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• W1963513125 abstract "TBX5 is the gene mutated in Holt-Oram syndrome, an autosomal dominant disorder with complex heart and limb deformities. Its protein product is a member of the T-box family of transcription factors and an evolutionarily conserved dosage-sensitive regulator of heart and limb development. Understanding TBX5 regulation is therefore of paramount importance. Here we uncover the existence of novel exons and provide evidence that TBX5 activity may be extensively regulated through alternative splicing to produce protein isoforms with differing N- and C-terminal domains. These isoforms are also present in human heart, indicative of an evolutionarily conserved regulatory mechanism. The newly identified isoforms have different transcriptional properties and can antagonize TBX5a target gene activation. Droplet Digital PCR as well as immunohistochemistry with isoform-specific antibodies reveal differential as well as overlapping expression domains. In particular, we find that the predominant isoform in skeletal myoblasts is Tbx5c, and we show that it is dramatically up-regulated in differentiating myotubes and is essential for myotube formation. Mechanistically, TBX5c antagonizes TBX5a activation of pro-proliferative signals such as IGF-1, FGF-10, and BMP4. The results provide new insight into Tbx5 regulation and function that will further our understanding of its role in health and disease. The finding of new exons in the Tbx5 locus may also be relevant to mutational screening especially in the 30% of Holt-Oram syndrome patients with no mutations in the known TBX5a exons. TBX5 is the gene mutated in Holt-Oram syndrome, an autosomal dominant disorder with complex heart and limb deformities. Its protein product is a member of the T-box family of transcription factors and an evolutionarily conserved dosage-sensitive regulator of heart and limb development. Understanding TBX5 regulation is therefore of paramount importance. Here we uncover the existence of novel exons and provide evidence that TBX5 activity may be extensively regulated through alternative splicing to produce protein isoforms with differing N- and C-terminal domains. These isoforms are also present in human heart, indicative of an evolutionarily conserved regulatory mechanism. The newly identified isoforms have different transcriptional properties and can antagonize TBX5a target gene activation. Droplet Digital PCR as well as immunohistochemistry with isoform-specific antibodies reveal differential as well as overlapping expression domains. In particular, we find that the predominant isoform in skeletal myoblasts is Tbx5c, and we show that it is dramatically up-regulated in differentiating myotubes and is essential for myotube formation. Mechanistically, TBX5c antagonizes TBX5a activation of pro-proliferative signals such as IGF-1, FGF-10, and BMP4. The results provide new insight into Tbx5 regulation and function that will further our understanding of its role in health and disease. The finding of new exons in the Tbx5 locus may also be relevant to mutational screening especially in the 30% of Holt-Oram syndrome patients with no mutations in the known TBX5a exons. Holt-Oram syndrome (HOS) 3The abbreviations used are: HOSHolt-Oram syndromeTBXT-boxTBET-box binding elementTADtranscriptional activation domainAAamino acid(s)Eembryonic dayPpostnatal day. is an autosomal dominant disorder characterized by upper limb and cardiac defects (1Basson 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. Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome.Nat. Genet. 1997; 15: 30-35Crossref PubMed Scopus (907) Google Scholar, 2Li 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. Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family.Nat. Genet. 1997; 15: 21-29Crossref PubMed Scopus (756) Google Scholar). The most common structural heart abnormalities include atrial septal defects and ventricular septal defects. Conduction defects have also been commonly seen, and they mostly involve atrioventricular blocks. Hypoplastic left ventricles, mitral valve problems, and endocardial cushion defects have also been reported in HOS patients (1Basson 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. Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome.Nat. Genet. 1997; 15: 30-35Crossref PubMed Scopus (907) Google Scholar, 2Li 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. Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family.Nat. Genet. 1997; 15: 21-29Crossref PubMed Scopus (756) Google Scholar). Genetic linkage analyses have mapped the disease to the chromosomal locus where TBX5 is located, and mutations in TBX5 have been found in patients with HOS. Moreover, Tbx5 expression pattern in the upper limb, atria, and left ventricle along with mouse genetics studies have strengthened the causative link between TBX5 and HOS (3Bruneau 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. A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease.Cell. 2001; 106: 709-721Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar). Over 70 mutations in the TBX5 locus have been identified so far in HOS patients (4Heinritz W. Shou L. Moschik A. Froster U.G. The human TBX5 gene mutation database.Hum. Mutat. 2005; 26: 397Crossref PubMed Scopus (31) Google Scholar). Many result in no protein production or in truncated proteins. Other more subtle mutations generate functionally impaired proteins with altered subcellular localization, DNA binding, transcriptional activity, and/or interaction with cofactors (5Fan C. Liu M. Wang Q. Functional analysis of TBX5 missense mutations associated with Holt-Oram syndrome.J. Biol. Chem. 2003; 278: 8780-8785Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 6Garg 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. GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5.Nature. 2003; 424: 443-447Crossref PubMed Scopus (969) Google Scholar7Ghosh T.K. Packham E.A. Bonser A.J. Robinson T.E. Cross S.J. Brook J.D. Characterization of the TBX5 binding site and analysis of mutations that cause Holt-Oram syndrome.Hum. Mol. Genet. 2001; 10: 1983-1994Crossref PubMed Scopus (122) Google Scholar). These findings led to the suggestion that haploinsufficiency may be the mechanism of pathogenesis, but this remains uncertain in many cases. Interestingly, in about 30–35% of HOS patients, no mutations in TBX5 coding sequences or intron-exon junctions are detected (8Borozdin W. Bravo Ferrer Acosta A.M. Bamshad M.J. Botzenhart E.M. Froster U.G. Lemke J. Schinzel A. Spranger S. McGaughran J. Wand D. Chrzanowska K.H. Kohlhase J. Expanding the spectrum of TBX5 mutations in Holt-Oram syndrome: detection of two intragenic deletions by quantitative real time PCR, and report of eight novel point mutations.Hum. Mutat. 2006; 27: 975-976Crossref PubMed Scopus (38) Google Scholar), which has raised the controversial suggestion of the existence of another as yet unidentified HOS-causing locus. An alternative explanation could be that unscreened mutations within presumed untranscribed regions of TBX5 account for this low detection rate. Consistent with this, we recently reported the existence of a new exon downstream of the T-box whose alternative splicing results in a TBX5 isoform lacking the entire C terminus, which contains several functional domains (9Georges R. Nemer G. Morin M. Lefebvre C. Nemer M. Distinct expression and function of alternatively spliced Tbx5 isoforms in cell growth and differentiation.Mol. Cell. Biol. 2008; 28: 4052-4067Crossref PubMed Scopus (43) Google Scholar). Holt-Oram syndrome T-box T-box binding element transcriptional activation domain amino acid(s) embryonic day postnatal day. TBX5 is a member of the large family of T-box transcription factors critical for early cellular commitment, differentiation, and organ development (10Hariri F. Nemer M. Nemer G. T-box factors: insights into the evolutionary emergence of the complex heart.Ann. Med. 2012; 44: 680-693Crossref PubMed Scopus (11) Google Scholar). T-box (or Tbx) proteins bind specific DNA motifs, called TBEs (T-box binding elements), to activate or repress target promoters. TBX5 appears to act essentially as a transcriptional activator and cooperates with other transcription factors such as GATA4 and NKX2.5 to synergistically regulate downstream targets (3Bruneau 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. A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease.Cell. 2001; 106: 709-721Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar, 6Garg 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. GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5.Nature. 2003; 424: 443-447Crossref PubMed Scopus (969) Google Scholar, 11Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Tbx5 associates with Nkx2–5 and synergistically promotes cardiomyocyte differentiation.Nat. Genet. 2001; 28: 276-280Crossref PubMed Scopus (474) Google Scholar). As such, TBX5 activity can be modulated at the DNA binding level and through protein-protein interactions. In addition to transcriptional regulators, TBX5 was shown to interact with the cytoskeleton-associated LIM protein LMP4, which represses its transcriptional activity, possibly by stimulating its cytoplasmic redistribution (12Camarata T. Bimber B. Kulisz A. Chew T.L. Yeung J. Simon H.G. LMP4 regulates Tbx5 protein subcellular localization and activity.J. Cell Biol. 2006; 174: 339-348Crossref PubMed Scopus (37) Google Scholar). TBX51–518 (referred to thereafter as TBX5a) resides largely if not exclusively in the nucleus, and two nuclear localization signals have been identified, one within the T-box DNA binding domain and another between amino acids (AA) 325 and 340 outside the T-box (13Zaragoza M.V. Lewis L.E. Sun G. Wang E. Li L. Said-Salman I. Feucht L. Huang T. Identification of the TBX5 transactivating domain and the nuclear localization signal.Gene. 2004; 330: 9-18Crossref PubMed Scopus (45) Google Scholar). A putative nuclear export signal within the T-box has also been suggested to mediate Crml-dependent nuclear export of TBX5 (14Kulisz A. Simon H.G. An evolutionarily conserved nuclear export signal facilitates cytoplasmic localization of the Tbx5 transcription factor.Mol. Cell. Biol. 2008; 28: 1553-1564Crossref PubMed Scopus (32) Google Scholar), but this has been challenged based on the crystal structure of the T-box domain of TBX5 in DNA-bound and unbound forms (15Stirnimann C.U. Ptchelkine D. Grimm C. Müller C.W. Structural basis of TBX5-DNA recognition: the T-box domain in its DNA-bound and -unbound form.J. Mol. Biol. 2010; 400: 71-81Crossref PubMed Scopus (37) Google Scholar). The crystal structure also identified the T-box residues that contact DNA as those toward the C terminus of the T-box. Interactions between TBX5 and other transcriptional regulators also require the T-box (3Bruneau 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. A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease.Cell. 2001; 106: 709-721Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar, 9Georges R. Nemer G. Morin M. Lefebvre C. Nemer M. Distinct expression and function of alternatively spliced Tbx5 isoforms in cell growth and differentiation.Mol. Cell. Biol. 2008; 28: 4052-4067Crossref PubMed Scopus (43) Google Scholar). In addition to DNA binding, transcriptional activation by TBX5 depends on sequences outside the T-box. Deletion analysis showed that removal of the N-terminal 50 AA decreases TBX5 transcriptional activity, albeit not as severely as removal of the C-terminal 100 AA. Another domain that contributes to transcriptional activation was localized between AA 255 and 316 just C-terminal of the T-box. Interestingly, the three domains are differentially required for physical and functional interaction with GATA4 and NKX2.5 (9Georges R. Nemer G. Morin M. Lefebvre C. Nemer M. Distinct expression and function of alternatively spliced Tbx5 isoforms in cell growth and differentiation.Mol. Cell. Biol. 2008; 28: 4052-4067Crossref PubMed Scopus (43) Google Scholar). Gal4-TBX5 chimera confirmed the presence of a potent transcriptional activation domain (TAD) in the last 250 AA of TBX5a (16Plageman Jr., T.F. Yutzey K.E. Differential expression and function of Tbx5 and Tbx20 in cardiac development.J. Biol. Chem. 2004; 279: 19026-19034Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar); an autonomous TAD was also mapped between AA 339 and 379 (13Zaragoza M.V. Lewis L.E. Sun G. Wang E. Li L. Said-Salman I. Feucht L. Huang T. Identification of the TBX5 transactivating domain and the nuclear localization signal.Gene. 2004; 330: 9-18Crossref PubMed Scopus (45) Google Scholar). In the present work, we report the existence of new Tbx5 exons and additional alternatively spliced TBX5 isoforms that differ from TBX5 in the N- or C-terminal domains. We show that the new isoforms are expressed in distinct domains that sometimes overlap with Tbx5a. We show that one of the novel isoforms plays a unique role in skeletal muscle differentiation where it suppresses proliferation signals and induces differentiation. The results provide important information on the Tbx5 locus and novel insight into Tbx5 regulation and function. Tbx5 transcripts with variable 3′ ends (Tbx5c and Tbx5d) were obtained using nested cDNA amplification of mRNA from embryonic hearts and limbs. cDNAs were amplified by RT-PCR using a 5′ oligonucleotide spanning the first codons of the T-box and at the 3′ end, an oligo(dT). The resulting products were cloned into the Bluescript plasmid, and individual clones were sequenced. Full-length clones containing the N-terminal sequences were then amplified using specific primers and subcloned in the pcDNA3 expression vector in phase with Kozak triple FLAG epitope. To isolate isoforms with variable 5′ end (Tbx5e), primer extension amplification was used with the primer sequence overlapping the 5′ end of the T-box sequences. Oligonucleotides used were: 5′-GGAGGTACCGCCGATACAGATGAGGGCTTTG-3′ (forward for Tbx5a, Tbx5c, Tbx5d, and Tbx5b); 5′-GCAGGTACCGAAGGAATCAAGGTGTTTCTTCATG-3′ (forward for Tbx5e); 5′-CCGGAATTCTTAGCTATTCTCACTCCACTCTGG-3′ (reverse for Tbx5a and Tbx5e); 5′-GCACTCGAGCTAATGAAAGGATGGTGAGAGAG-3′ (reverse for Tbx5c); 5′-CGCGAATTCCTATATTTCTGTGCCACTTACTT-3′ (reverse for Tbx5d); and 5′-CCCCTCGAGCTAAAGCAGAGGCCTTTGCATCCGAG-3′ (reverse for Tbx5b). GATA4 and NKX2.5 expression vectors as well as the luciferase reporters were previously described (9Georges R. Nemer G. Morin M. Lefebvre C. Nemer M. Distinct expression and function of alternatively spliced Tbx5 isoforms in cell growth and differentiation.Mol. Cell. Biol. 2008; 28: 4052-4067Crossref PubMed Scopus (43) Google Scholar, 17Durocher D. Charron F. Warren R. Schwartz R.J. Nemer M. The cardiac transcription factors Nkx2–5 and GATA-4 are mutual cofactors.EMBO J. 1997; 16: 5687-5696Crossref PubMed Scopus (549) Google Scholar, 18Nadeau M. Georges R.O. Laforest B. Yamak A. Lefebvre C. Beauregard J. Paradis P. Bruneau B.G. Andelfinger G. Nemer M. An endocardial pathway involving Tbx5, Gata4, and Nos3 required for atrial septum formation.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 19356-19361Crossref PubMed Scopus (53) Google Scholar19Lavallée G. Andelfinger G. Nadeau M. Lefebvre C. Nemer G. Horb M.E. Nemer M. The Kruppel-like transcription factor KLF13 is a novel regulator of heart development.EMBO J. 2006; 25: 5201-5213Crossref PubMed Scopus (66) Google Scholar). Cell lines were maintained in culture and transfected as described previously (9Georges R. Nemer G. Morin M. Lefebvre C. Nemer M. Distinct expression and function of alternatively spliced Tbx5 isoforms in cell growth and differentiation.Mol. Cell. Biol. 2008; 28: 4052-4067Crossref PubMed Scopus (43) Google Scholar, 20Aries A. Paradis P. Lefebvre C. Schwartz R.J. Nemer M. Essential role of GATA-4 in cell survival and drug-induced cardiotoxicity.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6975-6980Crossref PubMed Scopus (227) Google Scholar) C2C12 were maintained in 10% FBS for 24 h, and the medium was switched to 1% BSA or 2% horse serum for differentiation when indicated. For co-transfection assays, the total amount of DNA was maintained constant by adding the appropriate amount of empty DNA vector. Fgf10-luc reporter construct was a kind gift from Dr. Benoit Bruneau from the Gladstone Institute of Cardiovascular Disease and was previously described (21Agarwal P. Wylie J.N. Galceran J. Arkhitko O. Li C. Deng C. Grosschedl R. Bruneau B.G. Tbx5 is essential for forelimb bud initiation following patterning of the limb field in the mouse embryo.Development. 2003; 130: 623-633Crossref PubMed Scopus (227) Google Scholar). Nppa-luc, Bclx-luc, Ccnd1-luc, and Bmp4-luc reporters were previously described (19Lavallée G. Andelfinger G. Nadeau M. Lefebvre C. Nemer G. Horb M.E. Nemer M. The Kruppel-like transcription factor KLF13 is a novel regulator of heart development.EMBO J. 2006; 25: 5201-5213Crossref PubMed Scopus (66) Google Scholar, 20Aries A. Paradis P. Lefebvre C. Schwartz R.J. Nemer M. Essential role of GATA-4 in cell survival and drug-induced cardiotoxicity.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6975-6980Crossref PubMed Scopus (227) Google Scholar, 22Nemer G. Nemer M. Transcriptional activation of BMP-4 and regulation of mammalian organogenesis by GATA-4 and -6.Dev. Biol. 2003; 254: 131-148Crossref PubMed Scopus (134) Google Scholar). GAL4 fusion proteins were designed as described previously (23Charron F. Tsimiklis G. Arcand M. Robitaille L. Liang Q. Molkentin J.D. Meloche S. Nemer M. Tissue-specific GATA factors are transcriptional effectors of the small GTPase RhoA.Genes Dev. 2001; 15: 2702-2719Crossref PubMed Scopus (196) Google Scholar). Nuclear extracts were prepared from 293T cells. Binding reactions were done at room temperature using 1 μg of poly(dI-dC). The TBE probe used was from the Nppa promoter and previously described (9Georges R. Nemer G. Morin M. Lefebvre C. Nemer M. Distinct expression and function of alternatively spliced Tbx5 isoforms in cell growth and differentiation.Mol. Cell. Biol. 2008; 28: 4052-4067Crossref PubMed Scopus (43) Google Scholar). Production of the GST-TBX5a and MBP-NKX2.5 constructs (where MBP is maltose-binding protein) and the pulldown assays were done as described previously (24Yamak A. Latinkic B.V. Dali R. Temsah R. Nemer M. Cyclin D2 is a GATA4 cofactor in cardiogenesis.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 1415-1420Crossref PubMed Scopus (26) Google Scholar). Western blots were done on nuclear extracts from 293T cells overexpressing the relevant Tbx5 constructs and FLAG or TBX5 antibody as described previously (9Georges R. Nemer G. Morin M. Lefebvre C. Nemer M. Distinct expression and function of alternatively spliced Tbx5 isoforms in cell growth and differentiation.Mol. Cell. Biol. 2008; 28: 4052-4067Crossref PubMed Scopus (43) Google Scholar). Endogenous TBX5 proteins were analyzed in two relevant mouse lines, the TC13 cardiogenic cell line and in C2C12 myoblasts, as well as in 30-day-old mouse hearts. Protein extraction and analysis were as reported in Georges et al. (9Georges R. Nemer G. Morin M. Lefebvre C. Nemer M. Distinct expression and function of alternatively spliced Tbx5 isoforms in cell growth and differentiation.Mol. Cell. Biol. 2008; 28: 4052-4067Crossref PubMed Scopus (43) Google Scholar). The N-terminal TBX5a antibody was previously described. The C-terminal TBX5a antibody as well as TBX5c and TBX5d antibodies were similarly raised in rabbits against the unique C-terminal domains: AA 331–425 for TBX5a, AA 327–404 for TBX5c, and AA 327–376 for TBX5d. Immunocytochemistry was done on 293T cells overexpressing the relevant FLAG-TBX5 constructs as described previously (9Georges R. Nemer G. Morin M. Lefebvre C. Nemer M. Distinct expression and function of alternatively spliced Tbx5 isoforms in cell growth and differentiation.Mol. Cell. Biol. 2008; 28: 4052-4067Crossref PubMed Scopus (43) Google Scholar). Mouse anti-FLAG M2 was purchased from Sigma (F1804). Immunofluorescence was performed on C2C12 cells overexpressing the indicated HA-TBX5 constructs. Myosin heavy chain and myogenin antibodies were both obtained from mouse hybridoma cells (MF20 and F5D, respectively). HA antibody was purchased from Santa Cruz Biotechnology (SC-805). Immunohistochemistry was done on mouse tissues at different embryonic stages as described previously (25Yamak A. Temsah R. Maharsy W. Caron S. Paradis P. Aries A. Nemer M. Cyclin D2 rescues size and function of GATA4 haplo-insufficient hearts.Am. J. Physiol. Heart Circ. Physiol. 2012; 303: H1057-H1066Crossref PubMed Scopus (16) Google Scholar). Droplet Digital PCR was carried out on the QX200 Bio-Rad Droplet Digital PCR system using cDNA from embryonic and postnatal tissues as well as cardiac and muscle cells using sequence-specific primers as indicated and according to the manufacturer's protocol. Primer sequences are available on request. Quantitative PCR analysis was performed on C2C12 cells overexpressing the indicated TBX5 constructs as described previously (25Yamak A. Temsah R. Maharsy W. Caron S. Paradis P. Aries A. Nemer M. Cyclin D2 rescues size and function of GATA4 haplo-insufficient hearts.Am. J. Physiol. Heart Circ. Physiol. 2012; 303: H1057-H1066Crossref PubMed Scopus (16) Google Scholar). siRNAs were obtained from Sigma and transfected in C2C12 cells using the HiPerFect transfection reagent from Qiagen (catalog number 301705). siRNA sequences are available upon request. To analyze endogenous TBX5 protein, we developed a specific antibody against the N-terminal TBX5 region; in Western blots, this antibody detected, in addition to the expected TBX5a band, immunoreactive bands that co-migrated with a TBX5 protein truncated of the last 118 amino acids (9Georges R. Nemer G. Morin M. Lefebvre C. Nemer M. Distinct expression and function of alternatively spliced Tbx5 isoforms in cell growth and differentiation.Mol. Cell. Biol. 2008; 28: 4052-4067Crossref PubMed Scopus (43) Google Scholar). To determine whether these bands represent novel TBX5 isoforms, we used a PCR amplification strategy to isolate Tbx5 cDNAs from mouse heart and limb RNA. A 5′ oligonucleotide primer spanning the first codons of the T-box and a 3′ oligo(dT) primer allowed amplification of Tbx5 transcripts containing the T-domain together with any variable 3′ sequences. Isolation and sequencing of several independent cDNA clones revealed the presence of four distinct transcripts that may result in four different TBX5 proteins; in addition to sequences corresponding to Tbx5a and the previously described short isoform (9Georges R. Nemer G. Morin M. Lefebvre C. Nemer M. Distinct expression and function of alternatively spliced Tbx5 isoforms in cell growth and differentiation.Mol. Cell. Biol. 2008; 28: 4052-4067Crossref PubMed Scopus (43) Google Scholar), two new sequences were identified that would encode novel TBX5 isoforms of 404 and 376 AA, termed Tbx5c and Tbx5d, respectively (Fig. 1A). These two isoforms result from the use of two alternative exons (exon 9a or exon 10), which are mutually exclusive with exon 11. Tbx5c is generated from the splicing of exon 9 to exon 10 instead of exon 11, whereas Tbx5d originates from the usage of an alternative exon 9, named exon 9a, which emanates from the retention of additional intronic sequences 3′ of exon 9 (Fig. 1C). The result in both cases is the addition, after position 327, of a 77-AA (TBX5c) or 49-AA (TBX5d) divergent region (Fig. 1, B and D). Once the additional exons were sequenced, we cloned the entire cDNA coding sequence of the novel isoforms from mouse embryonic atria (Tbx5d) and forelimb (Tbx5c) using primers spanning the first codon (ATG) and the stop codon of the different isoforms. Sequence analysis of the genomic TBX5 locus in existing databases (NCBI, Ensembl) revealed the possibility of a fifth TBX5 isoform, TBX5e (Fig. 1A). This isoform would result from the splicing of an alternative exon 1 in mice (termed exon 1b) with exon 3; thus, skipping exon 2 leading to an N-terminal truncation of 50 AA (Fig. 1, C and D). Exon 1b is highly homologous to the human exon 1. To confirm the in vivo existence of such Tbx5e transcripts, a primer overlapping exon 1b and 3 was used in RT-PCR amplification together with a reverse primer spanning the stop codon of exon 11; this resulted in the isolation of Tbx5e cDNA clones from mouse heart RNA. Thus a N-terminally truncated TBX5 isoform is present in both humans and rodents. Next, we analyzed the tissue distribution of Tbx5c and Tbx5d, which contain novel exons, using Droplet Digital PCR. In the case of Tbx5a and Tbx5d, a forward primer corresponding to sequences in the common exon 7 was used along with a reverse primer specific to exon 11 (Tbx5a) or exon 9a (Tbx5d). Forward and reverse primers were chosen in exon 10 in the case of Tbx5c. Using this strategy, we detected all isoforms in E11.5 atria and forelimbs. E11.5 ventricles expressed mostly Tbx5a and lower levels of the other isoforms. Tbx5a and Tbx5c transcripts were also detected in the hindlimbs, albeit at much lower levels (Fig. 2A). A similar expression pattern was seen in the postnatal heart where Tbx5a was always the predominant isoform (Fig. 2B). We also verified the presence of these isoforms in cardiogenic cell lines, notably the atrium-like HL-1 cells and the endocardial progenitors TC13 cells, as well as in skeletal muscle progenitors such as C2C12 myoblasts. The Tbx5 expression pattern in HL1 resembled that of the E11.5 heart with the predominance of Tbx5a (Fig. 2C). Tbx5a was the only isoform present in TC13 cells but not in C2C12 myoblasts where Tbx5c was the predominant isoform and was dramatically up-regulated after differentiation. We then used the N-terminal TBX5 antibody (9Georges R. Nemer G. Morin M. Lefebvre C. Nemer M. Distinct expression and function of alternatively spliced Tbx5 isoforms in cell growth and differentiation.Mol. Cell. Biol. 2008; 28: 4052-4067Crossref PubMed Scopus (43) Google Scholar) to analyze the profile of TBX5 proteins in cardiac cells. Western blot analysis of nuclear extracts from TC13 cells revealed the existence of multiple immunoreactive bands (Fig. 2D) co-migrating with TBX5a, TBX5b, TBX5c, and TBX5d; TBX5a and TBX5c were also detected in C2C12 cells (Fig. 2D) although the TBX5a band was much weaker. To verify the presence of TBX5e, a similar blot was incubated with an antibody raised against a C-terminal epitope encoded by exon 11 (AA 331–425); this antibody can detect TBX5a and TBX5e but not TBX5b, TBX5c, and TBX5d. Consistent with the Droplet Digital PCR results, a strong band co-migrating with TBX5a was detected in TC13 nuclear extracts as well as a weaker band co-migrating with TBX5e. A 50-kDa band that may be TBX5e was also present in C2C12 cells (Fig. 2E). Next, we analyzed TBX5 complexes in postnatal hearts. Nuclear extracts were obtained using 300 mm NaCl extraction and dialyzed to 100 mm salt, and chromatin-binding proteins were enriched on a phosphocellulose column; chromatin-bound proteins were eluted at 300 mm salt and size-fractionated under non-denaturing conditions using a gel filtration Superose 6 column. Western blots on the resulting fractions were carried out using the N-terminal TBX5 antibody. Bands corresponding to TBX5a (∼64–80 kDa) were evident in several high molecular mass fractions. An immunoreactive band around 48 kDa was also present and would correspond to TBX5d (Fig. 2F). These results confirm the presence in the heart of TBX5a and TBX5d and suggest the existence of different protein complexes containing distinct TBX5 isoforms in Tbx5-expressing organs. Next, we wanted to verify whether similar splice isoforms exist in humans. In silico analysis of the mouse and human TBX5 genomic sequences revealed the presence of similar alternate splice site junctions for Tbx5c and TBX5d. For Tbx5d, this predicted the existence of alternate splicing within exon 9 to produce the splice variant shown in Fig. 3A, which turned out to be identical to the reported transcribed sequence MN_181486, which would produce a 350-AA isoform whose last 22 AA are divergent from TBX5a (Fig. 3B). The presence of this cDNA sequence was also reported by Basson et al. (1Basson 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. Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome.Nat. Genet. 1997; 15: 30-35Crossref Pub" @default.
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