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W2117277878 abstract "Combinatorial actions of transcription factors in multiprotein complexes dictate gene expression profiles in cardiac development and disease. The Hairy-related transcription factor (HRT) family of basic helix-loop-helix proteins is composed of transcriptional repressors highly expressed in the cardiovascular system. However, it has remained unclear whether HRT proteins modulate gene expression driven by cardiac transcriptional activators. Here, we have shown that HRT proteins inhibit cardiac gene transcription by interfering with GATA transcription factors that are implicated in cardiac development and hypertrophy. HRT proteins inhibited GATA-dependent transcriptional activation of cardiac gene promoters such as the atrial natriuretic factor (ANF) promoter. Adenovirus-mediated expression of Hrt2 suppressed mRNA expression of ANF and other cardiac-specific genes in cultured cardiomyocytes. Among various signaling molecules implicated in cardiomyocyte growth, constitutively active Akt1/protein kinase Bα relieved Hrt2-mediated inhibition of GATA-dependent transcription. HRT proteins physically interacted with GATA proteins, and the basic domain of HRT was critical for physical association as well as transcriptional inhibition. These results suggest that HRT proteins may regulate specific sets of cardiac genes by modulating the function of GATA proteins and other cardiac transcriptional activators in a signal-dependent manner. Combinatorial actions of transcription factors in multiprotein complexes dictate gene expression profiles in cardiac development and disease. The Hairy-related transcription factor (HRT) family of basic helix-loop-helix proteins is composed of transcriptional repressors highly expressed in the cardiovascular system. However, it has remained unclear whether HRT proteins modulate gene expression driven by cardiac transcriptional activators. Here, we have shown that HRT proteins inhibit cardiac gene transcription by interfering with GATA transcription factors that are implicated in cardiac development and hypertrophy. HRT proteins inhibited GATA-dependent transcriptional activation of cardiac gene promoters such as the atrial natriuretic factor (ANF) promoter. Adenovirus-mediated expression of Hrt2 suppressed mRNA expression of ANF and other cardiac-specific genes in cultured cardiomyocytes. Among various signaling molecules implicated in cardiomyocyte growth, constitutively active Akt1/protein kinase Bα relieved Hrt2-mediated inhibition of GATA-dependent transcription. HRT proteins physically interacted with GATA proteins, and the basic domain of HRT was critical for physical association as well as transcriptional inhibition. These results suggest that HRT proteins may regulate specific sets of cardiac genes by modulating the function of GATA proteins and other cardiac transcriptional activators in a signal-dependent manner. Cardiac transcription factors play essential roles in regulating tissue-specific gene expression during proper development and function of the heart (1Olson E.N Schneider M.D. Genes Dev. 2003; 17: 1937-1956Crossref PubMed Scopus (337) Google Scholar, 2Srivastava D. Olson E.N. Nature. 2000; 407: 221-226Crossref PubMed Scopus (525) Google Scholar, 3Akazawa H. Komuro I. Circ. Res. 2003; 92: 1079-1088Crossref PubMed Scopus (294) Google Scholar). The expression profiles of distinct sets of cardiac genes are altered in cardiac disease, indicating the importance of transcriptional regulation in response to disease stimuli (1Olson E.N Schneider M.D. Genes Dev. 2003; 17: 1937-1956Crossref PubMed Scopus (337) Google Scholar, 2Srivastava D. Olson E.N. Nature. 2000; 407: 221-226Crossref PubMed Scopus (525) Google Scholar, 3Akazawa H. Komuro I. Circ. Res. 2003; 92: 1079-1088Crossref PubMed Scopus (294) Google Scholar). Transcription factors form multiprotein complexes, and combinatorial actions of transcription factors in such complexes dictate the specificity of downstream gene expression. For example, the physical and functional interaction among various cardiac transcription factors including MEF2, NKX2.5, TBX5, and GATA4 can be regulated by upstream cellular signaling and is likely to be impaired in patients with congenital heart disease (4Wang L. Fan C. Topol S.E. Topol E.J. Wang Q. Science. 2003; 302: 1578-1581Crossref PubMed Scopus (300) Google Scholar, 5Bruneau B.G. Nemer G. Schmitt J.P. Charron F. Robitaille L. Caron S. Conner D.A. Gessler M. Nemer M. Seidman C.E. Seidman J.G. Cell. 2001; 106: 709-721Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 6Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Crossref PubMed Scopus (468) Google Scholar, 7Garg V. Kathiriya I.S. Barnes R. Schluterman M.K. King I.N. Butler C.A. Rothrock C.R. Eapen R.S. Hirayama-Yamada K. Joo K. Matsuoka R. Cohen J.C. Srivastava D. Nature. 2003; 424: 443-447Crossref PubMed Scopus (958) Google Scholar). Members of the Hairy-related transcription factor (HRT) 1The abbreviations used are: HRT, Hairy-related transcription factor; ANF, atrial natriuretic factor; bHLH, basic helix-loop-helix; GSK3β, glycogen synthase kinase 3β; HES, Hairy and Enhancer of split; MEF2, myocyte enhancer factor 2; MyHC, myosin heavy chain; SRF, serum response factor. 1The abbreviations used are: HRT, Hairy-related transcription factor; ANF, atrial natriuretic factor; bHLH, basic helix-loop-helix; GSK3β, glycogen synthase kinase 3β; HES, Hairy and Enhancer of split; MEF2, myocyte enhancer factor 2; MyHC, myosin heavy chain; SRF, serum response factor. family of repressors, also known as Hesr, Hey, CHF, gridlock, and HERP (8Nakagawa O. Nakagawa M. Richardson J.A. Olson E.N. Srivastava D. Dev. Biol. 1999; 216: 72-84Crossref PubMed Scopus (245) Google Scholar, 9Kokubo H. Lun Y. Johnson R.L. Biochem. Biophys. Res. Commun. 1999; 260: 459-465Crossref PubMed Scopus (126) Google Scholar, 10Leimeister C. Externbrink A. Klamt B. Gessler M. Mech. Dev. 1999; 85: 173-177Crossref PubMed Scopus (210) Google Scholar, 11Chin M.T. Maemura K. Fukumoto S. Jain M.K. Layne M.D. Watanabe M. Hsieh C.M. Lee M.E. J. Biol. Chem. 2000; 275: 6381-6387Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 12Zhong T.P. Rosenberg M. Mohideen M.A. Weinstein B. Fishman M.C. Science. 2000; 287: 1820-1824Crossref PubMed Scopus (352) Google Scholar, 13Iso T. Sartorelli V. Chung G. Shichinohe T. Kedes L. Hamamori Y. Mol. Cell. Biol. 2001; 21: 6071-6079Crossref PubMed Scopus (170) Google Scholar), are highly expressed in the heart and vasculature. HRT proteins have a basic helix-loop-helix (bHLH) motif, an orange domain, and a conserved C-terminal tetrapeptide motif and show the highest structural similarity to Hairy and Enhancer of split in flies and the mammalian HES family proteins (8Nakagawa O. Nakagawa M. Richardson J.A. Olson E.N. Srivastava D. Dev. Biol. 1999; 216: 72-84Crossref PubMed Scopus (245) Google Scholar, 9Kokubo H. Lun Y. Johnson R.L. Biochem. Biophys. Res. Commun. 1999; 260: 459-465Crossref PubMed Scopus (126) Google Scholar, 10Leimeister C. Externbrink A. Klamt B. Gessler M. Mech. Dev. 1999; 85: 173-177Crossref PubMed Scopus (210) Google Scholar, 11Chin M.T. Maemura K. Fukumoto S. Jain M.K. Layne M.D. Watanabe M. Hsieh C.M. Lee M.E. J. Biol. Chem. 2000; 275: 6381-6387Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 12Zhong T.P. Rosenberg M. Mohideen M.A. Weinstein B. Fishman M.C. Science. 2000; 287: 1820-1824Crossref PubMed Scopus (352) Google Scholar, 13Iso T. Sartorelli V. Chung G. Shichinohe T. Kedes L. Hamamori Y. Mol. Cell. Biol. 2001; 21: 6071-6079Crossref PubMed Scopus (170) Google Scholar). Expression of the HRT genes is activated by Notch signaling, suggesting a role for HRT proteins as transcriptional mediators of Notch signaling in the cardiovascular system (14Nakagawa O. McFadden D.G. Nakagawa M. Yanagisawa H. Hu T. Srivastava D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13655-13660Crossref PubMed Scopus (198) Google Scholar, 15Iso T. Sartorelli V. Poizat C. Iezzi S. Wu H.Y. Chung G. Kedes L. Hamamori Y. Mol. Cell. Biol. 2001; 21: 6080-6089Crossref PubMed Scopus (183) Google Scholar, 16Leimeister C. Dale K. Fischer A. Klamt B. Hrabe de Angelis M. Radtke F. McGrew M.J. Pourquie O. Gessler M. Dev. Biol. 2000; 227: 91-103Crossref PubMed Scopus (137) Google Scholar, 17Zhong T.P. Childs S. Leu J.P. Fishman M.C. Nature. 2001; 414: 216-220Crossref PubMed Scopus (461) Google Scholar). Mutations of the HRT2 ortholog in zebrafish, gridlock, result in defects of aortic development, and misexpression of gridlock favors the development of arteries over veins (12Zhong T.P. Rosenberg M. Mohideen M.A. Weinstein B. Fishman M.C. Science. 2000; 287: 1820-1824Crossref PubMed Scopus (352) Google Scholar, 17Zhong T.P. Childs S. Leu J.P. Fishman M.C. Nature. 2001; 414: 216-220Crossref PubMed Scopus (461) Google Scholar). In mice, targeted disruption of Hrt2/Hey2/CHF1 causes ventricular septal defects and other congenital cardiac anomalies (18Gessler M. Knobeloch K.P. Helisch A. Amann K. Schumacher N. Rohde E. Fischer A. Leimeister C. Curr. Biol. 2002; 12: 1601-1604Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 19Donovan J. Kordylewska A. Jan Y.N. Utset M.F. Curr. Biol. 2002; 12: 1605-1610Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 20Sakata Y. Kamei C.N. Nakagami H. Bronson R. Liao J.K. Chin M.T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16197-16202Crossref PubMed Scopus (147) Google Scholar), indicating that Hrt2 plays an essential role in cardiovascular development. HRT proteins preferentially bind to an E-box DNA element and negatively regulate transcription (14Nakagawa O. McFadden D.G. Nakagawa M. Yanagisawa H. Hu T. Srivastava D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13655-13660Crossref PubMed Scopus (198) Google Scholar, 15Iso T. Sartorelli V. Poizat C. Iezzi S. Wu H.Y. Chung G. Kedes L. Hamamori Y. Mol. Cell. Biol. 2001; 21: 6080-6089Crossref PubMed Scopus (183) Google Scholar). In addition, HRT proteins associate with other transcription factors and specifically inhibit their transactivation capacity independent of DNA binding (11Chin M.T. Maemura K. Fukumoto S. Jain M.K. Layne M.D. Watanabe M. Hsieh C.M. Lee M.E. J. Biol. Chem. 2000; 275: 6381-6387Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 14Nakagawa O. McFadden D.G. Nakagawa M. Yanagisawa H. Hu T. Srivastava D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13655-13660Crossref PubMed Scopus (198) Google Scholar, 21Sun J. Kamei C.N. Layne M.D. Jain M.K. Liao J.K. Lee M.E. Chin M.T. J. Biol. Chem. 2001; 276: 18591-18596Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Although HRT proteins are highly expressed in the developing and adult heart, it remains unknown whether HRT proteins modulate gene expression driven by cardiac transcriptional activators such as GATA proteins (22Gajewski K. Fossett N. Molkentin J.D. Schulz R.A. Development. 1999; 126: 5679-5688Crossref PubMed Google Scholar, 23Reiter J.F. Alexander J. Rodaway A. Yelon D. Patient R. Holder N. Stainier D.Y. Genes Dev. 1999; 13: 2983-2995Crossref PubMed Scopus (354) Google Scholar, 24Molkentin J.D. Lin Q. Duncan S.A. Olson E.N. Genes Dev. 1997; 11: 1061-1072Crossref PubMed Scopus (947) Google Scholar, 25Kuo C.T. Morrisey E.E. Anandappa R. Sigrist K. Lu M.M. Parmacek M.S. Soudais C. Leiden J.M. Genes Dev. 1997; 11: 1048-1060Crossref PubMed Scopus (860) Google Scholar), which are implicated in cardiac development and growth (1Olson E.N Schneider M.D. Genes Dev. 2003; 17: 1937-1956Crossref PubMed Scopus (337) Google Scholar, 2Srivastava D. Olson E.N. Nature. 2000; 407: 221-226Crossref PubMed Scopus (525) Google Scholar, 3Akazawa H. Komuro I. Circ. Res. 2003; 92: 1079-1088Crossref PubMed Scopus (294) Google Scholar). Here, we show that HRT proteins physically associate with cardiac-enriched GATA transcription factors and inhibit GATA-mediated cardiac gene transcription. The present study suggests that signal-dependent modulation of the interaction between HRT and GATA factors may regulate cardiac gene expression during development and disease. Plasmid Construction and Adenovirus Preparation—Plasmids encoding rat atrial natriuretic factor (ANF)-luciferase (26Sprenkle A.B. Murray S.F. Glembotski C.C. Circ. Res. 1995; 77: 1060-1069Crossref PubMed Scopus (97) Google Scholar), rat α-myosin heavy chain (MyHC)-luciferase (27Molkentin J.D. Kalvakolanu D.V. Markham B.E. Mol. Cell. Biol. 1994; 14: 4947-4957Crossref PubMed Google Scholar), mouse Nkx2.5-luciferase (28Lien C.L. Wu C. Mercer B. Webb R. Richardson J.A. Olson E.N. Development. 1999; 126: 75-84Crossref PubMed Google Scholar), mouse Hrt2-luciferase (14Nakagawa O. McFadden D.G. Nakagawa M. Yanagisawa H. Hu T. Srivastava D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13655-13660Crossref PubMed Scopus (198) Google Scholar), Myc-human HRT1/-2/-3, Myc-mouse Hrt2 and its deletion mutants (14Nakagawa O. McFadden D.G. Nakagawa M. Yanagisawa H. Hu T. Srivastava D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13655-13660Crossref PubMed Scopus (198) Google Scholar), and the constitutively active forms of calcineurin A (29Molkentin J.D. Lu J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2197) Google Scholar), ribosomal S6 kinase 2 (30Poteet-Smith C.E. Smith J.A. Lannigan D.A. Freed T.A. Sturgill T.W. J. Biol. Chem. 1999; 274: 22135-22138Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), protein kinase A (31Mellon P.L. Clegg C.H. Correll L.A. McKnight G.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4887-4891Crossref PubMed Scopus (200) Google Scholar), myr-Akt (32Ballou L.M. Cross M.E. Huang S. McReynolds E.M. Zhang B.X. Lin R.Z. J. Biol. Chem. 2000; 275: 4803-4809Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and Notch1 intracellular domain (14Nakagawa O. McFadden D.G. Nakagawa M. Yanagisawa H. Hu T. Srivastava D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13655-13660Crossref PubMed Scopus (198) Google Scholar) were described previously. An expression construct of the dominant negative form of glycogen synthase kinase 3β (GSK3β) (33He X. Saint-Jeannet J.P. Woodgett J.R. Varmus H.E. Dawid I.B. Nature. 1995; 374: 617-622Crossref PubMed Scopus (446) Google Scholar) was provided by Dr. G. Crabtree (Stanford University). A luciferase reporter harboring a single GATA-binding element ((GATA)-MIS-luciferase) was provided by Dr. R. Viger (Universite Lavel, Quebec, Canada) (34Tremblay J.J. Viger R.S. J. Biol. Chem. 2003; 278: 22128-22135Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Other expression constructs were generated by PCR as cassettes flanked by unique restriction sites and were cloned into pcDNA3.1-N-Myc or -N-FLAG vectors as described previously (14Nakagawa O. McFadden D.G. Nakagawa M. Yanagisawa H. Hu T. Srivastava D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13655-13660Crossref PubMed Scopus (198) Google Scholar). The recombinant adenovirus encoding Myc-Hrt2 or its viral packaging control was prepared by Cre/loxP-mediated recombination of a sub360 adenoviral cosmid in 911 cells as described previously (35Aoki K. Barker C. Danthinne X. Imperiale M.J. Nabel G.J. Mol. Med. 1999; 5: 224-231Crossref PubMed Google Scholar). Luciferase Assays—In 6-well plates, HeLa cells were transfected using FuGENE 6 (Roche Applied Science) with 300 ng of plasmid for luciferase reporters, Myc- or FLAG-Gata4 plasmid, and Myc- or FLAG-Hrt2 plasmid. Luciferase activity was measured 40 h after transient transfection. All experiments were accompanied by Western blot analysis to verify relevant protein expression. Plasmids encoding Hrt2 mutations were titrated (600–1200 ng) such that protein expression of the mutations was comparable with protein levels of wild type Hrt2. 100 ng of plasmids encoding various enzymes was used for the initial screen, and subsequent titration revealed that 5 ng of myr-Akt plasmid was sufficient for the maximal effect. Cardiomyocyte Preparation and Adenoviral Infection—Neonatal rat cardiomyocytes were cultured as described previously (36Simpson P. Savion S. Circ. Res. 1982; 50: 101-116Crossref PubMed Scopus (446) Google Scholar, 37Nakagawa O. Ogawa Y. Itoh H. Suga S. Komatsu Y. Kishimoto I. Nishino K. Yoshimasa T. Nakao K. J. Clin. Investig. 1995; 96: 1280-1287Crossref PubMed Scopus (577) Google Scholar). Eighteen hours after plating, cells were infected with adenovirus for Myc-Hrt2 and its control at a multiplicity of infection of 30/cell for 2 h and cultured in serum-free Dulbecco's modified Eagle's medium for 36 h. Total RNA was harvested using TRIzol (Invitrogen), and dot blot analysis was performed as described previously (38Zhang C.L. McKinsey T.A. Chang S. Antos C.L. Hill J.A. Olson E.N. Cell. 2002; 110: 479-488Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar). Expression of Myc-Hrt2 was confirmed by Western blot analysis. Expression of α-tubulin was used as a loading control. Co-immunoprecipitation and in Vitro Binding Assays—COS-1 cells were transiently transfected with expression constructs for Myc- and FLAG-tagged proteins using FuGENE 6. Whole cell lysate was incubated with polyclonal anti-Myc antibody (Santa Cruz Biotechnology) and protein A-Sepharose beads (Amersham Biosciences) in lysis buffer (1× phosphate-buffered saline, 1 mm EDTA, 0.5% Triton X-100). Immunoprecipitates were detected by Western blot analysis using anti-FLAG M2 antibody (Sigma) or by staining with monoclonal anti-Myc antibody (Santa Cruz Biotechnology). For in vitro binding assays of Hrt2 deletion mutations, 35S-labeled Hrt2 fragments were synthesized using the TnT Quick Coupled Transcription/Translation System (Promega), incubated with the lysate of COS-1 cells transfected with Myc-Gata4 plasmid, immunoprecipitated with polyclonal anti-Myc antibody, and visualized by autoradiography. The largest 35S-labeled fragments corresponded to the predicted sizes of wild type Hrt2 and its mutants, and they were used for the quantification of the phosphorimage using ImageQuant software (Amersham Biosciences). Values of output bands were divided by those of the corresponding input bands and were presented as arbitrary binding capacity units for Gata4. Electrophoretic Mobility Shift Assay—Electrophoretic mobility shift assay was performed using oligonucleotides representing a GATA-binding element (underlined) (5′-TCGAGGTAATTAACTGATAATGGTGC-3′) as described previously (39McFadden D.G. Charite J. Richardson J.A. Srivastava D. Firulli A.B. Olson E.N. Development. 2000; 127: 5331-5341Crossref PubMed Google Scholar). Hrt2 Inhibits Gata4-dependent ANF Gene Expression—To test whether HRT proteins modulate cardiac gene expression by interacting with other transcription factors, we examined the effects of Hrt2 on the transcriptional activity of a series of cardiac transcriptional activators. As depicted in Fig. 1A, Hrt2 significantly repressed Gata4-mediated transactivation of the ANF promoter, which contains two essential GATA binding sites (26Sprenkle A.B. Murray S.F. Glembotski C.C. Circ. Res. 1995; 77: 1060-1069Crossref PubMed Scopus (97) Google Scholar). In contrast, transcriptional activity of various luciferase reporters controlled by MEF2C, NFAT3, or Hand2 was not significantly inhibited by co-expression of Hrt2 (data not shown). GATA proteins are known to regulate the expression of various cardiac genes in normal and hypertrophic myocytes (7Garg V. Kathiriya I.S. Barnes R. Schluterman M.K. King I.N. Butler C.A. Rothrock C.R. Eapen R.S. Hirayama-Yamada K. Joo K. Matsuoka R. Cohen J.C. Srivastava D. Nature. 2003; 424: 443-447Crossref PubMed Scopus (958) Google Scholar,22Gajewski K. Fossett N. Molkentin J.D. Schulz R.A. Development. 1999; 126: 5679-5688Crossref PubMed Google Scholar, 23Reiter J.F. Alexander J. Rodaway A. Yelon D. Patient R. Holder N. Stainier D.Y. Genes Dev. 1999; 13: 2983-2995Crossref PubMed Scopus (354) Google Scholar, 24Molkentin J.D. Lin Q. Duncan S.A. Olson E.N. Genes Dev. 1997; 11: 1061-1072Crossref PubMed Scopus (947) Google Scholar, 25Kuo C.T. Morrisey E.E. Anandappa R. Sigrist K. Lu M.M. Parmacek M.S. Soudais C. Leiden J.M. Genes Dev. 1997; 11: 1048-1060Crossref PubMed Scopus (860) Google Scholar,38Zhang C.L. McKinsey T.A. Chang S. Antos C.L. Hill J.A. Olson E.N. Cell. 2002; 110: 479-488Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar). We therefore analyzed the effects of Hrt2 expression upon endogenous cardiac gene expression. Adenovirus-mediated Hrt2 expression markedly decreased ANF mRNA expression in cultured neonatal rat cardiomyocytes (Fig. 1B). The relative ANF mRNA levels with control and Hrt2 adenovirus infection were 105 ± 11% and 26.5 ± 5.2% (mean ± S.D.), respectively, compared with the levels without adenovirus infection. Hrt2 also down-regulated other cardiac hypertrophy markers, βMyHC and α-skeletal actin. The βMyHC promoter contains a GATA binding site that is essential for its transcriptional activation in cardiac hypertrophy (40Hasegawa K. Lee S.J. Jobe S.M. Markham B.E. Kitsis R.N. Circulation. 1997; 96: 3943-3953Crossref PubMed Scopus (153) Google Scholar), suggesting that HRT proteins may generally suppress the GATA-dependent cardiac gene program by interfering with GATA proteins. HRT1, -2, and -3 Specifically Suppress Transcription Driven by GATA Proteins—To further investigate the generality of the repressive effects of HRT on GATA proteins, we examined its effects on the transcription of other GATA-dependent cardiac regulatory elements. As shown in Fig. 2A, a similar pattern of Hrt2-mediated repression of Gata4-dependent transcription was observed for αMyHC- and Nkx2.5-luciferase reporters, both of which contain essential GATA sites (27Molkentin J.D. Kalvakolanu D.V. Markham B.E. Mol. Cell. Biol. 1994; 14: 4947-4957Crossref PubMed Google Scholar, 28Lien C.L. Wu C. Mercer B. Webb R. Richardson J.A. Olson E.N. Development. 1999; 126: 75-84Crossref PubMed Google Scholar). Hrt2 also displayed repressive activity against transcription driven by other cardiac-enriched GATA proteins, Gata5 and Gata6 (Fig. 2B). In addition, all of the human HRT proteins, HRT1, -2, and -3, were able to repress Gata4-dependent transactivation (Fig. 2C). Because Gata4 cooperates with SRF to activate ANF transcription (41Belaguli N.S. Sepulveda J.L. Nigam V. Charron F. Nemer M. Schwartz R.J. Mol. Cell. Biol. 2000; 20: 7550-7558Crossref PubMed Scopus (156) Google Scholar, 42Moore M.L. Wang G.L. Belaguli N.S. Schwartz R.J. McMillin J.B. J. Biol. Chem. 2001; 276: 1026-1033Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), we asked whether Hrt2 could repress transactivation induced by co-expression of Gata4 and SRF. Hrt2 attenuated Gata4-SRF transactivation to levels similar to the contribution by SRF alone, whereas Hrt2 did not repress SRF-dependent transcription in the absence of Gata4 (Fig. 2D). These results indicate that HRT family proteins specifically inhibit GATA-dependent gene transcription. Akt1 Relieves HRT-mediated Repression of GATA-dependent Transcription—Because GATA and HRT proteins are co-expressed both in the embryonic and adult heart, we hypothesized that functional interactions between these proteins could be modulated by developmental and disease signals. To begin to search for such regulatory pathways, we examined whether any protein kinases or phosphatases implicated in cardiac growth and hypertrophy could affect Hrt2-mediated repression of Gata4-dependent transcription. Of the various proteins tested, only a constitutively active form of Akt1/protein kinase Bα, myr-Akt (32Ballou L.M. Cross M.E. Huang S. McReynolds E.M. Zhang B.X. Lin R.Z. J. Biol. Chem. 2000; 275: 4803-4809Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), significantly relieved Hrt2-mediated repression of Gata4-dependent transactivation (Fig. 3A). Calcineurin A, ribosomal S6 kinase 2, and protein kinase A have been implicated in cardiac growth (43Pinna L.A. Ruzzene M. Biochim. Biophys. Acta. 1996; 1314: 191-225Crossref PubMed Scopus (403) Google Scholar, 44Aramburu J. Rao A. Klee C.B. Curr. Top. Cell. Regul. 2000; 36: 237-295Crossref PubMed Scopus (274) Google Scholar). Although they appeared to slightly increase GATA transcriptional activity, they did not relieve Hrt2-mediated repression (data not shown). As we showed previously (14Nakagawa O. McFadden D.G. Nakagawa M. Yanagisawa H. Hu T. Srivastava D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13655-13660Crossref PubMed Scopus (198) Google Scholar), Hrt2 can also attenuate transactivation of its own promoter driven by the Notch/RBP-Jκ complex. However, myr-Akt did not antagonize Hrt2 repression in this system (Fig. 3B), suggesting that the effect of Akt1 is specific to Hrt2-mediated Gata4 inhibition. Various effects of Akt1 are mediated by the inhibition of GSK3β, a downstream protein kinase (45Morisco C. Seta K. Hardt S.E. Lee Y. Vatner S.F. Sadoshima J. J. Biol. Chem. 2001; 276: 28586-28597Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). However, because dominant-negative GSK3β (33He X. Saint-Jeannet J.P. Woodgett J.R. Varmus H.E. Dawid I.B. Nature. 1995; 374: 617-622Crossref PubMed Scopus (446) Google Scholar) did not mimic the action of Akt1 (Fig. 3C), Akt1 appeared to act independent of the GSK3β pathway, either by directly phosphorylating Hrt2 and/or Gata4 or via intermediate molecules upstream of HRT- and GATA-mediated transcriptional regulation. HRT Physically Associates with GATA and Inhibits Transcription through a GATA-binding DNA Element—To clarify the mechanism underlying the repression of GATA by Hrt2, we tested whether Hrt2 could form a protein complex with GATA proteins. As shown in Fig. 4A, Hrt2 showed physical interaction with Gata4 in co-immunoprecipitation experiments. Association of human HRT1 and HRT3 with Gata4 was also detected (data not shown). Furthermore, Hrt2 significantly suppressed Gata4-dependent transcription of a luciferase reporter with a single GATA-binding element, (GATA)-MIS-luciferase (34Tremblay J.J. Viger R.S. J. Biol. Chem. 2003; 278: 22128-22135Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) (Fig. 4B). However, Hrt2 did not inhibit the binding of Gata4 to the GATA-binding element in an electrophoretic mobility shift assay (Fig. 4C). These results suggested that direct interaction between HRT and GATA proteins on a GATA-binding DNA element is important for this repression. The Basic Domain of Hrt2 Is Essential for Transcriptional Repression—To determine the Hrt2 domains that are necessary for repression of GATA-dependent transcription, we tested the effects of various Hrt2 mutations (Fig. 5A) on Gata4-dependent transactivation of the ANF promoter. In contrast to most mutations, a deletion of the basic domain, Hrt2Δ(33–37), failed to repress GATA-mediated transcription (Fig. 5B). Lack of transcriptional repression by Hrt2Δ(33–37) was also observed using the reporter with a GATA-binding element (Fig. 5C). Because the basic domain of Hrt2 can also act as a nuclear localization signal, mislocalization of Hrt2Δ(33–37) to the cytoplasm might contribute to the loss of repressive activity. However, Hrt2Δ(33–37) was observed in the nucleus as well as the cytoplasm in HeLa cells, and increasing amounts of Hrt2Δ(33–37) did not result in any repressive activity (data not shown). The Basic Domain of Hrt2 Is Necessary for Physical Interaction with Gata4 —We next tested the effects of Hrt2 mutations on Hrt2-Gata4 association. Because the expression levels of these Hrt2 mutations were variable in COS-1 cells, we synthesized 35S-labeled Hrt2 proteins using rabbit reticulocyte lysate and performed in vitro association assays with Gata4 expressed in COS-1 cells. Hrt2 fragments containing the bHLH and orange domains were sufficient for the interaction with Gata4 in vitro (data not shown). Although a deletion of the loop segment in the HLH domain (Hrt2Δ(47–52)) did not affect the association significantly, Hrt2Δ(33–37) showed a marked decrease in binding capacity for Gata4 (Fig. 5D). This result is consistent with the results of the luciferase assays and suggests that Hrt2 associates with Gata4 mainly through its basic domain to inhibit Gata4-dependent transcription. In this study, we demonstrated that the HRT family of bHLH transcriptional repressors forms a protein complex with the GATA family of transcriptional activators and inhibits cardiac gene transcription. HRT was identified as a novel subfamily of bHLH repressors similar to Drosophila Hairy, Enhancer of split, and mammalian HES (8Nakagawa O. Nakagawa M. Richardson J.A. Olson E.N. Srivastava D. Dev. Biol. 1999; 216: 72-84Crossref PubMed Scopus (245) Google Scholar, 9Kokubo H. Lun Y. Johnson R.L. Biochem. Biophys. Res. Commun. 1999; 260: 459-465Crossref PubMed Scopus (126) Google Scholar, 10Leimeister C. Externbrink A. Klamt B. Gessler M. Mech. Dev. 1999; 85: 173-177Crossref PubMed Scopus (210) Google Scholar, 11Chin M.T. Maemura K. Fukumoto S. Jain M.K. Layne M.D. Watanabe M. Hsieh C.M. Lee M.E. J. Biol. Chem. 2000; 275: 6381-6387Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 12Zhong T.P. Rosenberg M. Mohideen M.A. Weinstein B. Fishman M.C. 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However, Hrt2 does not appear to alter GATA DNA-binding affinity, at least in electrophoretic mobility shift assays in vitro. One mechanism for HRT-mediated repression may be via recruitment of its co-repressors, including class 1 and class 3 histone deacetylases (15Iso T. Sartorelli V. Poizat C. Iezzi S. Wu H.Y. Chung G. Kedes L. Hamamori Y. Mol. Cell. Biol. 2001; 21: 6080-6089Crossref PubMed Scopus (183) Google Scholar, 46Takata T. Ishikawa F. Biochem. Biophys. Res. Commun. 2003; 301: 250-257Crossref PubMed Scopus (123) Google Scholar), to the GATA complex. The bHLH domain of HRT family proteins is important for association with these co-factors (15Iso T. Sartorelli V. Poizat C. Iezzi S. Wu H.Y. Chung G. Kedes L. Hamamori Y. Mol. Cell. Biol. 2001; 21: 6080-6089Crossref PubMed Scopus (183) Google Scholar, 46Takata T. Ishikawa F. Biochem. Biophys. Res. Commun. 2003; 301: 250-257Crossref PubMed Scopus (123) Google Scholar). 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Lawitts J. Cantley L.C. Izumo S. EMBO J. 2000; 19: 2537-2548Crossref PubMed Scopus (517) Google Scholar, 56Shioi T. McMullen J.R. Kang P.M. Douglas P.S. Obata T. Franke T.F. Cantley L.C. Izumo S. Mol. Cell. Biol. 2002; 22: 2799-280966Crossref PubMed Scopus (446) Google Scholar, 57Condorelli G. Drusco A. Stassi G. Bellacosa A. Roncarati R. Iaccarino G. Russo M.A. Gu Y. Dalton N. Chung C. Latronico M.V. Napoli C. Sadoshima J. Croce C.M. Ross Jr., J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12333-12338Crossref PubMed Scopus (400) Google Scholar). Through inhibition of GSK3β (45Morisco C. Seta K. Hardt S.E. Lee Y. Vatner S.F. Sadoshima J. J. Biol. Chem. 2001; 276: 28586-28597Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), Akt1 activates GATA- and NFAT-mediated cardiac gene expression (45Morisco C. Seta K. Hardt S.E. Lee Y. Vatner S.F. Sadoshima J. J. Biol. Chem. 2001; 276: 28586-28597Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 58Antos C.L. McKinsey T.A. Frey N. Kutschke W. McAnally J. Shelton J.M. Richardson J.A. Hill J.A. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 907-912Crossref PubMed Scopus (412) Google Scholar). The attenuation of HRT-mediated repression of GATA-dependent gene expression might serve as an additional mechanism by which Akt1-mediated signaling provokes cardiac growth and hypertrophy. Akt1 did not have strong effects on the transactivation by Gata4 in luciferase assays, and Akt1 did not influence the repressive activity of Hrt2 in a Notch-related system, suggesting that the effects of Akt1 may be specific to HRT-GATA functional interaction. Akt1 is known to regulate the sublocalization and/or transcriptional activity of various transcription factors either by direct phosphorylation or via intermediate signaling molecules (59Kops G.J. Burgering B.M. J. Mol. Med. 1999; 77: 656-665Crossref PubMed Scopus (252) Google Scholar, 60Puigserver P. Rhee J. Donovan J. Walkey C.J. Yoon J.C. Oriente F. Kitamura Y. Altomonte J. Dong H. Accili D. Spiegelman B.M. Nature. 2003; 423: 550-555Crossref PubMed Scopus (1165) Google Scholar). Although Akt1 can phosphorylate Hrt2 and Gata4 fragments in vitro, 2M. Murakami, O. Nakagawa, and E. N. Olson, unpublished observation. it is still unknown whether Akt1 influences Hrt2-mediated Gata4 inhibition by phosphorylation of Hrt2 and/or Gata4. We await further studies to elucidate how Akt1 modulates functional interactions between HRT and GATA. In summary, we have demonstrated that HRT proteins physically associate with GATA proteins and inhibit GATA-dependent cardiac gene expression in an Akt1-sensitive manner. An understanding of such functional interactions among cardiac transcription factors and their responsiveness to cellular signals should provide further insight into the mechanisms of cardiac development and disease. We thank G. Crabtree and R. Viger for plasmids. We also thank J. Page for secretarial assistance." @default.
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W2117277878 title "Hairy-related Transcription Factors Inhibit GATA-dependent Cardiac Gene Expression through a Signal-responsive Mechanism" @default.
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