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.