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• W2083248934 abstract "The T-box transcription factors play critical roles in embryonic development including cell type specification, tissue patterning, and morphogenesis. Several T-box genes are expressed in the heart and are regulators of cardiac development. At the earliest stages of heart development, two of these genes, Tbx5 and Tbx20, are co-expressed in the heart-forming region but then become differentially expressed as heart morphogenesis progresses. Although Tbx5 and Tbx20 belong to the same gene family and share a highly conserved DNA-binding domain, their transcriptional activities are distinct. The C-terminal region of the Tbx5 protein is a transcriptional activator, while the C terminus of Tbx20 can repress transcription. Tbx5, but not Tbx20, activates a cardiac-specific promoter (atrial natriuretic factor (ANF)) alone and synergistically with other transcription factors. In contrast, Tbx20 represses ANF promoter activity and also inhibits the activation mediated by Tbx5. Of the two T-box binding consensus sequences in the promoter of ANF, only T-box binding element 1 (TBE1) is required for the synergistic activation of ANF by Tbx5 and GATA4, but TBE2 is required for repression by Tbx20. To elucidate upstream signaling pathways that regulate Tbx5 and Tbx20 expression, recombinant bone morphogenetic protein-2 was added to cardiogenic explants from chick embryos. Using real time reverse transcription-PCR, it was demonstrated that Tbx20, but not Tbx5, is induced by bone morphogenetic protein-2. Collectively these data demonstrate clear differences in both the expression and function of two related transcription factors and suggest that the modulation of cardiac gene expression can occur as a result of combinatorial regulatory interactions of T-box proteins. The T-box transcription factors play critical roles in embryonic development including cell type specification, tissue patterning, and morphogenesis. Several T-box genes are expressed in the heart and are regulators of cardiac development. At the earliest stages of heart development, two of these genes, Tbx5 and Tbx20, are co-expressed in the heart-forming region but then become differentially expressed as heart morphogenesis progresses. Although Tbx5 and Tbx20 belong to the same gene family and share a highly conserved DNA-binding domain, their transcriptional activities are distinct. The C-terminal region of the Tbx5 protein is a transcriptional activator, while the C terminus of Tbx20 can repress transcription. Tbx5, but not Tbx20, activates a cardiac-specific promoter (atrial natriuretic factor (ANF)) alone and synergistically with other transcription factors. In contrast, Tbx20 represses ANF promoter activity and also inhibits the activation mediated by Tbx5. Of the two T-box binding consensus sequences in the promoter of ANF, only T-box binding element 1 (TBE1) is required for the synergistic activation of ANF by Tbx5 and GATA4, but TBE2 is required for repression by Tbx20. To elucidate upstream signaling pathways that regulate Tbx5 and Tbx20 expression, recombinant bone morphogenetic protein-2 was added to cardiogenic explants from chick embryos. Using real time reverse transcription-PCR, it was demonstrated that Tbx20, but not Tbx5, is induced by bone morphogenetic protein-2. Collectively these data demonstrate clear differences in both the expression and function of two related transcription factors and suggest that the modulation of cardiac gene expression can occur as a result of combinatorial regulatory interactions of T-box proteins. Members of the T-box family of transcription factors are expressed in a variety of embryonic structures and their functions include regulation of cell type specification, tissue patterning, and morphogenesis (1Showell C. Binder O. Conlon F.L. Dev. Dyn. 2004; 229: 201-218Google Scholar, 2Papaioannou V.E. Silver L.M. Bioessays. 1998; 20: 9-19Google Scholar). In the human population, mutations of T-box genes are associated with several developmental disorders. The congenital heart defects of Holt-Oram syndrome and DiGeorge syndrome are associated with genetic aberrations in TBX5 (3Li Q.Y. Newbury-Ecob R.A. Terrett J.A. Wilson D.I. Curtis A.R. Yi C.H. Gebuhr T. Bullen P.J. Robson S.C. Strachan T. Bonnet D. Lyonnet S. Young I.D. Raeburn J.A. Buckler A.J. Law D.J. Brook J.D. Nat. Genet. 1997; 15: 21-29Google Scholar, 4Basson C.T. Bachinsky D.R. Lin R.C. Levi T. Elkins J.A. Soults J. Grayzel D. Kroumpouzou E. Traill T.A. Leblanc-Straceski J. Renault B. Kucherlapati R. Seidman J.G. Seidman C.E. Nat. Genet. 1997; 15: 30-35Google Scholar) and TBX1 (5Merscher S. Funke B. Epstein J.A. Heyer J. Puech A. Lu M.M. Xavier R.J. Demay M.B. Russell R.G. Factor S. Tokooya K. Jore B.S. Lopez M. Pandita R.K. Lia M. Carrion D. Xu H. Schorle H. Kobler J.B. Scambler P. Wynshaw-Boris A. Skoultchi A.I. Morrow B.E. Kucherlapati R. Cell. 2001; 104: 619-629Google Scholar), respectively. The role of T-box genes in heart development is supported by the cardiac expression of several T-box genes during cardiogenesis including Tbx5 and Tbx20 as well as Tbx1, Tbx2, and Tbx18 (6Papaioannou V.E. Int. Rev. Cytol. 2001; 207: 1-70Google Scholar, 7Ryan K. Chin A.J. Birth Defects Res. Part C Embryo Today. 2003; 69: 25-37Google Scholar). The overlapping but distinct expression patterns of many of these T-box genes suggest discrete transcriptional functions. Tbx5 and Tbx20 are co-expressed in the cardiac primordia; however, during chamber formation their expression patterns diverge (8Iio A. Koide M. Hidaka K. Morisaki T. Dev. Genes Evol. 2001; 211: 559-562Google Scholar, 9Liberatore C.M. Searcy-Schrick R.D. Yutzey K.E. Dev. Biol. 2000; 223: 169-180Google Scholar, 10Yamada M. Revelli J.P. Eichele G. Barron M. Schwartz R.J. Dev. Biol. 2000; 228: 95-105Google Scholar, 11Takeuchi J.K. Ohgi M. Koshiba-Takeuchi K. Shiratori H. Sakaki I. Ogura K. Saijoh Y. Ogura T. Development. 2003; 130: 5953-5964Google Scholar, 12Bruneau B.G. Logan M. Davis N. Levi T. Tabin C.J. Seidman J.G. Seidman C.E. Dev. Biol. 1999; 211: 100-108Google Scholar). Although Tbx5 and Tbx20 are differentially expressed, it has yet to be determined that they differ in their regulatory functions in the development of the heart. Many recent studies have focused on the function of Tbx5 because of its association with Holt-Oram syndrome (3Li Q.Y. Newbury-Ecob R.A. Terrett J.A. Wilson D.I. Curtis A.R. Yi C.H. Gebuhr T. Bullen P.J. Robson S.C. Strachan T. Bonnet D. Lyonnet S. Young I.D. Raeburn J.A. Buckler A.J. Law D.J. Brook J.D. Nat. Genet. 1997; 15: 21-29Google Scholar, 4Basson C.T. Bachinsky D.R. Lin R.C. Levi T. Elkins J.A. Soults J. Grayzel D. Kroumpouzou E. Traill T.A. Leblanc-Straceski J. Renault B. Kucherlapati R. Seidman J.G. Seidman C.E. Nat. Genet. 1997; 15: 30-35Google Scholar). Tbx5 is required for the normal development of the heart as homozygous null tbx5 mice have hypoplastic atria and consequently do not survive past E10.5 (13Bruneau 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: 709-721Google Scholar). Mice heterozygous for the null tbx5 allele phenocopy some cardiac abnormalities of Holt-Oram syndrome in humans, including atrial septal defects as well as first and second degree atrioventricular block (13Bruneau 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: 709-721Google Scholar). The importance of tbx20 for heart development is supported by studies in zebrafish embryos where reduced tbx20 expression results in abnormal heart morphogenesis (14Szeto D.P. Griffin K.J. Kimelman D. Development. 2002; 129: 5093-5101Google Scholar). Despite the requirement of Tbx5 and Tbx20 for normal heart development, limited information is available regarding their specific transcriptional functions during cardiogenesis. Initial evidence for Tbx5 transcriptional regulatory function demonstrated that the promoters of atrial natriuretic factor (ANF) 1The abbreviations used are: ANF, atrial natriuretic factor; BMP, bone morphogenetic protein; RT, reverse transcription; E, embryonic day; HOS, Holt-Oram syndrome; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AVC, atrioventricular canal; FGF, fibroblast growth factor; TBE, T-box binding element. and connexin40 are direct downstream targets of Tbx5 and are cooperatively regulated with Nkx2.5 (13Bruneau 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: 709-721Google Scholar, 15Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar, 16Fan C. Liu M. Wang Q. J. Biol. Chem. 2003; 278: 8780-8785Google Scholar, 17Ghosh T.K. Packham E.A. Bonser A.J. Robinson T.E. Cross S.J. Brook J.D. Hum. Mol. Genet. 2001; 10: 1983-1994Google Scholar). Tbx5 and GATA4 also activate the ANF promoter (11Takeuchi J.K. Ohgi M. Koshiba-Takeuchi K. Shiratori H. Sakaki I. Ogura K. Saijoh Y. Ogura T. Development. 2003; 130: 5953-5964Google Scholar, 18Garg 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: 443-447Google Scholar); however, the cis-elements required for the cooperative interaction have not been identified. Tbx20 contains multiple transcriptional regulatory domains (19Stennard F.A. Costa M.W. Elliott D.A. Rankin S. Haast S.J. Lai D. McDonald L.P. Niederreither K. Dolle P. Bruneau B.G. Zorn A.M. Harvey R.P. Dev. Biol. 2003; 262: 206-224Google Scholar), but its role as an activator or repressor of cardiac gene expression has not been clearly defined. To better understand the transcriptional regulatory functions of Tbx5 and Tbx20, their expression and function were evaluated simultaneously. Expression of Tbx5 and Tbx20 was examined in chick embryos to define their respective expression domains during cardiac development. The differential expression of Tbx5 and Tbx20 in the heart suggests that they have distinct regulatory roles in chamber formation. Reporter gene analysis performed using sequence from the ANF promoter demonstrated that Tbx5 and Tbx20 exhibit differential transcriptional regulatory functions. Additionally it was shown that the C termini of Tbx5 and Tbx20 have functionally distinct transcriptional regulatory domains. Relatively little is known about the pathways responsible for regulating expression of Tbx5 and Tbx20 during initial stages of cardiogenesis. In explanted cardiogenic regions from chicken embryos, Tbx20 but not Tbx5 expression was induced by bone morphogenetic protein-2 (BMP2) treatment. Collectively these studies define distinct expression, transcriptional function, and regulation of the related transcription factors Tbx5 and Tbx20 during heart development. In Situ Hybridization—Fertilized White Leghorn chicken eggs (Spafas Inc., Roanoke, IL) were incubated at 38 °C under high humidity, and embryos were collected at 1, 2, 5, and 10 days. Whole embryos or dissected hearts were fixed overnight in 4% paraformaldehyde, phosphate-buffered saline. Fixed embryos and hearts were dehydrated in a graded methanol, phosphate-buffered saline, 0.1% Tween 20 series and stored at -20 °C in 100% methanol. Whole mount in situ hybridizations were performed as described by Wilkinson (20Wilkinson D.G. Stern C.D. Holland P.W.H. Essential Developmental Biology. Oxford University Press, New York1993: 257-274Google Scholar) with reported modifications (21Ehrman L.A. Yutzey K.E. Dev. Biol. 1999; 207: 163-175Google Scholar). Day 10 hearts were bisected with a razor blade prior to hybridization to visualize the developing valves and conduction system. Proteinase K digestions were performed for 10-15 min, and color reactions were incubated for 1-5 h using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche Applied Science). Digoxigenin UTP-labeled antisense RNA probes were generated specifically for chicken Tbx5 and Tbx20. Generation of Tbx5 riboprobe has been described previously (9Liberatore C.M. Searcy-Schrick R.D. Yutzey K.E. Dev. Biol. 2000; 223: 169-180Google Scholar). The chicken Tbx20 sequence was amplified by RT-PCR from E3 heart RNA with degenerate primers 5′-TGCTGRAAGTARTGRTG-3′ and 5′-GTGGAYAAYAAGAGATA-3′ (where R represents purine and Y represents pyrimidine) and was to be identical to GenBank™ accession number AB070554 (8Iio A. Koide M. Hidaka K. Morisaki T. Dev. Genes Evol. 2001; 211: 559-562Google Scholar). The 820-bp fragment was subcloned into pBlueScript-SK, and the riboprobe was synthesized with T3 polymerase from plasmid linearized with XhoI. Expression and Reporter Plasmids—The pAC-CMV-Tbx5 plasmid was generated by ligating full-length mouse tbx5 cDNA sequence from pBluescript-SK-Tbx5 (9Liberatore C.M. Searcy-Schrick R.D. Yutzey K.E. Dev. Biol. 2000; 223: 169-180Google Scholar) into the BamHI site of the pAC-CMVpLpA(5)+ plasmid (22Gomez-Foix A.M. Coats W.S. Baque S. Alam T. Gerard R.D. Newgard C.B. J. Biol. Chem. 1992; 267: 25129-25134Google Scholar). The pAC-CMV-Tbx5(R237Q) and pAC-CMVTbx5(R279ter) expression plasmids were generated by performing site-directed mutagenesis (see below) on the pBluescript-SK-Tbx5 plasmid followed by insertion into the BamHI site of the CMVpLpA(5)+ plasmid. The mouse tbx20 sequence was isolated from E10.5 ventricle RNA by RT-PCR using the following primers: 5′-CCCAGTTCCGCTTTGCTTGCTCTC-3′ and 5′-CCCCACTTCCCACCCACCCTACTT-3′. The ∼1500-base pair sequence, corresponding to GenBank™ accession number AF30667 (23Kraus F. Haenig B. Kispert A. Mech. Dev. 2001; 100: 87-91Google Scholar), was subcloned into pBluescript-SK. The tbx20 sequence was removed from pBluescript-SK and subcloned into the XbaI and HindIII sites of pAC-CMV to generate pAC-CMV-Tbx20. Gal4-Tbx5-(266-518) was generated by amplifying the tbx5 sequence encoding the C-terminal 252 amino acids in a 25-cycle PCR (94 °C, 1 min; 58 °C, 1.5 min; 72 °C, 3 min) using the pAC-CMV-Tbx5 plasmid as a template and the primers 5′-ATGGATCCTCCAACCACAGCCCCTTCAG-3′ and 5′-AATCTAGAGCCTTTAGCTATTCTCACTCC-3′. The resulting PCR fragment was ligated into the XbaI and BamHI sites of the PM2-GAL4 plasmid (24Sadowski I. Bell B. Broad P. Hollis M. Gene (Amst.). 1992; 118: 137-141Google Scholar), and subsequent sequence analysis confirmed that the Tbx5 protein was in-frame with the Gal4 protein. Gal4-Tbx20-(294-445) was generated by amplifying the tbx20 sequence encoding the C-terminal 151 amino acids in a 30-cycle PCR (94 °C, 1 min; 60 °C, 1 min; 72 °C, 1 min) using the pAC-CMV-Tbx20 plasmid as a template and the primers 5′-TTGGATCCATTGAGAGGGAGAGTGTG-3′ and 5′-AGGTAGTTTGTCCAATTATG-3′. The resulting PCR fragment was ligated into the BamHI and HindIII sites of the PM2-GAL4 plasmid (24Sadowski I. Bell B. Broad P. Hollis M. Gene (Amst.). 1992; 118: 137-141Google Scholar), and sequence analysis confirmed that the Tbx20 protein was in-frame with the Gal4 protein. The (-288)ANF-luciferase reporter was generated by performing PCR on the rat (-3003)ANF-luciferase reporter (25Argentin S. Nemer M. Drouin J. Scott G.K. Kennedy B.P. Davies P.L. J. Biol. Chem. 1985; 260: 4568-4571Google Scholar, 26Knowlton K.U. Baracchini E. Ross R.S. Harris A.N. Henderson S.A. Evans S.M. Glembotski C.C. Chien K.R. J. Biol. Chem. 1991; 266: 7759-7768Google Scholar) (25 cycles of 95 °C, 1 min; 65 °C, 1 min; 72 °C, 1 min) using the primers 5′-GCGTCTTCCATTTTACCAAC-3′ and 5′-GCGAGCGCCCAGGAAGATAA-3′ to amplify sequence containing the 288 proximal base pairs of the ANF promoter. This fragment was digested using the restriction enzymes AvaII and XbaI, blunt-ended using DNA polymerase I large fragment (Klenow) (New England BioLabs), and ligated into the SmaI site of pGL3 (NewEngland Biosciences). Sequence analysis confirmed that the reporter contained 288 base pairs of the rat ANF promoter (25Argentin S. Nemer M. Drouin J. Scott G.K. Kennedy B.P. Davies P.L. J. Biol. Chem. 1985; 260: 4568-4571Google Scholar). The LexA-VP16 (27Zaman Z. Ansari A.Z. Koh S.S. Young R. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2550-2554Google Scholar) and Gal4-VP16 (28Sadowski I. Ma J. Triezenberg S. Ptashne M. Nature. 1988; 335: 563-564Google Scholar) expression plasmids and the G5E1b-luciferase (29Huang J. Weintraub H. Kedes L. Mol. Cell. Biol. 1998; 18: 5478-5484Google Scholar), Gal4x5-LexAx2-E1B-luciferase (30Saha S. Brickman J.M. Lehming N. Ptashne M. Nature. 1993; 363: 648-652Google Scholar), and CMV-β-gal (31MacGregor G.R. Caskey C.T. Nucleic Acids Res. 1989; 172365Google Scholar) reporter plasmids have been described previously. The pMT2-GATA4 and pEMSV-Nkx2.5 expression plasmids were provided by J. Molkentin. Transient Transfections and Reporter Assays—NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium (Cellgro), supplemented with 10% fetal bovine serum (Hyclone), 100 units/ml penicillin/streptomycin (Invitrogen), and 2 mml-glutamine (Invitrogen). Cells were co-transfected with 100-500 ng of each expression vector, unless specified otherwise, and 500 ng of the reporter plasmid using FuGENE 6 transfection reagent (Roche Applied Science). Each co-transfection also included 100 ng of CMV-LacZ, and the DNA concentrations were kept constant by the addition of empty expression vectors. Cells were harvested 48 h after transfection in 100 μl of lysis buffer (Tropix). Luciferase and β-galactosidase activity was measured using the Luciferase Assay kit (Tropix) and Galacto-Star reagents (Tropix) according to the manufacturer's instructions. Reporter activity was detected using a Monolight 3010 luminometer, and luciferase activity was normalized to the β-galactosidase activity. Each experiment was completed in duplicate and repeated at least three times. Statistical significance of observed differences was determined by Student's t test. Site-directed Mutagenesis—Nucleotide changes were generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions. To generate mutations in T-box binding element 1 (TBE1) and TBE2 of the (-288)ANF-luciferase reporter, the following primers were used in the initial PCR: mutTBE2, 5′-CTTTTCTGCTCTTCTCTTTGCTTTGAAGTGGGGGCCTCTTGAGGC-3′ and 5′-GCCTCAAGAGGCCCCCACTTCAAAGCAAAGAGAAGAGCAGAAAAG-3′; mutTBE1, 5′-ATCTTCTCCTGGCCGCCGCAACAAGCAGAATGGGGAGGGTTCCAG-3′ and 5′-CTGGAACCCTCCCCATTCTGCTTGTTGCGGCGGCCAGGAGAAGAT-3′ (16 cycles of 95 °C, 30 s; 55 °C, 1 min; 68 °C, 7 min). Nucleotide changes in tbx5 to create Holt-Oram syndrome (HOS) mutations were generated using the pBlueScript-SK-Tbx5 as template and the following primers: R237Q, 5′-CCCTTCGCCAAAGGCTTTCAGGGCAGTGATGAC-3′ and 5′-GTCATCACTGCCCTGAAAGCCTTTGGCGAAGGG-3′; R279ter, 5′-CTTCAGCAGCGAGACCTGAGCTCTCTCCACCTC3′ and 5′-GAGGTGGAGAGAGCTCAGGTCTCGCTGCTGAAG-3′. Sequence analysis confirmed that the plasmids contained the intended nucleotide changes. Chicken Explant Cultures and Quantitative Real Time RT-PCR—Mesendodermal tissue from Hamburger-Hamilton stage 5 chick embryos was explanted as described previously (32Searcy R.D. Yutzey K.E. Dev. Dyn. 1998; 213: 82-91Google Scholar, 33Yutzey K. Gannon M. Bader D. Dev. Biol. 1995; 170: 531-541Google Scholar) and cultured for 6 h in M199 medium (Invitrogen) supplemented with penicillin/streptomycin with or without the addition of 200 ng/ml recombinant BMP2 (R&D Systems). The lateral heart-forming regions explanted were defined previously (21Ehrman L.A. Yutzey K.E. Dev. Biol. 1999; 207: 163-175Google Scholar). RNA was extracted using TRIzol reagent (Invitrogen) and pooled from lateral or medial explants of four embryos (32Searcy R.D. Yutzey K.E. Dev. Dyn. 1998; 213: 82-91Google Scholar). cDNA was generated with oligo(dT) primers and the SuperScript first-strand synthesis kit (Invitrogen). Quantitative real time RT-PCR was performed using the MJ Research Opticon Monitor II system (94 °C, 1 min; 55 °C, 1.5 min; 72 °C, 3 min; 35 cycles). Reactions included 0.1× SYBR Green (Molecular Probes), and fluorescence was monitored at 72 °C. The following primers were used in the RT-PCR: Tbx5, 5′-GGGCTCCCAGTACCAGTGTGA-3′ and 5′-GTAGGGCTTCTTGTAGGGATG-3′; Tbx20, 5′-TTGGCATGTGGAAAGAAGG-3′ and 5′-CAGGCAACGCAAAGCAGAG-3′; Nkx2.5 and GAPDH primers were reported previously (34Schultheiss T.M. Xydas S. Lassar A.B. Development. 1995; 121: 4203-4214Google Scholar). Gene expression levels were quantified based on the threshold cycle (C(t)) calibrated to a standard curve generated using E7 chicken whole heart cDNA and normalized to GAPDH as described by the manufacturer (MJ Research). RT-PCR results were compiled from seven independent experiments with PCRs performed two to three times in triplicate. Statistical significance of observed differences was determined using Student's t test. Tbx5 and Tbx20 Are Differentially Expressed in the Developing Chicken Heart—To compare the temporal and spatial regulation of Tbx5 and Tbx20 mRNA expression in the heart, in situ hybridization was performed on chicken embryos and isolated hearts from 1-10 days of development (Fig. 1). Expression of both Tbx5 and Tbx20 is detected in the heart primordia of Hamburger-Hamilton stage 6 embryos (Fig. 1, A and B, black arrowheads). At this stage, Tbx5 is expressed at low levels in the anterior heart-forming region, whereas Tbx20 expression is apparent in the anterior heart-forming region and in the posterior lateral regions of the embryo (Fig. 1B, red arrowheads). By stage 8, Tbx5 and Tbx20 are co-expressed in the cardiac primordia immediately prior to cardiomyocyte differentiation and heart tube formation (Fig. 1, C and D, black arrowheads). Concurrently the posterior lateral expression of Tbx20 is reduced, and expression becomes restricted to the cardiac primordia. At later stages, Tbx5 and Tbx20 are differentially expressed in the primitive heart tube and during heart chamber morphogenesis. In stage 12 embryos, Tbx5 expression becomes restricted to the posterior, atrial, and left ventricular regions of the heart tube (Fig. 1E, red arrowhead), while Tbx20 is expressed throughout the entire heart tube including the anterior outflow tract (Fig. 1F, red arrowhead). Although Tbx5 and Tbx20 are co-expressed in the atria at E5 (Fig. 1, G and H, black arrowheads), their expression in the atrioventricular canal (AVC), ventricles, and outflow tract are distinct. Tbx5 is present in the atria and left ventricle but is not expressed in the right ventricle and outflow tract. Tbx20 expression, however, is enriched in the AVC, the outflow tract (Fig. 1H, red arrowheads), and right ventricle but is reduced in the left ventricle. Interestingly the expression of Tbx5 and Tbx20 in the ventricles are complementary with sharp boundaries of expression where the ventricular septum will form (Fig. 1, G and H, blue arrowheads). After 10 days of development, Tbx5 is strongly expressed in the atria (Fig. 1I, black arrowhead) and in the developing conduction system (Fig. 1I, red arrowhead). Tbx20 expression is present in the atria (Fig. 1J, black arrowhead), the AVC, and specifically in the tricuspid and mitral valves (Fig. 1J, black arrows). These data show that Tbx5 and Tbx20 share a similar expression pattern in the early heart primordia and developing atria. However, Tbx5 and Tbx20 are differentially expressed in the atrioventricular valves and specialized myocardial lineages. The C Termini of Tbx5 and Tbx20 Have Distinct Transcriptional Regulatory Functions—Differential expression of Tbx5 and Tbx20 may be related to diverse functions in the development of the heart. Although homologous in the T-box DNA binding region (63.0% identity), Tbx5 and Tbx20 share no obvious homology outside of this domain (15.1% identity in the N terminus and 10.1% in the C terminus). To determine the transcriptional regulatory functions of their divergent domains, fusion proteins were generated containing the C terminus of either Tbx5 or Tbx20 and the Gal4 DNA-binding domain. The amino acids used in the fusion proteins relative to the T-box region are shown in Fig. 2A. The Gal4-Tbx5 fusion protein contains amino acids 266-518 of Tbx5, and the Gal4-Tbx20 fusion protein contains amino acids 294-445 of Tbx20. These fusion constructs were co-transfected into NIH 3T3 cells with the G5E1b-luciferase reporter gene that contains five sequential Gal4 binding sites linked to the E1b promoter (29Huang J. Weintraub H. Kedes L. Mol. Cell. Biol. 1998; 18: 5478-5484Google Scholar). Co-transfection of the G5E1b-luciferase reporter with Gal4-Tbx5-(266-518) led to a more than 250-fold increase in expression relative to the reporter co-transfected with the Gal4 DNA-binding domain alone (Fig. 2B). In contrast, co-transfection with Gal4-Tbx20-(294-445) resulted in a significant decrease in the level of reporter activity relative to that observed with the Gal4 DNA-binding domain alone (Fig. 2B). Additional evidence for the repressor activity of Tbx20 was provided by co-transfection of Gal4-Tbx20-(294-445) with a reporter gene containing five sequential Gal4 and two LexA binding sites (5xGal4-2xLexA-E1B-luc) (35Zhou X. Richon V.M. Rifkind R.A. Marks P.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1056-1061Google Scholar). This reporter allows repressor activity of Gal4-Tbx20-(294-445) to be assessed by the ability to reduce the activation mediated by a strong activator. The 5xGal4-2xLexA-E1B-luc reporter was co-transfected with the Gal4-Tbx20-(294-445) and a fusion construct containing the LexA DNA-binding domain fused to VP16. Co-transfection of Gal4-Tbx20-(294-445) significantly decreased the high reporter activity mediated by the LexA-VP16 construct (Fig. 2C). These data provide additional evidence that the C terminus of the Tbx20 protein contains a transcriptional repressor domain. Taken together, these experiments demonstrate that the C terminus of Tbx5 acts as an activator, while the C terminus of Tbx20 acts as a repressor. Tbx20 Antagonizes Tbx5 Activation of ANF Reporter Gene Expression—The relative abilities of Tbx5 and Tbx20 to activate cardiac gene expression was assessed using a reporter gene consisting of the proximal 3003 base pairs of the rat ANF promoter linked to the luciferase gene ((-3003)ANF-luciferase) (25Argentin S. Nemer M. Drouin J. Scott G.K. Kennedy B.P. Davies P.L. J. Biol. Chem. 1985; 260: 4568-4571Google Scholar, 26Knowlton K.U. Baracchini E. Ross R.S. Harris A.N. Henderson S.A. Evans S.M. Glembotski C.C. Chien K.R. J. Biol. Chem. 1991; 266: 7759-7768Google Scholar). The ANF promoter contains the consensus binding sequences of several cardiac transcription factors and has been extensively used to examine cardiac gene-regulatory mechanisms (13Bruneau 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: 709-721Google Scholar, 15Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar, 17Ghosh T.K. Packham E.A. Bonser A.J. Robinson T.E. Cross S.J. Brook J.D. Hum. Mol. Genet. 2001; 10: 1983-1994Google Scholar, 36Durocher D. Chen C.Y. Ardati A. Schwartz R.J. Nemer M. Mol. Cell. Biol. 1996; 16: 4648-4655Google Scholar, 37Durocher D. Nemer M. Dev. Genet. 1998; 22: 250-262Google Scholar, 38Lee Y. Shioi T. Kasahara H. Jobe S.M. Wiese R.J. Markham B.E. Izumo S. Mol. Cell. Biol. 1998; 18: 3120-3129Google Scholar, 39Shiojima I. Komuro I. Oka T. Hiroi Y. Mizuno T. Takimoto E. Monzen K. Aikawa R. Akazawa H. Yamazaki T. Kudoh S. Yazaki Y. J. Biol. Chem. 1999; 274: 8231-8239Google Scholar, 40Durocher D. Charron F. Warren R. Schwartz R.J. Nemer M. EMBO J. 1997; 16: 5687-5696Google Scholar). NIH 3T3 cells were co-transfected with the (-3003)ANF-luciferase reporter and with pAC-CMVTbx5 or pAC-CMV-Tbx20 expression plasmids, and ANF transcriptional activation was assessed 48 h later. (-3003)ANF-luciferase expression significantly increased ∼2.3-fold compared with the empty vector control when co-transfected with Tbx5. ANF reporter activity, however, was significantly decreased (∼30%) when co-transfected with Tbx20 compared with the empty vector control (Fig. 3A). To determine whether Tbx20 can antagonize Tbx5 function, (-3003)ANF-luciferase was co-transfected with increasing amounts of pAC-CMVTbx20 (0.1-1.5 μg) and a constant amount of pAC-CMV-Tbx5 (0.5 μg). Tbx5 activation of (-3003)ANF-luciferase expression decreased as the quantity of Tbx20 expression plasmid transfected increased and was completely abrogated by the maximal transfected ratio (1.5:0.5 μg) of Tbx20:Tbx5 (Fig. 3A). Together these results indicate that Tbx20 alone can repress ANF promoter activity and also is able to inhibit the ability of Tbx5 to activate ANF gene expression. Tbx5, but Not Tbx20, Cooperatively Acts with GATA4 and Nkx2.5 to Activate ANF Expression—The ability of Tbx5 or Tbx20 to cooperate with GATA4 and Nkx2.5 in activating (-3003)ANF-luciferase was determined in transfected NIH 3T3 cells. pAC-CMV-Tbx5 and pAC-CMV-Tbx20 were co-transfected with either pMT2-GATA4 or pEMSV-Nkx2.5 expression plasmids and the (-3003)ANF-luciferase reporter. Tbx5, Nkx2.5, and GATA4 each activated (-3003)ANF-luciferase expression (∼1.9-, ∼7.4-, and ∼4.1-fold, respectively) (Fig. 3B). A synergistic activation of (-3003)ANF-luciferase was observed when Tbx5 was co-transfected with Nkx2.5 (∼28.0-fold) (Fig. 3B). However, when Tbx20 was co-transfected with Nkx2.5, this synergistic activation was not observed, and the level of (-3003)ANF-luciferase activation was comparable to that of Nkx2.5 alone (∼7.0-fold) (Fig. 3B). Synergistic activation of (-3003)ANF-luciferase was also observed when Tbx5 was co-transfected with GATA4 (∼36.5-fold); however, this synergy was not achieved with Tbx20 and GATA4 together (∼5.4-fold) (Fig. 3B). These data indicate th" @default.
• W2083248934 created "2016-06-24" @default.
• W2083248934 creator A5024272087 @default.
• W2083248934 creator A5030290137 @default.
• W2083248934 date "2004-04-01" @default.
• W2083248934 modified "2023-09-26" @default.
• W2083248934 title "Differential Expression and Function of Tbx5 and Tbx20 in Cardiac Development" @default.
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