA1, -2, -3, -4, and -6; 15 and 25 ng of GATA and 1 μg of CycD2 expression vectors were used in the case of GATA5. Note a statistically significant synergy of CycD2 with GATA4. Modest but reproducible synergy was seen in case of GATA5 and 6. No synergy was seen with the hematopoietic GATA members even at different doses. **P < 0.01; #P < 0.005 vs. GATA4. (C) Structure–function analysis of GATA4/CycD2 activation of the GATA-dependent promoter. Transient transfections were carried out in NIH 3T3 cells using the indicated GATA4 and/or CycD2 expression vectors and the GATA-Luc reporter; 250 and 500 ng of GATA4 and 4 μg of CycD2 were used. The results shown are those of one representative experiment of three carried out in duplicates with the SD of the mean. *P < 0.05; **P < 0.01; #P < 0.005; ##P < 0.0001 vs. GATA4.
	Fig. 3. CycD2 physically interacts with N-terminal GATA4 in vitro. (A) In vitro-translated radiolabeled CycD2 protein (or luciferase protein as a negative control) was incubated with glutathione Sepharose beads containing GST–N-terminal GATA4 or GST–C-terminal GATA4 fusion proteins. The bound proteins were then resolved by SDS/PAGE and revealed by autoradiography. Note that CycD2 binds to N-terminal GATA4 (second lane). The experiment is one representative of two. (B) Amino acids 130–170 are sufficient to interact with CycD2, and S160 is required for this interaction. In vitro-translated radiolabeled CycD2 protein were incubated with glutathione Sepharose beads containing GST alone, GST–N-terminal region 2–207 of GATA4, the same GST–N-terminal part of GATA4 harboring the S160G mutation or GST–130–170 GATA4 fusion proteins. The bound proteins were then resolved by SDS/PAGE and revealed by autoradiography. In vitro-translated luciferase protein was used as a negative control. Note that CycD2 binds to amino acids 2–207 of GATA4 and 130–170 but not S160G mutant. The experiment is one representative of two. (C) Schematic representation of GATA4 protein. Note that the 145- to 174-aa region, and particularly amino acid S160, is highly conserved among the mouse, rat, human, and Xenopus. (D) Alignment of the CycD2-interacting domain (145–174) of human GATA4 with the other GATA members as indicated. Note that this region is highly divergent among the different GATA members. (E) Fold synergy of CycD2 with WT (Wt) GATA4 or the indicated S160 GATA4 mutants on Nppa promoter (Left) or GATA-dependent promoter (Right). Note the reduced fold of synergy with both S160G and S160A. *P < 0.05 vs. Wt. (F) Transient transactivation of Nppa promoter by WT or S160A GATA4 with or without either CycD2 or TBX5; 250 ng of GATA4 was used with 4 μg of CycD2; 5 ng of GATA4 was used with 50 ng of TBX5. Note the S160 mutation attenuates synergy with CycD2 but not TBX5. *P < 0.05 vs. GATA4.
	Fig. 4. Phosphorylation of GATA4 at S160 is required for its transcriptional activity. (A) In vitro CDK4 or MAPK phosphorylation of GST-GATA4 fusion proteins. Active CDK4 kinase was able to phosphorylate N-terminal GST-GATA4 (2–207) fusion protein (black arrow) but not GST alone, nor C-terminal GST-GATA4 (329–440) nor N-terminal GATA4 harboring the S160G mutation. MAPK phosphorylation of N-terminal GATA4 was not affected by S160 mutation. The experiment is one representative of three. (B) Inhibition of CDK4 reduces GATA4 activity. (Left) NIH 3T3 cells were transiently transfected with the GATA-dependent promoter and increasing doses of GATA4 expression vector (5, 10, 50, 100, and 500 ng) with or without treatment with CDK4 inhibitor; 2 μM the indicated CDK4 inhibitor was used. The cells were treated the following day after transfection and kept for 18 h. (Right) Effect of the CDK4 inhibitor RO506220 on S160G GATA4 mutant. NIH 3T3 cells were transiently transfected with Nppa-Luc and increasing doses of the indicated GATA4 expression vector (10, 50, 100, and 250 ng) with or without treatment with CDK4 inhibitor as above. Note S160G activity is affected less prominently by CDK4 inhibitor RO506220 than its WT. *P < 0.05 vs. Wt. (C) Inhibition of CDK4 does not affect GATA4/Cyclin D twofold synergy. NIH 3T3 cells were transiently transfected with GATA-Luc, 50 or 100 ng of GATA4, and/or 1 μg of CycD2 expression vectors with or without treatment with CDK4 inhibitors as above. (D) CycD2/GATA4 synergy does not require CDK-binding site. NIH 3T3 cells were transiently transfected with GATA-Luc, GATA4, and/or CycD2 expression vectors. K112A CycD2 mutant was prepared by PCR-mediated mutagenesis; 50, 250, and 500 ng of GATA4 expression vector and 1 and 4 μg of CycD2 and K112A expression vectors were used. (E) Lack of triple synergy between CycD2, CDK4, and GATA4. NIH 3T3 cells with transiently transfected with GATA-Luc, CDK4 (50 ng), and/or GATA4 (25 ng) and/or CycD2 (50 ng) expression vectors. Note that fold synergy of GATA4/CycD2 does not increase with addition of CDK4.
	Fig. 5. CycD2 potentiates cardiogenic activity of GATA4. (A) CycD2 stimulates cardiogenic activity of the rat GATA4. Animal cap explants injected with 100 pg of CycD2 mRNA, 300 pg of Gata4 mRNA (G4), or the two combined were analyzed for expression of indicated markers at stage 34. Myl7 and myh6 are exclusively expressed in cardiac myocytes, endodermin (a2m) is an endodermal marker, smooth muscle actin (acta2) marks smooth muscle, and globin (hba1) is a marker of blood. Note that stimulation of GATA4 activity by CycD2 is restricted to induction of cardiac but not other cell fates. (B) S160 is important for cardiogenic activity of GATA4; 400 pg of indicated GATA4 constructs were injected. Animal explants were cultured as above, and expression of indicated markers were determined by RT-PCR. (C) S160 is required for stimulation of GATA4 activity by CycD2; 100 pg of CycD2 mRNA and 300 pg of the indicated GATA4 constructs were injected per embryo. Expression of myl7, myh6, and odc1 were determined by RT-PCR. The dotted lines between lanes in A and C indicate that the image shown was assembled either from two gels run in parallel or derives from the same gel after splicing out unnecessary lanes. (D) Immunofluorescence assay to determine the nuclear localization of the indicated GATA4 constructs in stage 6–7 embryos. Anti-GATA4 antibody was used. (Scale bar: 0.1 mm.)

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