GATA4 Is a Direct Transcriptional Activator of Cyclin D2 and Cdk4

MOLECULAR AND CELLULAR BIOLOGY, Sept. 2008, p. 5420–5431 Vol. 28, No. 170270-7306/08/$08.00�0 doi:10.1128/MCB.00717-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

GATA4 Is a Direct Transcriptional Activator of Cyclin D2 and Cdk4and Is Required for Cardiomyocyte Proliferation in Anterior

Heart Field-Derived Myocardium�

Anabel Rojas,1 Sek Won Kong,2 Pooja Agarwal,1 Brian Gilliss,1 William T. Pu,2 and Brian L. Black1,3*Cardiovascular Research Institute1 and Department of Biochemistry and Biophysics,3 University of California, San Francisco,

California 94143-2240, and Department of Cardiology, Children’s Hospital, Boston, Boston, Massachusetts 021152

Received 2 May 2008/Accepted 19 June 2008

The anterior heart field (AHF) comprises a population of mesodermal progenitor cells that are added to thenascent linear heart to give rise to the majority of the right ventricle, interventricular septum, and outflow tractin mammals and birds. The zinc finger transcription factor GATA4 functions as an integral member of thecardiac transcription factor network in the derivatives of the AHF. In addition to its role in cardiac differen-tiation, GATA4 is also required for cardiomyocyte replication, although the transcriptional targets of GATA4required for proliferation have not been previously identified. In the present study, we disrupted Gata4 functionexclusively in the AHF and its derivatives. Gata4 AHF knockout mice die by embryonic day 13.5 and exhibithypoplasia of the right ventricular myocardium and interventricular septum and display profound ventricularseptal defects. Loss of Gata4 function in the AHF results in decreased myocyte proliferation in the rightventricle, and we identified numerous cell cycle genes that are dependent on Gata4 by microarray analysis. Weshow that GATA4 is required for cyclin D2, cyclin A2, and Cdk4 expression in the right ventricle and that theCyclin D2 and Cdk4 promoters are bound and activated by GATA4 via multiple consensus GATA binding sitesin each gene’s proximal promoter. These findings establish Cyclin D2 and Cdk4 as direct transcriptional targetsof GATA4 and support a model in which GATA4 controls cardiomyocyte proliferation by coordinately regu-lating numerous cell cycle genes.

The cardiac lineage in mammals is initially specified fromthe anterior lateral mesoderm at embryonic day 7.5 (E7.5)in the mouse. The nascent cardiac mesoderm migrates ante-riolaterally and fuses ventrally in the embryo to form a lineartube. The linear tube elongates through the addition of cellsfrom the second heart field to the arterial and venous poles (1,12, 28). A more restricted, anterior subset of these cells areadded only to the arterial pole from the pharyngeal andsplanchnic mesoderm. These cells, referred as the anteriorheart field (AHF), give rise to the outflow tract, right ventricle,and ventricular septum (1, 9, 11, 27, 81). As cells from the AHFare added, the heart bends toward the ventral side, undergoesrightward looping, expands dramatically, and is eventually re-modeled into the mature, four-chambered organ (13, 66).

Embryonic cardiomyocytes differentiate as they continue toproliferate (48, 52). At early stages in development, cardiomyo-cytes have a high proliferation rate, which decreases progres-sively in late gestation (67). The high rate of cell cycle activityduring the early stages of cardiomyocyte differentiation con-tributes to the growth of the future chambers within the lineartube during looping morphogenesis (42). The trabecular myo-cardium has a high rate of proliferation at this stage. As ven-tricular volumes increase, the trabeculations become com-pressed within the ventricular wall, resulting in a significantincrease in the thickness of the compact myocardium (66). The

compact myocardium proliferates more rapidly than the tra-becular myocardium after chamber maturation has occurred(84), and several cell cycle genes have been shown to playimportant roles in cardiomyocyte proliferation (51, 76). D-cyclins and their catalytic partners, cyclin-dependent kinases(Cdks), are key components of the cell cycle machinery thatdetermine whether cells divide or remain quiescent (24). D-cyclins are regarded as sensors of the extracellular environ-ment that link mitogenic pathways to the cell cycle machinery(35). Once D-cyclins are induced by mitogenic signals, theyassociate with Cdks, resulting in the phosphorylation of theretinoblastoma suppressor RB and RB-related proteins p107and p130 (37). This phosphorylation causes the release of theE2F transcription factor and allows cells to progress from G1

to S phase (2, 3, 63, 68).GATA transcription factors comprise an evolutionarily con-

served family of zinc finger-containing proteins and recognizethe consensus binding site WGATAR (53). There are sixGATA factors; GATA1, -2, and -3 play key roles in hemato-poiesis, and GATA4, -5, and -6 are important for developmentof multiple mesoderm- and endoderm-derived tissues, includ-ing heart and liver (5, 38). Gata4 is one of the earliest genesexpressed in the cardiac crescent of the mouse, and Gata4-nullmice die around E10 as a result of severe defects in the ex-traembryonic endoderm and display defects in heart and fo-regut morphogenesis (31, 39). In humans, GATA4 mutationsare associated with defects in ventricular and atrial septation(22, 45). GATA4 regulates the expression of genes that areimportant for cardiac contraction as well as the expression ofother cardiac transcription factor genes, such as Mef2c, Hand2,and Nkx2-5 (18, 33, 36, 65).

* Corresponding author. Mailing address: Genentech Hall, 600 16thStreet, Mail Code 2240, University of California, San Francisco, SanFrancisco, CA 94158-2517. Phone: (415) 502-7628. Fax: (415) 476-8173. E-mail: [email protected].

� Published ahead of print on 30 June 2008.

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In addition to its role in cardiac differentiation, GATA4 isalso an important regulator of apoptosis and cell proliferation(29, 47, 74, 82, 86). The balance of these two processes controlscardiomyocyte number and ultimately the function of theworking myocardium, and several studies have shown the im-portance of GATA4 in myocardial development (47, 59, 62, 82,86). Tetraploid complementation, which circumvents extraem-bryonic defects in Gata4-null mice, revealed a role for GATA4in myocardial growth (82). A mutation in GATA4 that disruptsits interaction with its cofactor Friend-of-GATA 2 (FOG2)results in embryonic arrest around E12.5. Affected embryosdisplayed defects in vascular development and also had a thinventricular wall (15). More-recent studies showed that condi-tional inactivation of Gata4 using Nkx2-5Cre resulted in embry-onic lethality around E11.5, with decreased cardiomyocyte pro-liferation and major defects in the development of the rightventricle (86). However, the expression of Cre from the Nkx2-5Cre knock-in allele is broad, encompassing the derivatives ofboth the first and second heart fields, as well as the pharyngealendoderm, which leaves open the possibility that signals down-stream of GATA4 in the pharyngeal endoderm or from thefirst heart field may have affected the development of the rightventricle (86).

While previous studies have demonstrated a role forGATA4 in cardiomyocyte proliferation, the genes regulated byGATA4 that mediate this activity were not previously identi-fied. In the present study, we used whole-genome microarrayanalysis to identify misexpressed genes in myocytes lackingGata4 function. In addition, we address the function ofGATA4 in a more restricted myocardial region than previousstudies by inactivating Gata4 exclusively in the AHF and itsderivatives in the outflow tract, right ventricle, and interven-tricular septum (81). Gata4-null cardiomyocytes show down-regulation of a wide array of cell cycle-associated genes, con-sistent with significant alteration of proliferation. Cdk4, CyclinD2, and Cyclin A2 were among the most dramatically down-regulated genes in Gata4-null hearts, and we show that expres-sion of all three cell cycle proteins is decreased specifically inthe right ventricles of Gata4 AHF conditional knockout em-bryos. Furthermore, we show that GATA4 binds and directlyactivates the Cyclin D2 and Cdk4 promoters in vitro and invivo, which establishes for the first time a direct regulatoryrelationship between GATA4 and these two components ofthe cell cycle machinery. The broad downregulation of cellcycle-associated genes provides an explanation for the pro-found proliferation defects in the hearts of mice lackingGATA4 function and suggests a coordinated, GATA-depen-dent program for myocyte proliferation. Given the broad over-lap of GATA transcription factors with cyclin D2, Cdk4, andother cell cycle regulators, the studies presented here suggestthe possibility that GATA transcription factors function gen-erally to regulate G1/S transition and cellular proliferation.

MATERIALS AND METHODS

Generation of Gata4 AHF knockout mice. Gata4flox/flox, Nkx2-5Cre, and Mef2c-AHF-Cre mice have been described previously (44, 59, 81, 86). Mice harboringthe Gata4 floxed allele were crossed with Mef2c-AHF-Cre mice such that thesecond coding exon was removed specifically in the AHF by the action of Crerecombinase. The strategy for genotyping Gata4 wild-type and floxed alleles hasbeen described previously (86). The Cre transgene was detected by PCR using

the following primers: 5�-TGCCACGACCAAGTGACAGC-3� and 5�-CCAGGTTACGGATATAGTTCATG-3�. To obtain Gata4flox/flox; Mef2c-AHF-CreTg/0

embryos, timed matings between Gata4flox/�; Mef2c-AHF-CreTg/0 male mice andGata4flox/flox female mice were set up. All experiments using animals compliedwith federal and institutional guidelines and were reviewed and approved byUniversity of California, San Francisco, Institutional Animal Care and UseCommittee.

Immunohistochemistry and in situ hybridization. Embryos collected at dif-ferent stages were fixed in 4% paraformaldehyde, dehydrated with ethanol andxylene, and mounted in paraffin. Sections were cut at a thickness of 5 �m with aLeica RM 2155 microtome. Sections were dewaxed through a series of xyleneand ethanol washes and counterstained with hematoxylin and eosin to visualizeembryonic structures using standard procedures (25).

For immunohistochemistry, sections were dewaxed, incubated in phosphate-buffered saline (PBS) for 5 min, boiled in antigen retrieval solution (Biogenex),and blocked in 3% normal goat serum for 1 h. Incubation with primary rabbitanti-cyclin D2 (Santa Cruz; Sc-593), rabbit anti-Cdk4 (Santa Cruz; Sc-260),rabbit anti-cyclin A2 (Santa Cruz; Sc-751), mouse monoclonal anti-Ki67 (Novo-castra), or rabbit anti-phospho-histone H3 (Upstate Laboratories; catalog no.06-570) at a 1:300 dilution in each case was done overnight at 4°C in a humidchamber. Following incubation with the primary antibodies, sections werewashed three times with PBS and incubated with one of the following secondaryantibodies: Alexa Fluor 594 donkey anti-rabbit antibody (Invitrogen; no.A21207), Oregon Green 488 goat anti-rabbit antibody (Invitrogen; no. 0-11038),or biotinylated goat anti-mouse antibody (Vector Laboratories; BA-9200). Sec-ondary antibodies were diluted 1:300 in 3% normal goat serum and were incu-bated with the slides at room temperature for 1 h. Slides were then washed threetimes in PBS, mounted using SlowFade Light antifade with DAPI (4�,6-di-amidino-2-phenylindole; Molecular Probes), and photographed on a fluores-cence microscope. For Ki67 and cyclin A2 detection, immunoperoxidase stainingwas performed using the Vectastain Elite ABC kit (Vector Laboratories; PK-6102) and developed using the peroxidase substrate diaminobenzidine (VectorLaboratories; SK-4100). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were performed using the ApopTag kitfrom Chemicon (S-7110) by following the manufacturer’s recommendations.

To measure DNA synthesis, 2 mg of 5-bromo-2-deoxyuridine (BrdU; Sigma;B9285) dissolved in saline was injected intraperitoneally into pregnant mice andthe mice were euthanized 2 h later. Embryos collected from these mice wereprocessed as described above, and the sections were dewaxed and treated with 1M HCl for 7 min at 60°C. Antibody staining was performed using rat anti-BrdU(Serotec; MCA2060) and tetramethyl rhodamine isocyanate-conjugated anti-ratantibody (Sigma).

Whole-mount in situ hybridization was performed as described previously(64). A Gata4 in situ probe was generated from a pBluescript II SK(�) plasmidcontaining the first and second exons of the murine Gata4 gene, linearized withNotI, and transcribed with T3 polymerase.

Microarray. RNA was isolated and pooled from four or five E9.5 hearts frommice with each of the following genotypes: Gata4flox/�; Nkx2-5�/� (control; n �3), Gata4flox/�; Nkx2-5Cre/� (Gata4; Nkx2-5; doubly heterozygous, n � 3), andGata4flox/flox; Nkx2-5Cre/� (Gata4 CKONkx; n � 4). Total RNA (50 ng) wasamplified and converted to cDNA using the Ovation RNA labeling kit (NuGen).The cDNA was then hybridized to Affymetrix GeneChip Mouse 430.2 microar-rays, which have 45,101 probe sets. Gene expression data are available throughthe Gene Expression Omnibus database (accession number GSE9652). Probesets with absent calls in nine or more samples were excluded. Comparisonsbetween controls and the other two groups were made. Differentially expressedgenes were defined as those for which the nominal P was �0.005. Gene setanalysis was performed using the gene set enrichment analysis method withdefault parameters (http://www.broad.mit.edu/gsea). Cell cycle-related gene setsof size 10 to 250 were selected from the Molecular Signature Database(MSigDB). The C2 collection is available at http://www.broad.mit.edu/gsea/msigdb/collections.jsp#c2 (72).

EMSA. DNA binding reactions were performed as described previously (19).Briefly, double-stranded oligonucleotides were labeled with [32P]dCTP, usingKlenow fragments to fill in the overhanging 5� ends, and purified on a nonde-naturing polyacrylamide–Tris-borate-EDTA gel. Binding reaction mixtures werepreincubated at room temperature in 1� binding buffer (40 mM KCl, 15 mMHEPES [pH 7.9], 1 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol) containingrecombinant protein, 1 �g of poly(dI-dC), and competitor DNA for 10 min priorto probe addition. Reaction mixtures were incubated for an additional 20 min atroom temperature after probe addition and were then electrophoresed on a 6%nondenaturing polyacrylamide gel. The Gata4 cDNA was transcribed and trans-lated using the TNT coupled transcription-translation system (Promega), as

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described in the manufacturer’s directions. GATA4 protein was generated frompCITE-GATA4 plasmid, which has been described previously (18). The sensestrand sequences of the mouse Cyclin D2 and Cdk4 GATA sites and mutantGATA sites used for the electrophoretic mobility shift assay (EMSA) were asfollows: cyclin D2 Gata I, 5�-GGAACAGCTTGAAAGTTATCAGGAGTCTAAGCTTGAG-3�; cyclin D2 Gata Im, 5�-AACAGCTTGAAAGGTACCAGGAGTCTAAGCTTGAG-3�; cyclin D2 Gata II, 5�-GGGAGGGGCATAACCTTTATCCCTGGTTTGGCGAGGT-3�; cyclin D2 Gata IIm, 5�-GAGGGGCATAACCTCTAGACCTGGTTTGGCGAGGT-3�; cyclin D2 Gata III, 5�-GGACAGAATGTCAGAAAGGATAATCAATAGGAATCCAT-3�; cyclin D2 Gata IIIm,5�-ACAGAATGTCAGAAAGGATCCTCAATAGGAATCCAT-3�; Cdk4 GataI/II, 5�-GGAATTACCTATACTAGTTATCTTTATCATTCACTTCAAAGGGC-3�; Cdk4 GataI/IIm, 5�-AATTACCTATACTAGTAAGCTTTATAATTCACTTCAAAGGGC-3�; Cdk4 GataIII, 5�-GGCAAGGGGTCACGTGGGATAGCAACAGGTCACCGTGG-3�; Cdk4 GataIIIm, 5�-CAAGGGGTCACGTGGGTTAACAACAGGTCACCGTGG-3�.

Cell culture, transfections, and reporter assays. A 931-bp fragment containing671 bp upstream and 260 bp downstream of the transcriptional start site from themouse Cyclin D2 promoter region was amplified by PCR using the followingprimers: 5�-ACAGAAAGGTTTCTGCAGGAGGGTCATATTC-3� and 5�-GCCAGCCGGCGTCGACTCGGTCCCGAC-3�. An 827-bp fragment containing771 bp upstream and 56 bp downstream of the transcriptional start site from themouse Cdk4 promoter region was amplified by PCR using the following primers:5�-CTTTTAATATTCCGCGGGAGGTTTAC-3� and 5�-GGGCAGCTGGATCCTTCGGGCCAGAC-3�. Cyclin D2 and Cdk4 PCR products were cloned intothe pAUG-�-gal reporter vector (36). Plasmid pECE-GATA4-EnR has theDrosophila Engrailed repressor domain fused to the Gata4 cDNA and has beendescribed previously (32). The expression plasmid pRK5-GATA4-VP16 containsthe herpes simplex virus type 1 Vmw65.1 transcriptional activation domain fusedin frame to the 3� end of the GATA4 cDNA. Mutations of the GATA sites in theCyclin D2 and Cdk4 promoters were introduced by PCR to create the mutantsequences in the EMSA oligonucleotides described above.

C3H10T1/2 was maintained in Dulbecco modified Eagle medium supple-mented with 10% fetal bovine serum. P19CL6 cells were maintained in Dulbeccomodified Eagle medium supplemented with 10% fetal bovine serum in thepresence of 1% dimethyl sulfoxide (DMSO) for 7 days prior to transfection.Transient transfections were performed in 12-well plates using Fugene6 (Roche)for C3H10T1/2 cells and Lipofectamine XLT (Invitrogen) for P19CL6 cells, byfollowing the manufacturer’s recommendations. Each transfection mixture con-tained 0.5 �g of Cyclin D2 or Cdk4 reporter plasmids and 1.0 �g of repressor oractivator plasmids. In transfections without an expression construct, the parentexpression plasmid was added to keep the total amount of DNA in each trans-fection constant at 1.5 �g. Cells were cultured for 48 h after transfection andharvested, and cellular extracts were prepared by sonication and were normal-ized as described previously (14). Chemiluminescence �-galactosidase (�-Gal)assays were performed using the luminescent �-Gal detection system (Clontech)according to the manufacturer’s recommendations, and relative light units weredetected using a Tropix TR717 microplate luminometer (PE Applied Biosys-tems).

ChIP assays. Chromatin immunoprecipitation (ChIP) assays were performedusing the ChIP assay kit from Upstate Pharmaceuticals (catalog no. 17-295), inaccordance with the recommendations of the manufacturer. Briefly, a 10-cmplate containing approximately 1 � 106 P19CL6 cells, which had been differen-tiated into cardiomyocytes by treatment with 1% DMSO for 7 days, was treatedwith 1% paraformaldehyde at 37°C for 10 min to cross-link protein-DNA com-plexes. Cells were then lysed and sonicated to shear the DNA into fragments ofbetween 300 and 500 bp. The cleared supernatant was incubated with 4 �g ofanti-GATA4 antibody (Santa Cruz; Sc-1237) or 4 �g of anti-goat immunoglob-ulin G (IgG) (Santa Cruz; Sc-2020) overnight at 4°C. The DNA fragments werethen precipitated after incubating the lysate and antibody mixture with proteinA-agarose beads for 1 h. Reaction mixtures were incubated with NaCl at 65°C for4 h to reverse the cross-links, and DNA was recovered by phenol-chloroformextraction. The following primers were used to amplify the Cyclin D2 promoter,which contains three GATA sites, following ChIP: P1, 5�-CTCCACGCACGTGGCTCGGGGCGG-3�, and P2, 5�-TAGGGGAACCCACAAACCCCATGG-3�.Two different regions of the Cdk4 promoter, a distal region containing twoGATA sites and a proximal region containing one GATA site, were amplifiedfollowing ChIP using the following primers: P1, 5�-CATACAGTGGCTTATTATATTTCC-3�, P2, 5�-CTCCACCGCCATGGGGAAACATTC-3�, P3, 5�-GTTGGCCCGGTTGCCATGACACCG-3�, and P4, 5�-CTGGACACGTGATCTTCACCCTTG-3�. The Cyclin D2 second exon was amplified as a negative control inChIP experiments using the following primers: 5�-GCGGCCTTAGTGTGATG

FIG. 1. Inactivation of Gata4 in the AHF results in lethality dueto right ventricular hypoplasia and VSDs. (A and B) Whole-mountin situ hybridization showing expression of Gata4 mRNA in control(A) and Gata4 AHF knockout (B) hearts at E10.5. The excision ofthe Gata4 floxed allele by Mef2c-AHF-Cre results in loss of Gata4mRNA in the right ventricle (RV) and outflow tract (OFT) in Gata4AHF knockout embryos. LV, left ventricle. (C and D) Gata4 AHFknockout embryos (D) display obvious vascular hemorrhage (ar-rowheads) compared to littermate controls (C) at E13.5. (E to H)Hematoxylin- and eosin-stained transverse sections of littermatecontrol (E) and Gata4 AHF knockout (F) embryos show that theformation of the ventricular septum (arrowheads) is aberrant atE13.5 in Gata4 AHF knockout embryos compared to controls. LA,left atrium; RA, right atrium. (G and H) The compact wall myo-cardium of the right ventricle (asterisks) is thinner at E13.5 inGata4 AHF knockout embryos (H) than in littermate control em-bryos (G). Bars, 100 �m. Genotypes for control (Gata4flox/flox) andGata4 AHF knockout (CreTg/0; Gata4flox/flox) embryos are indicated.n was 4 for each genotype.

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GGGAAGG-3� and 5�-TCGGACCCTACCCCACTCTTGATTG-3�. ChIP PCRproducts were confirmed by sequencing.

RESULTS

Inactivation of Gata4 in the AHF results in right ventricularhypoplasia and ventricular septal defects (VSDs). To deter-mine the role of the GATA4 specifically in the development ofthe right ventricle and outflow tract, we inactivated Gata4 inthe progenitors of the right ventricle and outflow tract usingMef2c-AHF-Cre, which directs early excision in AHF progen-itors in the splanchnic and pharyngeal mesoderm (4, 50, 81).This resulted in specific loss of Gata4 expression in AHF de-rivatives in the right ventricle and outflow tract (Fig. 1A andB). These crosses did not produce any live Gata4 AHF knock-out animals, indicating that GATA4 is required in the deriva-tives of the AHF for embryonic development (Table 1). AtE10.5 and E11.5, Gata4 conditional knockout embryos werepresent at normal Mendelian frequencies (Table 1), and theappearance of AHF knockout embryos was normal at thesestages (data not shown). However, by E13.5, all Gata4 AHFconditional knockout embryos exhibited cardiovascular con-gestion and vascular hemorrhage, and the majority of the em-bryos lacked a heartbeat at this stage (Fig. 1C and D).

Histological analyses of knockout hearts at E12.5 to E13.5did not reveal any obvious defects in outflow tract alignment,and the septation into the pulmonary trunk and aorta ap-peared to be normal (data not shown). However, the rightventricles of all AHF knockout embryos were obviously hypo-plastic compared with those of littermate control embryos (Fig.1E to H). The compact zone of the myocardium in knockoutembryos contained fewer myocardial cell layers than those incontrol embryos, where the myocardial wall of the right ven-tricle was much thicker by this stage (Fig. 1G and H). Inaddition, the right ventricular trabecular myocardium of con-ditional knockout embryos appeared disorganized and not wellconnected with the compact myocardium (Fig. 1G and H). Theformation of the ventricular septum, which was almost completedby E13.5 in control embryos, was delayed in all Gata4 AHFknockout embryos (Fig. 1E and F). As expected, no abnormalitieswere observed in the left ventricle, which is outside of the Mef2c-AHF-Cre expression domain (Fig. 1E and F).

Gata4 AHF knockout mice display myocardial proliferationdefects in the right ventricle. GATA4 has been implicatedpreviously in both myocardial proliferation and apoptosis (74,85, 86), and the myocardial hypoplasia observed in the rightventricles of Gata4 AHF knockout embryos could be explained

by an increase in cell death or a decrease in proliferation. Todetermine whether cell death might be involved in the myo-cardial hypoplasia of Gata4 AHF knockout embryos, we per-formed TUNEL staining on cardiac sections from embryos atE10.5. We selected this developmental stage since it representsa time prior to embryonic lethality and hearts would be lesslikely to exhibit nonspecific apoptosis secondary to cardiacfailure. Results from these experiments showed no differencesin TUNEL staining between conditional knockout and controlembryos (Fig. 2I and J).

To determine if inactivation of Gata4 in the AHF resulted indefective myocyte replication, we examined the expression ofseveral markers of proliferation, including Ki67 and phospho-histone H3, and BrdU incorporation at E10.5 (Fig. 2). In eachcase, Gata4 AHF knockout embryos displayed significantlyreduced expression of the proliferation markers in the rightventricle compared to littermate control embryos (Fig. 2A toH). Similarly, Gata4 AHF knockout embryos displayed re-duced proliferation in the interventricular septum (Fig. 2A, B,and E to H), which is consistent with theVSDs observed inknockout embryos at E13.5 (Fig. 1F). The reduced prolifera-tion in the right ventricular myocardium in AHF knockoutscompared to littermate controls was especially evident whenKi67, which marks all stages of the cell cycle, was examined(Fig. 2, compare panels A and C to B and D). Quantificationof BrdU-labeled and phospho-histone H3-labeled nuclei as apercentage of the total number of DAPI-stained nuclei showedthat proliferation was significantly reduced in the right ventri-cle (Fig. 2K and L). By contrast, in the left ventricle, whereGata4 excision did not occur since the left ventricle is outsidethe expression domain of Mef2c-AHF-Cre, levels of expressionof these markers in knockout and control embryos were thesame (Fig. 2K and L). Taken together, the results presented inFig. 2 demonstrate that GATA4 is required in the derivativesof the AHF for proliferation, which supports previous studiesthat demonstrated a role for GATA4 in cardiomyocyte prolif-eration (82, 86).

GATA4 regulates the expression of numerous cell cyclegenes in the heart. The defects in myocyte proliferation inGata4 AHF knockout hearts (Fig. 2) suggested that GATA4was likely to regulate one or more genes involved in the cellcycle. Therefore, to investigate further the molecular changesunderlying these alterations in cardiomyocyte proliferation, wemeasured mRNA expression in E9.5 mouse hearts by microar-ray. To accomplish this, we used Nkx2-5Cre to inactivate Gata4(Gata4 CKONkx). As in Gata4 AHF knockout hearts, cardio-

TABLE 1. Loss of Gata4 function in the AHF results in embryonic lethality by E13.5a

Mouse genotype

No. of offspring at:

E10.5(0.83, 0.841)

E11.5(0.16, 0.980)

E12.5(4.44, 0.216)

E13.5(11.10, 0.011)

P0(44.47, �0.0001)

Gata4flox/� 28 13 33 17 44Gata4flox/flox 28 12 24 16 35CreTg/0; Gata4flox/� 34 11 22 21 47CreTg/0; Gata4flox/flox 29 12 19 4* 0**

a Gata4flox/�; Mef2c-AHF-CreTg/0 mice were crossed to Gata4flox/flox mice, and the offspring were collected at the indicated developmental stages. Offspring of eachgenotype from E10.5 to E12.5 were present at normal Mendelian frequencies. By E13.5, most of the conditional knockout embryos (�) lacked a heartbeat. No Gata4AHF knockouts were present at birth (P0; ��). The �2 and P values are in parentheses (�2, P) after the developmental stage.



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myocyte proliferation was decreased in Gata4 CKONkx cardio-myocytes (86). However, the expression domain of Nkx2-5Cre isbroader than that of Mef2c-AHF-Cre (44, 81), allowing us touse the entire hearts from Gata4 CKONkx embryos at E9.5 formicroarray analyses.

We used the microarray expression data to determine if estab-lished sets of cell cycle genes showed statistically discordant dif-ferences between Gata4flox/� (control) and Gata4flox/flox; Nkx2-5Cre/� (Gata4 CKONkx) hearts. We used curated gene setsavailable from the Molecular Signature Database (http://www.broad.mit.edu/gsea) and the gene set enrichment analysismethod (72). Seven out of 12 cell cycle-related gene sets,including the Brentani cell cycle gene set (10), were signifi-cantly altered in Gata4 conditional knockout hearts (P �

0.001; data not shown), suggesting that Gata4 inactivationleads to a broad, coordinate perturbation of genes involved incell cycle regulation (Fig. 3). By comparison, no cell cycle genesets were significantly altered between Gata4flox/�; Nkx2-5Cre/�

and control mice, indicating that double heterozygosity forGata4 and Nkx2-5 does not result in significant alteration in theexpression of cell cycle gene sets (data not shown).

Next, we looked for individual genes that when misregulatedmight contribute to abnormal expression of cell cycle gene setsand abnormal cardiomyocyte proliferation. We found that1,302 probe sets were differentially expressed between controland Gata4 CKONkx embryos (P � 0.005). In contrast, only 68probe sets were differentially expressed between Gata4flox/flox

control and Gata4flox/�; Nkx2-5Cre/� doubly heterozygous

FIG. 2. Gata4 AHF knockout embryos have profound myocardial proliferation defects. (A to H) Immunohistochemical analyses of prolifer-ation markers on transverse sections show that Gata4 AHF knockout embryos (B, D, F, and H) have reduced proliferation compared to controlembryos (A, C, E, and G) at E10.5. (A and B) Gata4 AHF knockout embryos display decreased staining of the nuclear antigen Ki67 (brown) inthe 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 notinactivated, such as the epicardium (epi). (E and F) BrdU incorporation is diminished in the myocardium of the right ventricle of Gata4 AHFknockout embryos compared to control embryos (asterisks). (G and H) Expression of the mitotic marker phospho-histone H3 (pHH3) is reducedin the right ventricle in Gata4 AHF knockout embryos compared to control littermates (arrowheads). No differences in the staining of any of theseproliferation markers between Gata4 AHF knockout and control embryos was observed in the left ventricle (LV). (I and J) TUNEL staining ontransverse sections of embryonic hearts shows no difference in apoptosis between Gata4 AHF knockout and control embryos at E10.5. Genotypesfor control (Gata4flox/flox) and Gata4 AHF knockout (Gata4flox/flox; Mef2c-AHF-CreTg/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) embryoscompared to littermate controls. The total number of DAPI-labeled cells and the number of BrdU- or pHH3-labeled cells were determined bycounting cells in a series of sections from three CKO and three control hearts. Data are presented as the mean percentages of cells labeled withBrdU 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.



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hearts, indicating that Gata4 inactivation rather than Nkx2-5and Gata4 heterozygosity is responsible for the majority of theobserved changes in gene expression (data not shown). Nota-bly, many genes that are known to play fundamental roles incellular proliferation, including several cyclin genes and nu-merous other cell cycle genes, were significantly downregu-lated in the absence of GATA4 function (Fig. 3). Interestingly,Cyclin D2 (CCND2) and Cyclin A2 (CCNA2) were among themost dramatically downregulated genes in the absence ofGATA4 function in the heart (Fig. 3). Cyclin function dependson the activity of Cdks, including Cdk2 and Cdk4, and micelacking multiple Cdk genes die during embryonic developmentwith thin myocardial walls (8). Our microarray data indicatedthat several Cdk genes, including Cdk4, also had reduced ex-pression in the absence of GATA4 (Fig. 3).

Gata4 is required for Cyclin D2, Cdk4, and Cyclin A2 expres-sion in the right ventricle. Because Cyclin D2, Cyclin A2, andCdk4 were among the most significantly downregulated tran-scripts according to microarray data (Fig. 3), we further exam-ined the expression of the corresponding gene products inGata4flox/flox; Mef2c-AHF-CreTg/0 embryos by immunohisto-chemistry at E10.5. These analyses showed that the expressionof all three cell cycle proteins was substantially reduced in themyocardium and endocardium of the right ventricle in Gata4AHF knockouts (Fig. 4B, D, and F) compared with controlembryos (Fig. 4A, C, and E). No differences in expression inthe left ventricle between Gata4 AHF knockouts and litter-mate controls were observed, consistent with the specific inac-tivation of Gata4 in the AHF. Similarly, expression was unper-turbed in the epicardial cell layer, where the Gata4 floxedallele also was not excised by Cre recombinase (Fig. 4). Takentogether, these immunohistochemistry data strongly supportour microarray studies, which indicate that GATA4 is requiredfor expression of multiple cell cycle control genes. In particu-lar, our results demonstrate the requirement of GATA4 func-tion for cyclin D2, cyclin A2, and Cdk4 expression in myocytesin the embryonic right ventricle (Fig. 4).

GATA4 binds to the Cyclin D2 and Cdk4 promoters in vitroand in vivo. To determine if the regulation of these cell cyclegenes by GATA4 was direct, we examined the upstream re-gions for evolutionarily conserved GATA binding sites. Thesebioinformatic analyses identified three perfect consensusGATA sites upstream of both the Cyclin D2 and Cdk4 genes,and therefore we examined these two genes in detail to deter-mine if they were regulated by direct GATA4 binding to theirpromoter regions in cardiomyocytes (Fig. 5). The Cyclin D2promoter contains two conserved, consensus GATA sites atpositions 558 and 525 relative to the transcriptional startsite (Fig. 5A). These two sites, referred to as D2 Gata I and D2Gata II, were each found to be bound efficiently by GATA4 byEMSA (Fig. 5C, lanes 2 and 6). Binding of GATA4 to these

FIG. 3. GATA4 regulates multiple cell cycle control genes. Af-fymetrix gene expression data were analyzed by gene set enrichment(72). Several sets of genes with known roles in cell cycle regulationshowed statistically significant, concordant differences between control(Gata4flox/�) and Gata4 CKONkx (Gata4flox/flox; Nkx2-5Cre/�) hearts.The heat map of genes comprising the cell cycle gene set with the mostsignificant 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 scaleat the bottom). Numerous cell cycle control genes were significantlydownregulated in Gata4 CKONkx hearts compared to controls, includ-ing Cyclin D2 (CCND2), Cyclin A2 (CCNA2), and Cdk4, which aredenoted by arrows.



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sites in the Cyclin D2 promoter was specific because it wasefficiently competed by an excess of unlabeled self probe (Fig.5C, lanes 3 and 7) but not by mutant versions of the Cyclin D2Gata I or Gata II sites (Fig. 5C, lanes 4 and 8). In addition tothese two conserved GATA sites, another candidate site at bp299 (D2 Gata III) was also bound robustly by GATA4 (Fig.5C, lane 10). Binding to this site was also specific as it wasinhibited by the addition of excess unlabeled self probe but notby the addition of a mutant version of itself (Fig. 5C, lanes 11and 12).

Within the proximal Cdk4 promoter, a perfectly conservedGATA site (Cdk4 Gata III) is present at position 180 relativeto the transcriptional start site (Fig. 5B). This GATA site wasefficiently bound by GATA4 in EMSA (Fig. 5D, lane 6). Thebinding to this site was competed by an excess of unlabeled selfprobe but not by a mutant version of the self probe (Fig. 5D,lanes 7 and 8). In addition, the Cdk4 upstream region alsocontains two nonconserved, consecutive candidate sites at po-sitions 607 and 601 relative to the transcriptional start site(Fig. 5B). These two Cdk4 GATA sites, referred as Cdk4 GataI/II, were also found to be robustly bound by GATA4 byEMSA (Fig. 5D, lane 2). The binding to these sites was also

specifically competed by an excess of unlabeled probe contain-ing both sites but not by a probe in which both GATA siteswere mutated (Fig. 5D, lanes 3 and 4).

The data presented in Fig. 5C and D demonstrate that theCyclin D2 and Cdk4 promoter regions each contain multiplebona fide GATA sites that are efficiently bound in vitro byGATA4. To determine the ability of GATA4 to bind to theCyclin D2 and Cdk4 promoters in cardiomyocytes, we per-formed ChIP assays with differentiated P19CL6 cardiomyo-cytes (Fig. 5E). P19CL6 is a clonal derivative from the pluri-potent P19 mouse embryonal carcinoma cell line, whichefficiently differentiates into functional, contractile cardiacmyocytes in the presence of 1% DMSO, and these myocytesexpress numerous cardiac transcription factors, includingGATA4, Nkx2-5, and MEF2C (40, 41, 54, 80, 83). Anti-GATA4 antibodies specifically precipitated DNA fragmentsencompassing the GATA sites in the endogenous Cyclin D2promoter (Fig. 5E, lane 3). This product was specific to theGATA4 antibody since the addition of nonspecific anti-IgG inthe ChIP reaction did not result in the detection of Cyclin D2by PCR (Fig. 5E, lane 1). Similarly, the anti-GATA4 antibodyspecifically precipitated promoter fragments from the endog-enous Cdk4 gene that encompassed the proximal Gata III siteand the more distal Gata I and II sites (Fig. 5E, lanes 6 and 9).These results demonstrate that GATA4 directly interacts withthe endogenous Cyclin D2 and Cdk4 promoters in cardiacmyocytes via multiple bona fide, consensus GATA sites.

Transcriptional activation of the Cyclin D2 and Cdk4 pro-moters requires GATA sites. The observations that cyclin D2and Cdk4 expression required GATA4 and that GATA4bound directly to the Cyclin D2 and Cdk4 promoters in vitroand in vivo suggested that the promoters of these two cell cyclegenes might require GATA4 for activation. Therefore, we ex-amined the requirement of the GATA sites in the Cyclin D2and Cdk4 promoters for activation in P19CL6 cardiomyocytesin vivo by fusing the promoters to the lacZ reporter gene andtesting them in a luminescent �-Gal assay (Fig. 6). Both theCyclin D2 and Cdk4 promoters exhibited significant activationin differentiated P19CL6 cells compared to the parent reporterconstruct, pAUG-�-gal (Fig. 6A and B), due to the presence ofendogenous GATA4 factors in P19CL6 cells (40, 41, 54, 80,83). Consistent with this notion, we observed a dramatic in-crease in GATA4 protein in P19CL6 cells by Western blottingafter 7 days of culture in the presence of DMSO (data notshown). Importantly, the activation of both the Cyclin D2 andCdk4 promoters significantly decreased in P19CL6 cardiomy-ocytes when the GATA sites in the promoters were mutated,indicating that GATA factors are important in the transcrip-tional activation of both promoters (Fig. 6A and B).

We also observed a significant level of activation of theCyclin D2 promoter in C3H10T1/2 cells, suggesting that thisfibroblast cell line also expresses GATA factors endogenously(Fig. 6C, lane 2). Consistent with this notion, the activation ofthe Cyclin D2 reporter by endogenous factors in C3H10T1/2cells was also dependent on the presence of intact GATA sites(Fig. 6C, lane 3). In addition, the activity of the Cyclin D2promoter in C3H10T1/2 was inhibited by coexpression of arepressor form of GATA4, GATA4-EnR, which has the re-pressor domain from the Drosophila Engrailed protein fused tothe Gata4 cDNA (Fig. 6D). In spite of the activation of the

FIG. 4. Gata4 inactivation leads to decreased expression of cellcycle proteins. Immunohistochemical staining of transverse sectionswith 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 cycleproteins is dramatically reduced in the right ventricular myocardium inGata4 AHF knockout embryos (B, D, and F) compared to that inlittermate control embryos (A, C, and E) at E10.5 (asterisks). In panelsA to D, staining for cyclin D2 and Cdk4 is red and nuclei have beencounterstained with DAPI (blue). In panels E and F, cyclin A2 isstained in brown. No differences in cyclin D2, Cdk4, or cyclin A2protein expression between knockout and control embryos were ob-served in regions outside the Mef2c-AHF-Cre domain, such as the leftventricle (LV). Genotypes for control (Gata4flox/flox) and Gata4 AHFknockout (CreTg/0;Gata4flox/flox) embryos are indicated. RV, rightventricle.



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Cyclin D2 promoter by endogenous GATA factors inC3H10T1/2 cells, the activation was significantly less in thisfibroblast cell line than in differentiated P19CL6 cardiomyo-cytes, prompting us to test the ability of exogenous GATA4 toactivate the Cyclin D2 promoter in this cell line (Fig. 6E).GATA4 has weak intrinsic transactivation ability and is widelyappreciated for interacting with transcriptional coregulators toactivate target genes in the heart (16, 20, 43, 71). Therefore, weused an activator form of GATA4, GATA4-VP16, to over-come the requirement for GATA4 cofactors that may not beabundant in C3H10T1/2 cells. GATA4-VP16 strongly transac-tivated the Cyclin D2-lacZ reporter construct (Fig. 6E, lane 6),and this activation was dependent on the presence of intactGATA binding sites since mutation of the three consensusGATA elements in the Cyclin D2 promoter ablated transacti-vation (Fig. 6E, lane 4).

Taken together, the data presented in Fig. 5 and 6 demon-strate that GATA4 is a direct transcriptional activator of theCyclin D2 and Cdk4 genes through direct promoter bindingand activation. These data support a model in which GATA4

regulates myocyte proliferation, at least in part, through directregulation of Cyclin D2 and Cdk4.

DISCUSSION

GATA4 is an essential regulator of mesodermal andendodermal organ formation and is a key component of thecore cardiac transcription factor network (38, 56, 57). Recentstudies using conditional-inactivation approaches in mice haveshown that Gata4 is required for proper cardiomyocyte prolif-eration, although the pathways downstream of GATA4 thatcontrol myocyte division have not been elucidated previously(85, 86). In this paper, we show that inactivation of Gata4 inthe AHF, prior to the formation of the right ventricle, resultsin hypoplasia of the right ventricle and VSDs resulting fromdiminished cardiac proliferation. We also show for the firsttime that GATA4 regulates the expression of numerous cellcycle control genes, including Cyclin D2 and Cdk4, via directpromoter binding and activation. Interestingly, later inactiva-tion of Gata4 using -myosin heavy chain–Cre, which does not

FIG. 5. GATA4 binds directly to the Cyclin D2 and Cdk4 promoters in vivo and in vitro. (A and B) Schematic representations of the mouseCyclin D2 and Cdk4 promoters. The Cyclin D2 construct encompasses nucleotides 671 to �260 relative to the transcriptional start site (bentarrow). The Cdk4 construct encompasses nucleotides 771 to �56 relative to the transcriptional start site (bent arrow). Boxes denote consensusGATA 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 thelocations 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 oligonucleotidesencompassing 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, lanes1 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) GATA4binds to the endogenous Cyclin D2 and Cdk4 promoters in vivo. Differentiated P19CL6 cardiomyocytes were subjected to ChIP to detectendogenous GATA4 bound to the Cyclin D2 and Cdk4 promoters using anti-GATA4 antibody. Following ChIP, the Cyclin D2 promoter wasdetected 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 andP4 (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 productswere analyzed by agarose gel electrophoresis. Lanes 3, 6, 9, and 12 contain PCR products obtained following ChIP using anti-GATA4 antibody(-G4). Lanes 1, 4, 7, and 10 contain PCR products obtained following ChIP using a nonspecific anti-IgG (-IgG). Lanes 2, 5, 8, and 11 containPCR products from input DNA (Inp) amplified prior to immunoprecipitation. ChIP products were detected only from promoter regions in sampleswhere anti-GATA4 antibody was used. Sizes in bp are shown at the left.



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become fully active until after E10.5, when the right ventriclehas already formed, does not result in loss of Cyclin D2 ex-pression (86), suggesting a requirement for GATA4 regulationof cyclin D2 expression early in the development of the rightventricle.

Mouse models have been developed in order to understandthe cell cycle and the interplay of cyclin/Cdk complexes. CyclinD2 is a member of the D-cyclin family of cell cycle regulators(61). D-cyclins are intracellular sensors that integrate mito-genic signals to direct G1/S cell cycle transition (68). Threemammalian D-cyclins are expressed in overlapping patterns inall proliferating cell types (30). Consistent with the overlappingexpression of D-cyclin proteins, mice lacking any single D-cyclin are viable and display only narrow, highly tissue-re-stricted phenotypes with no obvious cardiac defects (21, 26, 69,70). However, compound mutation of all three D-cyclin genesresults in embryonic lethality due to cellular proliferation de-fects, including reduced cardiomyocyte cell division (30). Sim-ilarly, individual knockout of either Cdk4 or Cdk2 did notreveal any obvious defects, and neither gene is required forviability in mice (7, 49, 60, 79). However, compound mutationof Cdk4 and Cdk2 results in impaired proliferation and heartgrowth (8). The microarray studies presented here demon-strate that GATA4 regulates a large number of cell cyclegenes, including multiple cyclin and cyclin-dependent kinasegenes (Fig. 3). These observations suggest that GATA4 con-trols cardiomyocyte proliferation through coordinate regula-tion of numerous cell cycle genes. In support of that notion, weshow that GATA4 directly binds to and activates the Cyclin D2and Cdk4 promoters (Fig. 5 and 6). It is likely that GATA4 alsodirectly regulates other cell cycle genes.

GATA factors have an important function in either enhanc-ing or inhibiting cell cycle progression in tissues other than themyocardium (73, 75, 78). For example, it has been proposedthat GATA6 maintains the quiescent state of vascular smoothmuscle cells, probably through induction of p21, a Cdk inhib-itor (55). In pulmonary smooth muscle cells, GATA4 appearsto be important for cell proliferation, and overexpression of arepressor form of GATA4 suppresses cyclin D2 expression(73), which supports the direct activation of Cyclin D2 byGATA4 observed in our studies. Similarly, GATA1 inducesthe sustained expression of cyclin D1 in a myeloid cell line (77),and GATA4 cooperates with the Kruppel-like factor KLF13 toactivate Cyclin D1 in Xenopus laevis (46). All of these studies,taken together with the work presented here, support a model

FIG. 6. The GATA sites in the Cyclin D2 and Cdk4 promoters arerequired for activation. (A and B) The Cyclin D2 (A) and Cdk4(B) promoters were significantly activated by endogenous GATA fac-tors in differentiated P19CL6 cardiomyocytes (lane 2) compared to theactivity of the parent reporter construct, pAUG-�-gal (lane 1), andmutation of the GATA sites in each promoter significantly attenuatedactivity (lane 3). RLU, relative light units. (C) The Cyclin D2 promoterwas significantly activated in C3H10T1/2 fibroblasts (lane 2) comparedto the activity of the parent reporter (lane 1), and mutation of theGATA sites significantly attenuated promoter activation (lane 3).

(D) GATA4-EnR inhibits activation of the Cyclin D2 promoter inC3H10T1/2 cells. Cotransfection of GATA4-EnR expression plasmidresulted in potent repression of the Cyclin D2 reporter construct (com-pare lanes 3 and 4). (E) Cotransfection of a GATA4-VP16 expressionplasmid with the Cyclin D2-lacZ reporter plasmid resulted in potenttransactivation of the Cyclin D2 promoter in C3H10T1/2 cells (lane 6).Mutation of the GATA sites (mGATA) in the Cyclin D2 promoterdisrupted transactivation by GATA4-VP16 (lane 4). In all cases, thetotal amount of transfected plasmid DNA was held constant by addi-tion of the appropriate amount of the parent expression plasmid. Errorbars represent the standard errors of the means for at least threeindependent triplicate sets of transfections and analyses for eachpanel. P values were calculated by two-tailed, unpaired t test.



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in which GATA factors may function generally as regulators ofthe cell cycle in multiple tissues. It will be interesting to deter-mine if additional cell cycle genes are also direct transcrip-tional targets of GATA4 and other GATA factors in the heartand other tissues.

In addition to its role in proliferation, GATA4 is widelyappreciated as a key regulator of cardiomyocyte differentiationthrough the activation of other transcription factor and down-stream structural genes (38, 56, 57). Our data suggest thatGATA4 is dispensable for myocyte specification and differen-tiation in the AHF and that it is not essential for the patterningor alignment of the outflow tracts. AHF-derived structuresappear to be normal in Gata4 AHF knockouts except for aVSD, which is probably secondary to myocyte proliferationdefects in the muscular septum. Lack of Gata4 in the endo-cardium has been previously shown to affect the proliferationof the membranous portion of the septum, also leading toVSDs (62). Gata4 AHF conditional knockout mice also haveGATA4 depleted in the endocardium, which may contribute tothe observed membranous VSDs in the conditional knockoutmice described in the present study (Fig. 1). Previous work hasshown that Gata4 is not broadly expressed in the pharyngealmesoderm (86), which may explain why the outflow tracts formnormally and have normal alignment in Gata4 AHF knockoutembryos. Alternatively, Gata5 and Gata6 may be able to com-pensate for Gata4 in myocyte specification and differentiationin the AHF and its derivatives.

GATA4 may regulate the balance between differentiationand proliferation through cofactor interactions or by integrat-ing and interpreting distinct upstream signals into unique out-puts. Consistent with this idea, numerous GATA4 differenti-ation partners have been identified previously, includingMEF2C, Nkx2-5, HAND2, SRF, and Tbx5 (6, 16, 20, 43, 58).Interestingly, Tbx5 regulates cell cycle genes that control G1/Sphase transition in Xenopus (23). We show here that GATA4also regulates numerous cell cycle genes, including several thatcontrol G1/S transition (Fig. 3). GATA4 interacts with Tbx5 inthe activation of the Nppa, p204, and connexin40 promotersduring cardiomyocyte differentiation in vitro (17, 22, 34, 58),and disruption of the Tbx5-GATA4 interaction in humansresults in congenital septation defects (22). It will be importantto determine if Tbx5 and GATA4 also cooperatively regulatecell cycle genes and whether other core cardiac transcriptionfactors also participate in a complex with GATA4 for cell cyclecontrol.

ACKNOWLEDGMENTS

We thank Jeff Molkentin and Evie Dodou for providing plasmidsused in these studies and Benoit Bruneau for helpful comments on themanuscript.

A.R. was supported in part by a postdoctoral fellowship from theAmerican Heart Association, Western States Affiliate. S.W.K. andW.T.P. were supported by NIH SCCOR grant P01 HL074734. Thiswork was supported by grants HL64658 and AR52130 from the NIH toB.L.B.

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GATA4 Is a Direct Transcriptional Activator of Cyclin D2 and Cdk4