MCB Accepts, published online ahead of print on 30 June 2008 Mol. Cell. Biol. doi:10.1128/MCB.00717-08 Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

1 GATA4 is a direct transcriptional activator of Cyclin D2 and Cdk4 and is required for

2 cardiomyocyte proliferation in anterior heart field-derived myocardium

3

4 Anabel Rojas1, Sek Won Kong2, Pooja Agarwal1,

5 Brian Gilliss1, William T. Pu2, and Brian L. Black1,3,*

6

7 Cardiovascular Research Institute1 and Department of Biochemistry and Biophysics3, University of

8 California, San Francisco, CA 94143-2240; and Department of Cardiology, Children's Hospital, Downloaded from

9 Boston2, Boston, MA 02115

10 mcb.asm.org

11 Running Title: GATA4 regulates Cyclin D2 and Cdk4 in the heart

12 at Harvard Libraries on August 1, 2008

13 *Corresponding author:

14 Genentech Hall, 600 16th Street, Mail Code 2240

15 University of California, San Francisco 16 San ACCEPTEDFrancisco, CA 94158-2517 17 Tel: 415-502-7628

18 Fax: (415) 476-8173

19 E-mail: [email protected]

20

21 Word Count (Materials and Methods): 1440 words

22 Word Count (Introduction, Results, and Discussion): 4367 words

23

1 23 Abstract

24

25 The anterior heart field (AHF) comprises a population of mesodermal progenitor cells that are added

26 to the nascent linear heart to give rise to the majority of the right ventricle, interventricular septum,

27 and outflow tract of mammals and birds. The zinc finger transcription factor GATA4 functions as

28 an integral member of the cardiac transcription factor network in the derivatives of the AHF. In

29 addition to its role in cardiac differentiation, GATA4 is also required for cardiomyocyte replication,

30 although the transcriptional targets of GATA4 required for proliferation have not been previously Downloaded from

31 identified. In the present study, we disrupted Gata4 function exclusively in the AHF and its

32 derivatives. Gata4 AHF knockout mice die by embryonic day 13.5 and exhibit hypoplasia of the mcb.asm.org

33 right ventricular myocardium and interventricular septum and display profound ventricular septal

34 defects. Loss of Gata4 function in the AHF results in decreased myocyte proliferation in the right at Harvard Libraries on August 1, 2008

35 ventricle, and we identified numerous cell cycle genes that are dependent on Gata4 by microarray

36 analysis. We show that GATA4 is required for Cyclin D2, Cyclin A2, and Cdk4 expression in the

37 right ventricle and that the Cyclin D2 and Cdk4 promoters are bound and activated by GATA4 via 38 multipleACCEPTED consensus GATA binding sites in each gene's proximal promoter. These findings establish 39 Cyclin D2 and Cdk4 as direct transcriptional targets of GATA4 and support a model in which

40 GATA4 controls cardiomyocyte proliferation by coordinately regulating numerous cell cycle genes.

2 41 Introduction

42

43 The cardiac lineage in mammals is initially specified from the anterior lateral mesoderm at

44 embryonic day (E) 7.5 in the mouse. The nascent cardiac mesoderm migrates anteriolaterally, where

45 it fuses ventrally in the embryo to form a linear tube. The linear tube elongates through the addition

46 of cells from the second heart field to the arterial and venous poles (1, 12, 28). A more restricted,

47 anterior subset of these cells are added only to the arterial pole from the pharyngeal and splanchnic

48 mesoderm. These cells, referred as the anterior heart field (AHF), give rise to the outflow tract, right Downloaded from

49 ventricle, and ventricular septum (1, 9, 11, 27, 81). As cells from the AHF are added, the heart

50 bends toward the ventral side, undergoes rightward looping, expands dramatically, and is eventually mcb.asm.org

51 remodeled into the mature, four-chambered organ (13, 66).

52 at Harvard Libraries on August 1, 2008

53 Embryonic cardiomyocytes differentiate as they continue to proliferate (48, 52). At early stages in

54 development, cardiomyocytes have a high proliferation rate, which decreases progressively in late

55 gestation (67). The high rate of cell cycle activity during the early stages of cardiomyocyte 56 differentiationACCEPTED contributes to the growth of the future chambers within the linear tube during looping 57 morphogenesis (42). The trabecular myocardium has a high rate of proliferation at this stage. As

58 ventricular volumes increase, the trabeculations become compressed within the ventricular wall,

59 resulting in a significant increase in thickness of the compact myocardium (66). The compact

60 myocardium proliferates more rapidly than the trabecular myocardium after chamber maturation has

61 occurred (84), and several cell cycle genes have been shown to play important roles in

62 cardiomyocyte proliferation (51, 76). D-cyclins and their catalytic partners, cyclin-dependent

63 kinases (Cdks), are key components of the cell cycle machinery that determine whether cells divide

3 64 or remain quiescent (24). D-cyclins are regarded as sensors of the extracellular environment that

65 link mitogenic pathways to the cell cycle machinery (35). Once D-cyclins are induced by mitogenic

66 signals, they associate with Cdks, resulting in the phosphorylation of the retinoblastoma suppressor

67 RB and RB-related proteins p107 and p130 (37). This phosphorylation causes the release of the E2F

68 transcription factor and allows cells to progress from G1 to S phase (2, 3, 63, 68).

69

70 GATA transcription factors comprise an evolutionary-conserved family of zinc finger-containing

71 proteins and recognize the consensus binding site WGATAR (53). There are six GATA factors; Downloaded from

72 GATA 1-3 play key roles in hematopoiesis, and GATA 4-6 are important for development of

73 multiple mesoderm- and endoderm-derived tissues, including heart and liver (5, 38). Gata4 is one of mcb.asm.org

74 the earliest genes expressed in the cardiac crescent of the mouse, and Gata4-null mice die around

75 E10 as a result of severe defects in extraembryonic endoderm and display defects in heart and at Harvard Libraries on August 1, 2008

76 foregut morphogenesis (31, 39). In humans, GATA4 mutations are associated with defects in

77 ventricular and atrial septation (22, 45). GATA4 regulates the expression of genes that are important

78 for cardiac contraction as well as the expression of other cardiac transcription factor genes, such as 79 Mef2cACCEPTED, Hand2, and Nkx2-5 (18, 33, 36, 65). 80

81 In addition to its role in cardiac differentiation, GATA4 is also an important regulator of apoptosis

82 and cell proliferation (29, 47, 74, 82, 86). The balance of these two processes controls

83 cardiomyocyte number and ultimately the function of the working myocardium, and several studies

84 have shown the importance of GATA4 in myocardial development (47, 59, 62, 82, 86). Tetraploid

85 complementation, which circumvents extra-embryonic defects in Gata4-null mice, revealed a role

86 for GATA4 in myocardial growth (82). A mutation in GATA4 that disrupts its interaction with its

4 87 cofactor Friend-of-GATA 2 (FOG2) results in embryonic arrest around E12.5. Affected embryos

88 displayed defects in vascular development and also had a thin ventricular wall (15). More recent

89 studies showed that conditional inactivation of Gata4 using Nkx2-5Cre resulted in embryonic lethality

90 around E11.5 with decreased cardiomyocyte proliferation and major defects in the development of

91 the right ventricle (86). However, the expression of Cre from the Nkx2-5Cre knock-in allele is broad,

92 encompassing the derivatives of both first and second heart fields, as well as the pharyngeal

93 endoderm, which leaves open the possibility that signals downstream of GATA4 in the pharyngeal

94 endoderm or from the first heart field may have affected the development of the right ventricle (86). Downloaded from

95

96 While previous studies have demonstrated a role for GATA4 in cardiomyocyte proliferation, the mcb.asm.org

97 genes regulated by GATA4 that mediate this activity were not previously identified. In the present

98 study, we used whole genome microarray analysis to identify mis-expressed genes in myocytes at Harvard Libraries on August 1, 2008

99 lacking Gata4 function. In addition, we address the function of GATA4 in a more restricted

100 myocardial region than previous studies by inactivating Gata4 exclusively in the AHF and its

101 derivatives in the outflow tract, right ventricle, and interventricular septum (81). Gata4-null 102 cardiomyocytesACCEPTED show down regulation of a wide array of cell cycle-associated genes, consistent with 103 significant alteration of proliferation. Cdk4, Cyclin D2, and Cyclin A2 were among the most

104 dramatically down regulated genes in Gata4-null hearts, and we show that expression of all three

105 cell cycle proteins is decreased specifically in the right ventricle of Gata4 AHF conditional knockout

106 embryos. Furthermore, we show that GATA4 binds and directly activates the Cyclin D2 and Cdk4

107 promoters in vitro and in vivo, which establishes for the first time a direct regulatory relationship

108 between GATA4 and these two components of the cell cycle machinery. The broad down regulation

109 of cell cycle-associated genes provides an explanation for the profound proliferation defects in the

5 110 hearts of mice lacking GATA4 function and suggests a coordinated, GATA-dependent program for

111 myocyte proliferation. Given the broad overlap of GATA transcription factors with Cyclin D2,

112 Cdk4, and other cell cycle regulators, the studies presented here suggest the possibility that GATA

113 transcription factors function generally to regulate G1/S transition and cellular proliferation.

114

115 Material and Methods

116

117 Generation of Gata4 AHF knockout mice Downloaded from

118 Gata4flox/flox, Nkx2-5Cre, and Mef2c-AHF-Cre mice have been described previously (44, 59, 81, 86).

119 Mice harboring the Gata4 floxed allele were crossed with Mef2c-AHF-Cre mice such that the second mcb.asm.org

120 coding exon was removed specifically in the AHF by the action of Cre recombinase. The strategy for

121 genotyping Gata4 wild-type and floxed alleles has been described previously (86). The Cre at Harvard Libraries on August 1, 2008

122 transgene was detected by PCR using the following primers: 5-tgccacgaccaagtgacagc-3 and 5-

123 ccaggttacggatatagttcatg-3. To obtain Gata4flox/flox; Mef2c-AHF-CreTg/0 embryos, timed matings

124 were set up between Gata4flox/+; Mef2c-AHF-CreTg/0 male mice and Gata4flox/flox female mice. All 125 experimentsACCEPTED using animals complied with federal and institutional guidelines and were reviewed and 126 approved by UCSF Institutional Animal Care and Use Committee.

127

128 Immunohistochemistry and in situ hybridization

129 Embryos collected at different stages were fixed in 4% paraformaldehyde, dehydrated with ethanol

130 and xylene and mounted in paraffin. Sections were cut at a thickness of 5 m with a Leica RM 2155

131 microtome. Sections were dewaxed through a series of xylene and ethanol washes and

6 132 counterstained with hematoxylin and eosin to visualize embryonic structures using standard

133 procedures (25).

134

135 For immunohistochemistry, sections were dewaxed, incubated in PBS for 5 min, boiled in antigen

136 retrieval solution (Biogenex), and blocked in 3% normal goat serum for 1 h. Incubation with

137 primary rabbit anti-Cyclin D2 (Santa Cruz Sc-593), rabbit anti-Cdk4 (Santa Cruz Sc-260), rabbit

138 anti-Cyclin A2 (Santa Cruz Sc-751), mouse monoclonal anti-Ki67 (Novocastra), or rabbit anti-

139 phospho-Histone H3 (Upstate Laboratories, Cat. # 06-570) at a 1:300 dilution in each case was done Downloaded from

140 overnight at 4°C in a humid chamber. Following incubation with the primary antibodies, sections

141 were washed three times with PBS and incubated with one of the following secondary antibodies: mcb.asm.org

142 Alexa Fluor 594 donkey anti-rabbit (Invitrogen #A21207), Oregon Green 488 goat anti-rabbit

143 (Invitrogen #0-11038), or biotinylated goat anti-mouse (Vector Laboratories BA-9200). Secondary at Harvard Libraries on August 1, 2008

144 antibodies were diluted 1:300 in 3% normal goat serum and were incubated with the slides at room

145 temperature for 1 h. Slides were then washed three times in PBS, mounted using SlowFade Light

146 antifade with DAPI (Molecular Probes) and photographed on a fluorescence microscope. For Ki67 147 and ACCEPTEDCyclin A2 detection, immunoperoxidase staining was performed using the Vectastain Elite ABC 148 kit (Vector Laboratories PK-6102) and developed using the peroxidase substrate DAB (Vector

149 Laboratories SK-4100). TUNEL assays were performed using ApopTag kit from Chemicon (S-

150 7110), following the manufacturer’s recommendations.

151

152 To measure DNA synthesis, 2 mg of 5-bromo-2-deoxy-uridine (BrdU, Sigma B9285) dissolved in

153 saline was injected intraperitoneally into pregnant mice, and the mice were euthanized 2 h later.

154 Embryos collected from these mice were processed as described above, and the sections were

7 155 dewaxed and treated with 1M HCl for 7 min at 60°C. Antibody staining was performed using rat

156 anti-BrdU (Serotec MCA2060) and tetramethyl rhodamine isocyanate (TRITC)-conjugated anti-rat

157 (Sigma).

158

159 Whole mount in situ hybridization was performed as described previously (64). A Gata4 in situ

160 probe was generated from a pBluescript (SKII+) plasmid containing the first and second exons of the

161 murine Gata4 gene, linearized with NotI and transcribed with T3 polymerase.

162 Downloaded from

163 Microarray

164 RNA was isolated and pooled from 4-5 E9.5 mouse hearts of each of the following genotypes: mcb.asm.org

165 Gata4flox/+; Nkx2-5+/+ (control, n=3), Gata4flox/+; Nkx2-5Cre/+ (Gata4; Nkx2-5 double heterozygous,

166 n=3), and Gata4flox/flox; Nkx2-5Cre/+ (Gata4 CKONkx, n=4). Total RNA (50 ng) was amplified and at Harvard Libraries on August 1, 2008

167 converted to cDNA using the Ovation RNA labeling kit (NuGen). The cDNA was then hybridized

168 to Affymetrix GeneChip Mouse 430.2 microarrays, which have 45101 probe sets. Gene expression

169 data are available through the Gene Expression Omnibus (GEO) database: accession number 170 GSE9652.ACCEPTED Probe sets with absent calls in 9 or more samples were excluded. Comparisons were 171 made between control and the other two groups. Differentially expressed genes were defined as

172 those with nominal p < 0.005. Gene set analysis was performed using the Gene Set Enrichment

173 Analysis method with default parameters (http://www.broad.mit.edu/gsea). Cell cycle-related gene

174 sets of size 10-250 were selected from the Molecular Signature Database (MSigDB). The C2

175 collection is available at http://www.broad.mit.edu/gsea/msigdb/cards/c2_cards_index.html (72).

176

177

8 178 Electrophoretic Mobility Shift Assay (EMSA)

179 DNA binding reactions were performed as described previously (19). Briefly, double-stranded

180 oligonucleotides were labeled with [32P]-dCTP, using Klenow to fill in the overhanging 5 ends, and

181 purified on a nondenaturing polyacrylamide-TBE gel. Binding reactions were pre-incubated at room

182 temperature in 1x binding buffer (40mM KCl, 15mM HEPES [pH 7.9], 1mM EDTA, 0.5 mM DTT,

183 5% glycerol) containing recombinant protein, 1g of poly-dI-dC and competitor DNA for 10 min

184 prior to probe addition. Reactions were incubated for an additional 20 min at room temperature after

185 probe addition and were then electrophoresed on a 6% nondenaturing polyacrylamide gel. The Downloaded from

186 Gata4 cDNA was transcribed and translated using the TNT Coupled Transcription-Translation

187 System (Promega), as described in the manufacturer’s directions. GATA4 protein was generated mcb.asm.org

188 from pCITE-GATA4 plasmid, which has been described previously(18). The sense strand

189 sequences of the mouse Cyclin D2 and Cdk4 GATA sites and mutant GATA sites used for EMSA at Harvard Libraries on August 1, 2008

190 were:

191 Cyclin D2 Gata I, 5-ggaacagcttgaaagttatcaggagtctaagcttgag-3;

192 Cyclin D2 Gata Im, 5-aacagcttgaaaggtaccaggagtctaagcttgag-3; 193 CyclinACCEPTED D2 Gata II, 5-gggaggggcataacctttatccctggtttggcgaggt-3; 194 Cyclin D2 Gata IIm, 5-gaggggcataacctctagacctggtttggcgaggt-3;

195 Cyclin D2 Gata III, 5-ggacagaatgtcagaaaggataatcaataggaatccat-3;

196 Cyclin D2 Gata IIIm, 5-acagaatgtcagaaaggatcctcaataggaatccat-3;

197 Cdk4 GataI/II, 5-ggaattacctatactagttatctttatcattcacttcaaagggc-3;

198 Cdk4 GataI/IIm, 5-aattacctatactagtaagctttataattcacttcaaagggc-3;

199 Cdk4 GataIII, 5-ggcaaggggtcacgtgggatagcaacaggtcaccgtgg-3;

200 Cdk4 GataIIIm, 5-caaggggtcacgtgggttaacaacaggtcaccgtgg-3.

9 201

202 Cell culture, transfections, and reporter assays

203 A 931-bp fragment containing 671 bp upstream and 260 bp downstream of the transcriptional start

204 site from the mouse Cyclin D2 promoter region was amplified by PCR using the following primers:

205 5- acagaaaggtttctgcaggagggtcatattc-3 and 5-gccagccggcgtcgactcggtcccgac-3. An 827-bp fragment

206 containing 771 bp upstream and 56 bp downstream of the transcriptional start site from the mouse

207 Cdk4 promoter region was amplified by PCR using the following primers: 5- Downloaded from 208 cttttaatattccgcgggaggtttac-3 and 5-gggcagctggatccttcgggccagac-3. Cyclin D2 and Cdk4 PCR

209 products were cloned into the pAUG--gal reporter vector (36). Plasmid pECE-GATA4-EnR has

210 the Drosophila Engrailed repressor domain fused to the Gata4 cDNA and has been described mcb.asm.org

211 previously (32). The expression plasmid pRK5-GATA4-VP16 contains the herpes simplex virus I at Harvard Libraries on August 1, 2008 212 Vmw65.1 transcriptional activation domain fused in-frame to the 3 end of the GATA4 cDNA.

213 Mutations of the GATA sites in the Cyclin D2 and Cdk4 promoters were introduced by PCR to

214 create the mutant sequences indicated in the EMSA oligonucleotides described above.

215 216 C3H10T1/2ACCEPTED were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 217 10% fetal bovine serum (FBS). P19CL6 cells were maintained in DMEM supplemented with 10%

218 fetal bovine serum (FBS) in the presence of 1% DMSO for 7 days prior to transfection. Transient

219 transfections were performed in 12-well plates using Fugene6 (Roche) for C3H10T1/2 cells and

220 Lipofectamine XLT (Invitrogen) for P19CL6 cells, following the manufacturer’s recommendations.

221 Each transfection mixture contained 0.5 g of Cyclin D2 or Cdk4 reporter plasmids and 1.0 g of the

222 indicated repressor or activator plasmids. In transfections without an expression construct, the

223 parent expression plasmid was added to keep the total amount of DNA in each transfection constant

10 224 at 1.5 g. Cells were cultured for 48 h after transfection, harvested, and cellular extracts were

225 prepared by sonication and were normalized as described previously (14). Chemiluminescent -

226 galactosidase assays were performed using the Luminescent -gal Detection System (Clontech)

227 according to manufacturer’s recommendations, and relative light units (RLU) were detected using a

228 Tropix TR717 microplate luminometer (PE Applied Biosystems).

229

230 Chromatin Immunoprecipitation (ChIP) Assays Downloaded from 231 ChIP assays were performed using the ChIP assay kit from Upstate Pharmaceuticals (Cat. #17-295),

232 following the recommendations of the manufacturer. Briefly, a 10 cm plate containing

6

233 approximately 1 x 10 P19CL6 cells, which had been differentiated into cardiomyocytes by mcb.asm.org

234 treatment with 1% DMSO for 7 days, was treated with 1% paraformaldehyde at 37°C for 10 min to at Harvard Libraries on August 1, 2008 235 crosslink protein-DNA complexes. Cells were then lysed and sonicated to shear the DNA into

236 fragments between 300 and 500 bp in size. The cleared supernatant was incubated with 4 g of anti-

237 GATA4 antibody (Santa Cruz Sc-1237) or 4 g of anti-goat IgG (Santa Cruz Sc-2020) overnight at

238 4°C. The DNA fragments were then precipitated after incubating the lysate and antibody mixture 239 withACCEPTED protein A-agarose beads for 1 h. Reactions were incubated with NaCl at 65°C for 4 h to reverse 240 the crosslinks, and DNA was recovered by phenol-chloroform extraction. The following primers

241 were used to amplify the Cyclin D2 promoter, which contains three GATA sites, following ChIP: P1,

242 5-ctccacgcacgtggctcggggcgg-3 and P2, 5-taggggaacccacaaaccccatgg-3. Two different regions of

243 the Cdk4 promoter, a distal region containing two GATA sites and a proximal region containing one

244 GATA site, were amplified following ChIP using the following primers: P1, 5-

245 catacagtggcttattatatttcc-3 and P2, 5-ctccaccgccatggggaaacattc-3; P3, 5-gttggcccggttgccatgacaccg-

246 3 and P4, 5-ctggacacgtgatcttcacccttg-3. The Cyclin D2 second exon was amplified as a negative

11 247 control in ChIP experiments, using the following primers: 5-gcggccttagtgtgatggggaagg-3 and 5-

248 tcggaccctaccccactcttgattg-3. ChIP PCR products were confirmed by sequencing.

249

250 Results

251

252 Inactivation of Gata4 in the AHF results in right ventricular hypoplasia and ventricular septal

253 defects Downloaded from 254 To determine the role of the GATA4 specifically in the development of the right ventricle and

255 outflow tract, we inactivated Gata4 in the progenitors of the right ventricle and outflow tract using

256 Mef2c-AHF-Cre, which directs early excision in AHF progenitors in the splanchnic and pharyngeal mcb.asm.org

257 mesoderm (4, 50, 81). This resulted in specific loss of Gata4 expression in AHF derivatives in the

258 right ventricle and outflow tract (Fig. 1A, B). These crosses did not produce any live Gata4 AHF at Harvard Libraries on August 1, 2008

259 knockout animals, indicating that GATA4 is required in the derivatives of the AHF for embryonic

260 development (Table 1). At E10.5 and E11.5, Gata4 conditional knockout embryos were present at

261 normal Mendelian frequency (Table 1), and the appearance of AHF knockout embryos was normal 262 at theseACCEPTED stages (data not shown). However, by E13.5, all Gata4 AHF conditional knockout embryos 263 exhibited cardiovascular congestion and vascular hemorrhage, and the majority of the embryos

264 lacked a heartbeat at this stage (Fig.1C, D).

265

266 Histological analyses of knockout hearts at E12.5 to E13.5 did not reveal any obvious defects in

267 outflow tract alignment, and the septation into the pulmonary trunk and aorta appeared to be normal

268 (data not shown). However, the right ventricle of all AHF knockout embryos was obviously

269 hypoplastic when compared with littermate control embryos (Fig.1E-H). The compact zone of the

12 270 myocardium in knockout embryos contained fewer myocardial cell layers compared to control

271 embryos, where the myocardial wall of the right ventricle was much thicker by this stage (Fig. 1G,

272 H). In addition, the right ventricular trabecular myocardium of conditional knockout embryos

273 appeared disorganized and not well connected with the compact myocardium (Fig.1G, H). The

274 formation of the ventricular septum, which was almost completed by E13.5 in control embryos, was

275 delayed in all Gata4 AHF knockout embryos (Fig. 1E, F). As expected, no abnormalities were

276 observed in the left ventricle, which is outside of the Mef2c-AHF-Cre expression domain (Fig.1E,

277 F). Downloaded from

278

279 Gata4 AHF knockout mice display myocardial proliferation defects in the right ventricle mcb.asm.org

280 GATA4 has been implicated previously in both myocardial proliferation and apoptosis (74, 85, 86),

281 and the myocardial hypoplasia observed in the right ventricle of Gata4 AHF knockout embryos at Harvard Libraries on August 1, 2008

282 could be explained by an increase in cell death or a decrease in proliferation. To determine whether

283 cell death might be involved in the myocardial hypoplasia of Gata4 AHF knockout embryos, we

284 performed TUNEL staining on cardiac sections from embryos at E10.5. We selected this 285 developmentalACCEPTED stage since it represents a time prior to embryonic lethality, and hearts would be less 286 likely to exhibit nonspecific apoptosis secondary to cardiac failure. Results from these experiments

287 showed no differences in TUNEL staining between conditional knockout and control embryos (Fig.

288 2I, J).

289

290 To determine if inactivation of Gata4 in the AHF resulted in defective myocyte replication, we

291 examined the expression of several markers of proliferation, including Ki67, and phospho-histone

292 H3, and by BrdU incorporation at E10.5 (Fig. 2). In each case, Gata4 AHF knockout embryos

13 293 displayed significantly reduced expression of the proliferation markers in the right ventricle

294 compared to littermate control embryos (Fig. 2A-H). Similarly, Gata4 AHF knockout embryos

295 displayed reduced proliferation in the interventricular septum (Fig. 2A, B, E-H), which is consistent

296 with the VSDs observed in knockout embryos at E13.5 (Fig. 1F). The reduced proliferation in right

297 ventricular myocardium in AHF knockouts compared to littermate controls was especially evident

298 when Ki67, which marks all stages of the cell cycle, was examined (Fig. 2, compare panels A, C to

299 panels B, D). Quantification of BrdU-labeled and pHH3-labeled nuclei as a percentage of the total

300 number of DAPI-stained nuclei showed that proliferation was significantly reduced in the right Downloaded from

301 ventricle (Fig. 2K, L). By contrast, expression of these markers was the same in knockout and

302 control embryos in the left ventricle where Gata4 excision did not occur since the left ventricle is mcb.asm.org

303 outside the expression domain of Mef2c-AHF-Cre (Fig. 2K, L). Taken together, the results

304 presented in Fig. 2 demonstrate that GATA4 is required in the derivatives of the AHF for at Harvard Libraries on August 1, 2008

305 proliferation, which supports previous studies that demonstrated a role for GATA4 in cardiomyocyte

306 proliferation (82, 86).

307 308 GATA4ACCEPTED regulates the expression of numerous cell cycle genes in the heart 309 The defects in myocyte proliferation in Gata4 AHF knockout hearts (Fig. 2) suggested that GATA4

310 was likely to regulate one or more genes involved in the cell cycle. Therefore, to investigate further

311 the molecular changes underlying these alterations in cardiomyocyte proliferation, we measured

312 mRNA expression in E9.5 mouse hearts by microarray. To accomplish this, we used Nkx2-5Cre to

313 inactivate Gata4 (Gata4 CKONkx). As in Gata4 AHF knockout hearts, cardiomyocyte proliferation

314 was decreased in Gata4 CKONkx cardiomyocytes (86). However, the expression domain of Nkx2-

14 315 5Cre is broader than Mef2c-AHF-Cre (44, 81), allowing us to use the entire heart from Gata4 CKONkx

316 embryos at E9.5 for microarray analyses.

317

318 We used the microarray expression data to determine if established sets of cell cycle genes showed

319 statistically discordant differences between Gata4flox/+ (control) and Gata4flox/flox; Nkx2-5Cre/+ (Gata4

320 CKONkx) hearts. We used curated gene sets available from the Molecular Signature Database

321 (http://www.broad.mit.edu/gsea), and the Gene Set Enrichment Analysis method (72). Seven out of

322 12 cell cycle related gene sets, including the Brentani cell cycle gene set (10) were significantly Downloaded from

323 altered in Gata4 conditional knockout hearts (P < 0.001; Supplemental Material, Table S1),

324 suggesting that Gata4 inactivation leads to a broad, coordinate perturbation of genes involved in cell mcb.asm.org

325 cycle regulation (Fig. 3). By comparison, no cell cycle gene sets were significantly altered between

326 Gata4flox/+; Nkx2-5Cre/+ and control, indicating that double heterozygosity for Gata4 and Nkx2-5 does at Harvard Libraries on August 1, 2008

327 not result in significant alteration in the expression of cell cycle gene sets (Supplemental Material,

328 Table S1).

329 330 Next,ACCEPTED we looked for individual genes that when misregulated might contribute to abnormal 331 expression of cell cycle gene sets and abnormal cardiomyocyte proliferation. We found that 1302

332 probe sets were differentially expressed between control and Gata4 CKONkx embryos (P < 0.005).

333 In contrast, only 68 probe sets were differentially expressed between Gata4flox/flox control and

334 Gata4flox/+; Nkx2-5Cre/+ double heterozygous hearts, indicating that Gata4 inactivation rather than

335 Nkx2-5 and Gata4 heterozygosity is responsible for the majority of the observed changes in gene

336 expression (data not shown). Notably, many genes that are known to play fundamental roles in

337 cellular proliferation, including several cyclins and numerous other cell cycle genes, were

15 338 significantly down regulated in the absence of GATA4 function (Fig. 3). Interestingly, Cyclin D2

339 (CCND2) and Cyclin A2 (CCNA2) were among the most dramatically down regulated genes in the

340 absence of GATA4 function in the heart (Fig. 3). Cyclin function depends on the activity of Cdks,

341 including Cdk2 and Cdk4, and mice lacking multiple Cdk genes die during embryonic development

342 with thin myocardial walls (8). Our microarray data indicated that several Cdk genes, including

343 Cdk4, also had reduced expression in the absence of GATA4 (Fig. 3).

344

345 Gata4 is required for Cyclin D2, Cdk4, and Cyclin A2 expression in the right ventricle Downloaded from

346 Because Cyclin D2, Cyclin A2, and Cdk4 were among the most significantly down regulated

347 transcripts by microarray (Fig. 3), we further examined the expression of those gene products in mcb.asm.org

348 Gata4flox/flox; Mef2c-AHF-CreTg/0 embryos by immunohistochemistry at E10.5. These analyses

349 showed that the expression of all three cell cycle proteins was substantially reduced in the at Harvard Libraries on August 1, 2008

350 myocardium and endocardium of the right ventricle in Gata4 AHF knockouts (Fig. 4B, D, F) when

351 compared with control embryos (Fig. 4A, C, E). No differences in expression between Gata4 AHF

352 knockouts and littermate controls was observed in the left ventricle, consistent with the specific 353 inactivationACCEPTED of Gata4 in the AHF. Similarly, expression was unperturbed in the epicardial cell layer, 354 where the Gata4 floxed allele also was not excised by Cre recombinase (Fig.4). Taken together,

355 these immunohistochemistry data strongly support our microarray studies, which indicate that

356 GATA4 is required for expression of multiple cell cycle control genes. In particular, our results

357 demonstrate the requirement of GATA4 function for Cyclin D2, Cyclin A2, and Cdk4 expression in

358 myocytes in the embryonic right ventricle (Fig. 4).

359

360

16 361 GATA4 binds to the Cyclin D2 and Cdk4 promoters in vitro and in vivo

362 To determine if the regulation of these cell cycle genes by GATA4 was direct, we examined the

363 upstream regions for evolutionary-conserved GATA binding sites. These bioinformatic analyses

364 identified three perfect consensus GATA sites upstream of both the Cyclin D2 and Cdk4 genes, and

365 therefore, we examined these two genes in detail to determine if they were regulated by direct

366 GATA4 binding to their promoter regions in cardiomyocytes (Fig. 5). The Cyclin D2 promoter

367 contains two conserved, consensus GATA sites at positions -558 and -525 relative to the

368 transcriptional start site (Fig. 5A). These two sites, referred to as D2 Gata I (GI) and D2 Gata II Downloaded from

369 (GII), were each bound efficiently by GATA4 in EMSA (Fig. 5C, lanes 2, 6). Binding of GATA4 to

370 these sites in the Cyclin D2 promoter was specific because it was efficiently competed by an excess mcb.asm.org

371 of unlabeled self-probe (Fig. 5C, lanes 3, 7), but not by mutant versions of the Cyclin D2 (D2) Gata I

372 or Gata II sites (Fig. 5C, lanes 4, 8). In addition to these two conserved GATA sites, another at Harvard Libraries on August 1, 2008

373 candidate site at -299 bp (GIII) was also bound robustly by GATA4 (Fig. 5C, lane 10). Binding to

374 this site was also specific as it was inhibited by the addition of excess unlabeled self-probe but not

375 by the addition of a mutant version of itself (Fig. 5C, lanes 11, 12). 376 ACCEPTED 377 Within the proximal Cdk4 promoter, a perfectly conserved GATA site (Cdk4 Gata III [GIII]) is

378 present at position –180 relative to the transcriptional start site (Fig. 5B). This GATA site was

379 efficiently bound by GATA4 in EMSA (Fig. 5D, lane 6). The binding to this site was competed by

380 an excess of unlabeled self-probe but not by a mutant version of the self-probe (Fig. 5D, lanes 7, 8).

381 In addition, the Cdk4 upstream region also contains two non-conserved, consecutive candidate sites

382 at position –607 and –601 relative to the transcriptional start site (Fig. 5B). These two Cdk4 GATA

383 sites, referred as Cdk4 Gata I-II (GI-GII), were also robustly bound by GATA4 in EMSA (Fig. 5D,

17 384 lane 2). The binding to these sites was also specifically competed by an excess of unlabeled probe

385 containing both sites but not by a probe in which both GATA sites were mutated (Fig. 5D, lanes 3,

386 4).

387

388 The data presented in Figs. 5C and 5D demonstrate that the Cyclin D2 and Cdk4 promoter regions

389 each contain multiple bona fide GATA sites that are efficiently bound in vitro by GATA4. To

390 determine the ability of GATA4 to bind to the Cyclin D2 and Cdk4 promoters in cardiomyocytes, we

391 performed chromatin immunoprepitation (ChIP) assays from differentiated P19CL6 cardiomyocytes Downloaded from

392 (Fig. 5E). P19CL6 is a clonal derivative from the pluripotent P19 mouse embryonal carcinoma cell

393 line, which efficiently differentiates into functional, contractile cardiac myocytes in the presence of mcb.asm.org

394 1% DMSO, and these myocytes express numerous cardiac transcription factors, including GATA4,

395 Nkx2-5 and MEF2C (40, 41, 54, 80, 83). Anti-GATA4 antibodies specifically precipitated DNA at Harvard Libraries on August 1, 2008

396 fragments encompassing the GATA sites in the endogenous Cyclin D2 promoter (Fig. 5E, lane 3).

397 This product was specific to the GATA4 antibody since the addition of nonspecific anti-IgG in the

398 ChIP reaction did not result in the detection of Cyclin D2 by PCR (Fig. 5E, lane 1). Similarly, the 399 anti-ACCEPTEDGATA4 antibody specifically precipitated promoter fragments from the endogenous Cdk4 gene 400 that encompassed the proximal Gata III site and the more distal Gata I/II sites (Fig. 5E, lanes 6, 9).

401 These results demonstrate that GATA4 directly interacts with the endogenous Cyclin D2 and Cdk4

402 promoters in cardiac myocytes via multiple bona fide, consensus GATA sites.

403

404 Transcriptional activation of the Cyclin D2 and Cdk4 promoters requires GATA sites

405 The observations that Cyclin D2 and Cdk4 expression required GATA4 and that GATA4 bound

406 directly to the Cyclin D2 and Cdk4 promoters in vitro and in vivo suggested that the promoters of

18 407 these two cell cycle genes might require GATA4 for activation. Therefore, we examined the

408 requirement of the GATA sites in the Cyclin D2 and Cdk4 promoters for activation in P19CL6

409 cardiomyocytes in vivo by fusing the promoters to the lacZ reporter gene and testing them in a

410 luminescent -galactosidase assay (Fig. 6). Both the Cyclin D2 and Cdk4 promoters exhibited

411 significant activation in differentiated P19CL6 cells when compared to the activity of the parent

412 reporter construct pAUG--gal (Figs. 6A, B), due to the presence of endogenous GATA4 factors in

413 P19CL6 cells (40, 41, 54, 80, 83). Consistent with this notion, we observed a dramatic increase in Downloaded from 414 GATA4 protein in P19CL6 cells by western blot after 7 days of culture in the presence of DMSO

415 (data not shown). Importantly, the activation of both the Cyclin D2 and Cdk4 promoters

416 significantly decreased in P19CL6 cardiomyocytes when the GATA sites in the promoters were mcb.asm.org

417 mutated, indicating that GATA factors are important in the transcriptional activation of both

418 promoters (Fig. 6A, B). at Harvard Libraries on August 1, 2008

419

420 We also observed a significant level of activation of the Cyclin D2 promoter in C3H10T1/2 cells,

421 suggesting that this fibroblast cell line also expresses GATA factors endogenously (Fig. 6C, lane 2). 422 ConsistentACCEPTED with this notion, the activation of the Cyclin D2 reporter by endogenous factors in 423 C3H10T1/2 cells was also dependent on the presence of intact GATA sites (Fig. 6C, lane 3). In

424 addition, the activity of the Cyclin D2 promoter in C3H10T1/2 was inhibited by coexpression of a

425 repressor form of GATA4, GATA4-EnR, which has the repressor domain from the Drosophila

426 Engrailed protein fused to the Gata4 cDNA (Fig. 6D). In spite of the activation of the Cyclin D2

427 promoter by endogenous GATA factors in C3H10T1/2 cells, the activation was significantly less in

428 this fibroblast cell line than in differentiated P19CL6 cardiomyocytes, prompting us to test the

429 ability of exogenous GATA4 to activate the Cyclin D2 promoter in this cell line (Fig. 6E). GATA4

19 430 has weak intrinsic transactivation ability and is widely appreciated for interacting with

431 transcriptional coregulators to activate target genes in the heart (16, 20, 43, 71). Therefore, we used

432 an activator form of GATA4, GATA4-VP16, to overcome the requirement for GATA4 cofactors

433 that may not be abundant in C3H10T1/2 cells. GATA4-VP16 strongly transactivated the Cyclin D2-

434 lacZ reporter construct (Fig. 6E, lane 6), and this activation was dependent on the presence of intact

435 GATA binding sites since mutation of the three consensus GATA elements in the Cyclin D2

436 promoter ablated transactivation (Fig. 6E, lane 4).

437 Downloaded from

438 Taken together, the data presented in Figs. 5 and 6 demonstrate that GATA4 is a direct

439 transcriptional activator of the Cyclin D2 and Cdk4 genes through direct promoter binding and mcb.asm.org

440 activation. These data support a model in which GATA4 regulates myocyte proliferation, at least in

441 part, through direct regulation of Cyclin D2 and Cdk4. at Harvard Libraries on August 1, 2008

442

443 Discussion

444 445 GATA4ACCEPTED is an essential regulator of mesodermal and endodermal organ formation and is a key 446 component of the core cardiac transcription factor network (38, 56, 57). Recent studies using

447 conditional inactivation approaches in mice have shown that Gata4 is required for proper

448 cardiomyocyte proliferation, although the pathways downstream of GATA4 that control myocyte

449 division have not been elucidated previously (85, 86). In this manuscript, we show that inactivation

450 of Gata4 in the AHF, prior to the formation of the right ventricle, results in hypoplasia of the right

451 ventricle and ventricular septal defects resulting from diminished cardiac proliferation. We also

452 show for the first time that GATA4 regulates the expression of numerous cell cycle control genes,

20 453 including Cyclin D2 and Cdk4 via direct promoter binding and activation. Interestingly, later

454 inactivation of Gata4 using -MHC-Cre, which does not become fully active until after E10.5 when

455 the right ventricle has already formed, does not result in loss of Cyclin D2 expression (86),

456 suggesting a requirement for GATA4 regulation of Cyclin D2 expression early in the development

457 of the right ventricle.

458

459 Mouse models have been developed in order to understand the cell cycle and the interplay of

460 Cyclin/Cdk complexes. Cyclin D2 is a member of the D-cyclin family of cell cycle regulators (61). Downloaded from

461 D-cyclins are intracellular sensors that integrate mitogenic signals to direct G1/S cell cycle transition

462 (68). Three mammalian D-cyclins are expressed in overlapping patterns in all proliferating cell mcb.asm.org

463 types (30). Consistent with the overlapping expression of D-cyclin proteins, mice lacking any single

464 D-cyclin are viable and display only narrow, highly tissue-restricted phenotypes with no obvious at Harvard Libraries on August 1, 2008

465 cardiac defects (21, 26, 69, 70). However, compound mutation of all three D-cyclin genes results in

466 embryonic lethality due to cellular proliferation defects, including reduced cardiomyocyte cell

467 division (30). Similarly, individual knockout of either Cdk4 or Cdk2 did not reveal any obvious 468 defectsACCEPTED, and neither gene is required for viability in mice (7, 49, 60, 79). However, compound 469 mutation of Cdk4 and Cdk2 results in impaired proliferation and heart growth (8). The microarray

470 studies presented here demonstrate that GATA4 regulates a large number of cell cycle genes,

471 including multiple cyclins and cyclin-dependent kinases (Fig. 3). These observations suggest that

472 GATA4 controls cardiomyocyte proliferation through coordinate regulation of numerous cell cycle

473 genes. In support of that notion, we show that GATA4 directly binds to and activates the Cyclin D2

474 and Cdk4 promoters (Figs. 5 and 6). It is likely that GATA4 also directly regulates other cell cycle

475 genes as well.

21 476

477 GATA factors have an important function in either enhancing or inhibiting cell cycle progression in

478 tissues other than the myocardium (73, 75, 78). For example, GATA6 has been proposed to

479 maintain the quiescent state of vascular smooth muscle cells, probably through induction of p21, a

480 Cdk inhibitor (55). In pulmonary smooth muscle cells, GATA4 appears to be important for cell

481 proliferation, and overexpression of a repressor form of GATA4 suppresses Cyclin D2 expression

482 (73), which supports the direct activation of Cyclin D2 by GATA4 observed in our studies.

483 Similarly, GATA1 induces the sustained expression of Cyclin D1 in a myeloid cell line (77), and Downloaded from

484 GATA4 cooperates with the Kruppel-like factor KLF13 to activate Cyclin D1 in Xenopus (46). All

485 of these studies, taken together with the work presented here, support a model in which GATA mcb.asm.org

486 factors may function generally as regulators of the cell cycle in multiple tissues. It will be

487 interesting to determine if additional cell cycle genes are also direct transcriptional targets of at Harvard Libraries on August 1, 2008

488 GATA4 and other GATA factors in the heart and other tissues.

489

490 In addition to its role in proliferation, GATA4 is widely appreciated as a key regulator of 491 cardiomyocyteACCEPTED differentiation through the activation of other transcription factor and downstream 492 structural genes (38, 56, 57). Our data suggest that GATA4 is dispensable for myocyte specification

493 and differentiation in the AHF and that it is not essential for the patterning or alignment of the

494 outflow tracts. AHF-derived structures appear to be normal in Gata4 AHF knockouts except for a

495 VSD, which is probably secondary to myocyte proliferation defects in the muscular septum. Lack of

496 Gata4 in the endocardium has been previously shown to affect the proliferation of the membranous

497 portion of the septum, also leading to VSD (62). Gata4 AHF conditional knockout mice also have

498 GATA4 depleted in the endocardium, which may contribute to the observed membranous VSD in

22 499 the conditional knockout mice described in the present study (Fig. 1). Previous work has shown that

500 Gata4 is not broadly expressed in the pharyngeal mesoderm (86), which may explain why the

501 outflow tracts form normally and have normal alignment in Gata4 AHF knockout embryos.

502 Alternatively, Gata5 and Gata6 may be able to compensate for Gata4 in myocyte specification and

503 differentiation in the AHF and its derivatives.

504

505 GATA4 may regulate the balance between differentiation and proliferation through cofactor

506 interactions or by integrating and interpreting distinct upstream signals into unique outputs. Downloaded from

507 Consistent with this idea, numerous GATA4 differentiation partners have been identified previously,

508 including MEF2C, Nkx2-5, HAND2, SRF, and Tbx5 (6, 16, 20, 43, 58). Interestingly, Tbx5 mcb.asm.org

509 regulates cell cycle genes that control G1/S phase transition in Xenopus (23). We show here that at Harvard Libraries on August 1, 2008 510 GATA4 also regulates numerous cell cycle genes, including several that control G1/S transition (Fig.

511 3). GATA4 interacts with Tbx5 in the activation of the Nppa, p204, and connexin40 promoters

512 during cardiomyocyte differentiation in vitro (17, 22, 34, 58), and disruption of Tbx5-GATA4

513 interaction in humans results in congenital septation defects (22). It will be important to determine if 514 Tbx5ACCEPTED and GATA4 also cooperatively regulate cell cycle genes and whether other core cardiac 515 transcription factors also participate in a complex with GATA4 in cell cycle control.

516

23 516

517 Acknowledgements

518

519 We thank Jeff Molkentin and Evie Dodou for providing plasmids used in these studies and Benoit

520 Bruneau for helpful comments on the manuscript. AR was supported in part by a postdoctoral

521 fellowship from the American Heart Association, Western States Affiliate. SWK and WTP were

522 supported by NIH SCCOR grant P01 HL074734. This work was supported by grants HL64658 and

523 AR52130 from the NIH to BLB. Downloaded from

524 mcb.asm.org at Harvard Libraries on August 1, 2008 ACCEPTED

24 524 References

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776 man. Physiol Genomics 15:165-76.

35 777 85. Xin, M., C. A. Davis, J. D. Molkentin, C. L. Lien, S. A. Duncan, J. A. Richardson, and E. N.

778 Olson. 2006. A threshold of GATA4 and GATA6 expression is required for cardiovascular

779 development. Proc Natl Acad Sci U S A 103:11189-94.

780 86. Zeisberg, E. M., Q. Ma, A. L. Juraszek, K. Moses, R. J. Schwartz, S. Izumo, and W. T. Pu.

781 2005. Morphogenesis of the right ventricle requires myocardial expression of Gata4. J Clin

782 Invest 115:1522-31.

783 Downloaded from mcb.asm.org at Harvard Libraries on August 1, 2008 ACCEPTED

36 783

E10.5 E11.5 E12.5 E13.5 P0

Gata4flox/+ 28 13 33 17 44

Gata4flox/flox 28 12 24 16 35

CreTg/0; Gata4flox/+ 34 11 22 21 47

CreTg/0;Gata4flox/flox 29 12 19 4* 0**

2=0.83 2=0.16 2=4.44 2=11.10 2=44.47

Downloaded from P=0.841 P=0.980 P=0.216 P=0.011 P<0.0001

784

flox/+ 785 Table 1. Loss of Gata4 function in the AHF results in embryonic lethality by E13.5. Gata4 ; mcb.asm.org

786 Mef2c-AHF-CreTg/0 mice were crossed to Gata4flox/flox mice, and the offspring were collected at at Harvard Libraries on August 1, 2008 787 the indicated developmental stages. Offspring of each genotype from E10.5 to E12.5 were

788 present at normal Mendelian frequency. By E13.5, most of the conditional knockout embryos

789 (*) lacked a heartbeat. No Gata4 AHF knockouts were present at birth (P0, **). The 2 and P

790 values are given below each stage. 791 ACCEPTED

37 791 Figure legends

792

793 FIG. 1. Inactivation of Gata4 in the AHF results in lethality due to right ventricular hypoplasia and

794 ventricular septal defect. (A, B) Whole mount in situ hybridization showing expression of Gata4

795 mRNA in control (A) and Gata4 AHF knockout (B) hearts at E10.5. The excision of the Gata4

796 floxed allele by Mef2c-AHF-Cre results in loss of Gata4 mRNA in the right ventricle and outflow

797 tract of Gata4 AHF knockout embryos. (C, D) Gata4 AHF knockout embryos (D) display obvious

798 vascular hemorrhage (arrowheads) compared to littermate controls (C) at E13.5. (E-H) Hematoxylin Downloaded from

799 and Eosin stained transverse sections of littermate control (E) and Gata4 AHF knockout (F) embryos

800 show that the formation of the ventricular septum (arrowheads) is aberrant at E13.5 in Gata4 AHF mcb.asm.org

801 knockout embryos compared to controls. (G, H) The compact wall myocardium of the right

802 ventricle (asterisks) is thinner at E13.5 in Gata4 AHF knockout embryos (H) than in littermate at Harvard Libraries on August 1, 2008

803 control embryos (G). LA, left atrium; LV, left ventricle; OFT, outflow tract; RA, right atrium; RV,

804 right ventricle. The bars are equal to 100 m. Genotypes for control (Gata4flox/flox) and Gata4 AHF

805 knockout (CreTg/0;Gata4flox/flox) embryos are indicated. n= 4 for each genotype. 806 ACCEPTED 807 FIG. 2. Gata4 AHF knockout embryos have profound myocardial proliferation defects. (A-H)

808 Immunohistochemical analyses of proliferation markers on transverse sections show that Gata4

809 AHF knockout embryos (B, D, F, H) have reduced proliferation compared to control embryos (A, C,

810 E, G) at E10.5. (A, B) Gata4 AHF knockout embryos display decreased staining of the nuclear

811 antigen Ki67 (brown) in the right ventricular myocardium and interventricular septum compared to

812 control embryos (asterisks). (C, D) A closer view of the right ventricle shows that Ki67 staining in

813 Gata4 AHF knockout hearts is reduced in the myocardium (myo) but not in other regions where

38 814 Gata4 was not inactivated, such as the epicardium (epi). (E, F) BrdU incorporation is diminished in

815 the myocardium of the right ventricle of Gata4 AHF knockout embryos compared to control

816 embryos (asterisks). (G, H) Expression of the mitotic marker phospho-Histone H3 (pHH3) is

817 reduced in the right ventricle of Gata4 AHF knockout embryos compared to control littermates

818 (arrowheads). No differences in the staining of any of these proliferation markers between Gata4

819 AHF knockout and control embryos was observed in the left ventricle. (I, J) TUNEL staining on

820 transverse sections of embryonic hearts shows no difference in apoptosis between Gata4 AHF

821 knockout and control embryos at E10.5. LV, left ventricle; RV, right ventricle. Genotypes for Downloaded from

822 control (Gata4flox/flox) and Gata4 AHF knockout (Gata4flox/flox; Mef2c-AHF-CreTg/0) embryos are

823 indicated above panels A and B. (K, L) Quantification of BrdU (K) and pHH3 (L) labeled cells mcb.asm.org

824 shows a significant decrease in proliferation in the right ventricle of conditional knockout (CKO)

825 embryos compared to littermate controls. The total number of DAPI-labeled cells and the number of at Harvard Libraries on August 1, 2008

826 BrdU or pHH3-labeled cells was counted from a series of sections from 3 CKO and 3 control hearts.

827 Data are presented as the mean percentage of cells labeled with BrdU or pHH3 plus the SEM from

828 three hearts of each genotype. P values were calculated using a two-tailed, unpaired t test. 829 ACCEPTED 830 FIG. 3. GATA4 regulates multiple cell cycle control genes. Affymetrix gene expression data were

831 analyzed by Gene Set Enrichment Analysis (72). Several sets of genes with known roles in cell

832 cycle regulation showed statistically significant, concordant differences between control (Gata4flox/+)

833 and Gata4 CKONkx (Gata4flox/flox; Nkx2-5Cre/+) hearts. The heat map of genes comprising the cell

834 cycle gene set with the most significant statistical score (Brentani cell cycle gene set) is shown (10).

835 Color indicates degree of up (red) or down (blue) regulation relative to the mean expression across

836 all samples and is indicated by the color scale at the bottom. Numerous cell cycle control genes

39 837 were significantly down regulated in Gata4 CKONkx hearts compared to controls, including Cyclin

838 D2 (CCND2), Cyclin A2 (CCNA2), and Cdk4, which are denoted by arrows.

839

840 FIG. 4. Gata4 inactivation leads to decreased expression of cell cycle proteins.

841 Immunohistochemical staining of transverse sections with anti-Cyclin D2 (A, B), anti-Cdk4 (C,D),

842 and anti-Cyclin A2 (E, F) antibodies shows that the expression of all three cell cycle proteins is

843 dramatically reduced in right ventricular myocardium in Gata4 AHF knockout (B, D, F) compared

844 to littermate control (A, C, E) embryos at E10.5 (asterisks). In A-D, staining for Cyclin D2 and Downloaded from

845 Cdk4 is red, and nuclei have been counterstained with DAPI (blue). In E and F, Cyclin A2 is stained

846 in brown. No differences in Cyclin D2, Cdk4, or Cyclin A2 protein expression between knockout mcb.asm.org

847 and control embryos were observed in regions outside the Mef2c-AHF-Cre domain, such as the left

848 ventricle (LV). Genotypes for control (Gata4flox/flox) and Gata4 AHF knockout (CreTg/0;Gata4flox/flox) at Harvard Libraries on August 1, 2008

849 embryos are indicated.

850

851 FIG. 5. GATA4 binds directly to the Cyclin D2 and Cdk4 promoters in vivo and in vitro. (A, B) 852 SchematicACCEPTED representations of the mouse Cyclin D2 and Cdk4 promoters are shown. The Cyclin D2 853 construct encompasses nucleotides -671 to +260 relative to the transcriptional start site (bent arrow).

854 The Cdk4 construct encompasses nucleotides -771 to +56 relative to the transcriptional start site

855 (bent arrow). Boxes denote consensus GATA binding sites in the Cyclin D2 (Gata I [GI], Gata II

856 [GII], and Gata III [GIII]) and Cdk4 (Gata I/II [GI/II] and Gata III [GIII]) promoters. Arrowheads

857 indicate the location of primers used to amplify regions of the Cyclin D2 and Cdk4 promoters,

858 containing consensus GATA sites, in ChIP assays. (C, D) Recombinant GATA4 proteins were

859 transcribed and translated in vitro and used in EMSA with radiolabeled double-stranded

40 860 oligonucleotides encompassing the CyclinD2 Gata I (C, lanes 1-4), Gata II (C, lanes 5-8) and Gata

861 III (C, lanes 9-12) sites and the Cdk4 Gata I/II (D, lanes 1-4) and Gata III (D, lanes 5-8) sites. Lanes

862 1, 5, and 9 (Panel C) and lanes 1 and 5 (Panel D) contain reticulocyte lysate without recombinant

863 GATA4 (represented by a minus sign). GATA4 efficiently bound to all GATA sites in the Cyclin

864 D2 and Cdk4 promoters in vitro. (E) GATA4 binds to the endogenous Cyclin D2 and Cdk4

865 promoters in vivo. Differentiated P19CL6 cardiomyocytes were subjected to ChIP to detect

866 endogenous GATA4 bound to the Cyclin D2 and Cdk4 promoters using anti-GATA4 antibody.

867 Following ChIP, the Cyclin D2 promoter was detected using primers P1 and P2 (lanes 1-3), and the Downloaded from

868 Cdk4 promoter was detected using primers P1 and P2 (lanes 4-6) and primers P3 and P4 (lanes 7-9).

869 In addition, primers were used to detect the second exon of Cyclin D2 as a nonspecific control (lanes mcb.asm.org

870 10-12). PCR products were analyzed by agarose gel electrophoresis. Lanes 3, 6, 9, and 12 contain

871 PCR products following ChIP using anti-GATA4 antibody (-G4). Lanes 1, 4, 7, and 10 contain at Harvard Libraries on August 1, 2008

872 PCR products following ChIP using a nonspecific anti-IgG (-IgG). Lanes 2, 5, 8, and 11 contain

873 PCR products from input DNA (Inp), amplified prior to immunoprecipitation. ChIP products were

874 only detected from promoter regions in samples where anti-GATA4 antibody was used. Size in bp 875 is shownACCEPTED at the left. 876

877 FIG. 6. The GATA sites in the Cyclin D2 and Cdk4 promoters are required for activation. (A, B)

878 The Cyclin D2 (A) and Cdk4 (B) promoters were significantly activated by endogenous GATA

879 factors in differentiated P19CL6 cardiomyocytes (lane 2) when compared to the activity of the

880 parent reporter construct pAUG--gal (lane 1), and mutation of the GATA sites in each promoter

881 significantly attenuated activity (lane 3). (C) The Cyclin D2 promoter was significantly activated in

882 C3H10T1/2 fibroblasts (lane 2) compared to the activity of the parent reporter (lane 1), and mutation

41 883 of the GATA sites significantly attenuated promoter activation (lane 3). (D) GATA4-EnR inhibits

884 activation of the Cyclin D2 promoter in C3H10T1/2 cells. Cotransfection of GATA4-EnR

885 expression plasmid resulted in potent repression of the Cyclin D2 reporter construct (compare lanes

886 3 and 4). (E) Cotransfection of a GATA4-VP16 expression plasmid with the Cyclin D2-lacZ

887 reporter plasmid resulted in potent transactivation of the Cyclin D2 promoter in C3H10T1/2 cells

888 (lane 6). Mutation of the GATA sites (mGATA) in the Cyclin D2 promoter disrupted transactivation

889 by GATA4-VP16 (lane 4). In all cases, the total amount of transfected plasmid DNA was held

890 constant by addition of the appropriate amount of the parent expression plasmid. Error bars Downloaded from

891 represent the standard error of the means for at least three independent triplicate sets of transfections

892 and analyses for each panel. P-values were calculated by two-tailed, unpaired t test. mcb.asm.org at Harvard Libraries on August 1, 2008 ACCEPTED

42 flox/flox Gata4 CreTg/0; Gata4flox/flox Gata4 Downloaded from D mcb.asm.org at Harvard Libraries on August 1, 2008

E13.5

ACCEPTEDG RV *

E13.5

Rojas, Fig. 1 flox/flox Gata4 CreTg/0; Gata4flox/flox K

30

P=0.033 Ki67 20 Downloaded from

10

Ki67 0 Control CKO Control CKO mcb.asm.org Left Ventricle Right Ventricle

L at Harvard Libraries on August 1, 2008 BrdU 7.5 P=0.042 G

5.0

RV LV pHH3 pHH3 ACCEPTED2.5

0.0 Control CKO Control CKO TUNEL Left Ventricle Right Ventricle

Rojas, Fig. 2 Control Gata4CKONkx

MAD2L1 GSPT1 CDK4 CCND2 BUB1B CDC20 CCNA2 BUB3 CDC25C BCCIP CCNF CENPA CKS2 MYBL2 POLR3D CDC45L CCND1 DDX11 CCNE1 CDC37 CDC2L1 CENPF

SKP1A Downloaded from CKS1B ZW10 CDK5 CDC16 CDK2 CDC6 CDC7

ERH mcb.asm.org BUB1 MDM2 SKP2 TTK CCNH

CDK7 at Harvard Libraries on August 1, 2008 CHEK1 CDC42 CENPC1 CDC27 CCNE2 HUS1 NPM1 RBL1 CDC2L5 TFDP2 CDKN2A GAS1 CDC25A CDK8 ORC1L CDK6 ACCEPTEDNUMA1 CENPE MAD1L1 CCND3 E2F1 MNAT1 CDKN1A CDKN1C BTG2 MXI1 E2F2 MAX FRK CDC14A CDC25B CDKN1B GADD45B CHES1 RB1 MYC GAS2

4.0 0.0 -4.0

Rojas, Fig.3 Gata4flox/flox CreTg/0; Gata4flox/flox Cyclin D2 Downloaded from

D Cdk4 mcb.asm.org LV * RV * at Harvard Libraries on August 1, 2008 Cyclin A2

ACCEPTEDRojas, Fig. 4 A B GI GII GIII GI/II GIII Cyclin D2 Cdk4 -671 P1 P2 +260 -771 P1 P2 P3 P4 +56 Downloaded from

C D2 Gata I D2 Gata II D2 Gata III D Cdk4 Gata I/II Cdk4 Gata III GATA4 + + + +++ + + + GATA4 + + + +++ competitor ImI II mII III mIII competitor I/IImI/II III mIII

GATA4 mcb.asm.org GATA4 at Harvard Libraries on August 1, 2008

free probe free probe 123 4 56 7 8 9101112 1 2 3 4 5 6 7 8

E -IgG Inp -G4 -IgG Inp -G4 -IgGInp -G4 -IgG Inp -G4 600 ACCEPTED400 300 200 M 1 2 3 4 5 6 7 8 9 10 11 12 Cyclin D2 Gata sites Cdk4 Gata III site Cdk4 Gata I/II sites Cyclin D2 nonspecific

Rojas, Fig. 5 A Lane Reporter P19CL6 1 AUG-ß-gal

2 Cyclin D2

3 D2 (mGATA) P=0.006

0 75 150 225 300 ß-galactosidase activity (RLU x 1000)

B Lane Reporter P19CL6 1 AUG-ß-gal

2 Cdk4

3 Cdk4 (mGATA) P<0.001

0 250 500 750 Downloaded from ß-galactosidase activity (RLU x 1000)

C Lane Reporter C3H10T1/2 1 AUG-ß-gal mcb.asm.org

2 Cyclin D2

3 D2 (mGATA) P=0.011 at Harvard Libraries on August 1, 2008 0 50 100 150 200 ß-galactosidase activity (RLU x 1000)

D Lane GATA4-EnR Reporter C3H10T1/2

1 AUG-ß-gal

2 + AUG-ß-gal

3 Cyclin D2 ACCEPTED4 + Cyclin D2 P<0.001 0 25 50 75 100 ß-galactosidase activity (RLU x 1000)

E Lane GATA4-VP16 Reporter C3H10T1/2 1 AUG-ß-gal

2 + AUG-ß-gal

3 D2 (mGATA)

4 + D2 (mGATA)

5 Cyclin D2 P=0.002

6 + Cyclin D2

0 250 500 750 1000 1250 ß-galactosidase activity (RLU x 1000)

Rojas, Fig. 6