Functional Analysis of TBX5 Missense Mutations Associated with Holt-Oram Syndrome

Abstract

TBX5 is a T-box transcription factor that plays a critical role in organogenesis. Seven missense mutations in TBX5 have been identified in patients with Holt-Oram syndrome characterized by congenital heart defects and upper limb abnormalities. However, the functional significance and molecular pathogenic mechanisms of these mutations are not clear. In this study we describe functional defects in DNA binding, transcriptional activity, protein-protein interaction, and cellular localization of mutant TBX5 with these missense mutations (Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I). Mutations G80R, R237Q, and R237W represent a group of mutations that dramatically reduce DNA-binding activity of TBX5, leading to reduced transcription activation by TBX5 and the loss of synergy in transcriptional activation between TBX5 and NKX2.5. The second group of mutations includes Q49K, I54T, G169R, and S252I, which have no or moderate effect on DNA-binding activity and the function of transcription activation of TBX5 but cause the complete loss of synergistic transcription activity between TBX5 and NKX2.5. All seven missense mutations greatly reduced the interaction of TBX5 with NKX2.5in vivo and in vitro. Immunofluorescent staining showed that wild type TBX5 was localized completely into the nucleus, but mutants were localized in both nucleus and cytoplasm. These results demonstrate that all seven missense mutations studied here are functional mutations with a spectrum of defects ranging from decreases in DNA-binding activity and transcriptional activation to the dramatic reduction of interaction between TBX5 and NKX2.5, and loss of synergy in transcriptional activation between these two proteins, as well as impairment in the nuclear localization of TBX5. These defects are likely central to the pathogenesis of Holt-Oram syndrome. TBX5 is a T-box transcription factor that plays a critical role in organogenesis. Seven missense mutations in TBX5 have been identified in patients with Holt-Oram syndrome characterized by congenital heart defects and upper limb abnormalities. However, the functional significance and molecular pathogenic mechanisms of these mutations are not clear. In this study we describe functional defects in DNA binding, transcriptional activity, protein-protein interaction, and cellular localization of mutant TBX5 with these missense mutations (Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I). Mutations G80R, R237Q, and R237W represent a group of mutations that dramatically reduce DNA-binding activity of TBX5, leading to reduced transcription activation by TBX5 and the loss of synergy in transcriptional activation between TBX5 and NKX2.5. The second group of mutations includes Q49K, I54T, G169R, and S252I, which have no or moderate effect on DNA-binding activity and the function of transcription activation of TBX5 but cause the complete loss of synergistic transcription activity between TBX5 and NKX2.5. All seven missense mutations greatly reduced the interaction of TBX5 with NKX2.5in vivo and in vitro. Immunofluorescent staining showed that wild type TBX5 was localized completely into the nucleus, but mutants were localized in both nucleus and cytoplasm. These results demonstrate that all seven missense mutations studied here are functional mutations with a spectrum of defects ranging from decreases in DNA-binding activity and transcriptional activation to the dramatic reduction of interaction between TBX5 and NKX2.5, and loss of synergy in transcriptional activation between these two proteins, as well as impairment in the nuclear localization of TBX5. These defects are likely central to the pathogenesis of Holt-Oram syndrome. the atrial natriuretic factor gene wild type luciferase electrophoretic mobility shift assay hemagglutinin glutathione S-transferase phosphate-buffered saline nickel-nitrilotriacetic acid 4′,6-diamidino-2-phenylindole T-box transcription factors belong to a conserved family of proteins that contribute to early cell fate determination, differentiation, and organogenesis (1Papaioannou V.E. Int. Rev. Cytol. 2001; 207: 1-70Google Scholar). Genetic mutations in human T-box transcription factors cause a number of human congenital disorders such as Holt-Oram syndrome (TBX5) (2Basson C.T. Bachinsky D.R. Lin R.C. Nat. Genet. 1997; 15: 30-35Google Scholar, 3Li Q.Y. Newbury-Ecob R.A. Terrett J.A. Nat. Genet. 1997; 15: 21-29Google Scholar), ulnar-mammary syndrome (TBX3) (4Bamshad M. Aoki V.T. Adkison M.G. Warren W.S. Bartness T.J. Nat. Genet. 1997; 16: 311-315Google Scholar), X-linked cleft palate and ankyloglossia (TBX22) (5Braybrook C. Doudney K. Marcano A.C. Arnason A. Bjornsson A. Patton M.A. Goodfellow P.J. Moore G.E. Stanier P. Nat. Gene. 2001; 29: 179-183Google Scholar), and DiGeorge syndrome (TBX1) (6Lindsay E.A. Vitelli F. Su H. Morishima M. Huynh T. Pramparo T. Jurecic V. Ogunrinu G. Sutherland H.F. Scambler P.J. Bradley A. Baldini A. Nature. 2001; 410: 97-101Google Scholar, 7Jerome L.A. Papaioannou V.E. Nat. Genet. 2001; 27: 286-291Google Scholar, 8Merscher 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). T-box genes have been identified in animals from ctenophores (comb jellies) and nematodes to mammals, and may account for ∼0.1% of animal genomes. The Tbx5 gene is located on chromosome 12q24 and encodes a protein of 518 amino acids (2Basson C.T. Bachinsky D.R. Lin R.C. Nat. Genet. 1997; 15: 30-35Google Scholar, 3Li Q.Y. Newbury-Ecob R.A. Terrett J.A. Nat. Genet. 1997; 15: 21-29Google Scholar). TBX5 contains a highly conserved DNA-binding domain, T-box domain. Tbx5 is expressed in embryonic heart and limb tissues (9Sepulveda J.L. Vlahopoulos S. Iyer D. Belaguli N. Schwartz R.J. J. Biol. Chem. 2002; 277: 25775-25782Google Scholar, 10Liberatore C.M. Searcy-Schrick R.D. Yutzey K.E. Dev. Biol. 2000; 223: 169-180Google Scholar, 11Hatcher C.J. Kim M.S. Mah C.S. Dev. Biol. 2001; 230: 177-188Google 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). During embryogenesis,Tbx5 expression starts at the earliest stages of heart formation and is co-localized with genes encoding other important cardiac transcription factors, Nkx2.5 and Gata4(10Liberatore C.M. Searcy-Schrick R.D. Yutzey K.E. Dev. Biol. 2000; 223: 169-180Google Scholar). Over-expression of Tbx5 under the control of the β-myosin heavy chain promoter in mice inhibits ventricular chamber development and loss of ventricular-specific gene expression (10Liberatore C.M. Searcy-Schrick R.D. Yutzey K.E. Dev. Biol. 2000; 223: 169-180Google Scholar). Homozygous knockout mice deficient in Tbx5 die early in embryogenesis at D9.5, and heterozygous mice showed the forelimb and congenital heart defects (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). These results indicate thatTbx5 is critical to development of the heart and skeletal structures. Mutations in Tbx5 have been found to cause Holt-Oram syndrome, an autosomal-dominant disease characterized by upper limb malformations and congenital heart defects including atrial and ventricular septal defects or cardiac conduction disease (2Basson C.T. Bachinsky D.R. Lin R.C. Nat. Genet. 1997; 15: 30-35Google Scholar, 3Li Q.Y. Newbury-Ecob R.A. Terrett J.A. Nat. Genet. 1997; 15: 21-29Google Scholar, 14Holt M. Oram S. Br. Heart J. 1960; 22: 236Google Scholar,15Newbury-Ecob R.A. Leanage R. Raeburn J.A. Young I.D. J. Med. Genet. 1996; 33: 300-307Google Scholar). To date, twenty-four different Tbx5 mutations (15Newbury-Ecob R.A. Leanage R. Raeburn J.A. Young I.D. J. Med. Genet. 1996; 33: 300-307Google Scholar, 16Basson C.T. Huang T. Lin R.C. Bachinsky D.R. Weremowicz S. Vaglio A. Bruzzone R. Quadrelli R. Lerone M. Romeo G. Silengo M. Pereira A. Krieger J. Mesquita S.F. Kamisago M. Morton C.C. Pierpont M.E. Muller C.W. Seidman J.G. Seidman C.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2919-2924Google Scholar, 17Cross S.J. Ching Y.H. Li Q.Y. Armstrong-Buisseret L. Spranger S. Lyonnet S. Bonnet D. Penttinen M. Jonveaux P. Leheup B. Mortier G. van Ravenswaaij C. Gardiner C.A. J. Med. Genet. 2000; 37: 785-787Google Scholar, 18Yang J. Hu D. Xia J. Yang Y. Ying B. Hu J. Zhou X. Am. J. Med. Genet. 2000; 92: 237-240Google Scholar, 19Vaughan C.J. Basson C.T. Am. J. Med. Genet. 2000; 97: 304-309Google Scholar) have been described in patients with Holt-Oram syndrome. ManyTbx5 mutations are nonsense, frameshift, and splice site mutations, or large chromosomal rearrangements (translocations, deletions) that are expected to produce truncated TBX5 proteins or no TBX5 at all (e.g. nonsense-mediated mRNA decay, translocation in intron 1). Based on these findings, Tbx5haploinsufficiency has been proposed as a mechanism underlying pathogenesis of Holt-Oram syndrome. This hypothesis is further supported by the finding that heterozygousTbx5del/+ mice modeled Holt-Oram syndrome (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). It is interesting to note that increased Tbx5 dosage, such as a chromosome 12q2 duplication, also results in Holt-Oram syndrome (19Vaughan C.J. Basson C.T. Am. J. Med. Genet. 2000; 97: 304-309Google Scholar), suggesting that Tbx5 dosage is critical to the development of the heart and limbs. However, seven missense mutations in the Tbx5 gene, which would not be expected to alter the dosage of Tbx5, have been reported in patients with Holt-Oram syndrome. It remains to be determined whether theseTbx5 missense mutations are functional mutations, and if they are, what are the molecular pathogenic mechanisms by which these mutations act? Biochemical studies have demonstrated that TBX5 protein interacts with the homeobox transcription factor NKX2.5 to synergistically transactivate expression of target genes (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, 20Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar). The homeodomain of NKX2.5 was shown to be necessary and sufficient for its interaction with TBX5, although the NK domain (a17 amino acid domain specific to NK-class proteins) located C-terminal to the homeodomain can significantly enhance the interaction (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, 20Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar). For TBX5, the region including the N-terminal domain and the N-terminal portion of the T-domain (amino acids 1–90) is sufficient for its association with NKX2.5 (20Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar). The target genes identified for Tbx5 include the genes for atrial natriuretic factor (ANF)1 (20Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar) and connexin 40 (Cx40) (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, 20Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar). Synergistic activation of bothANF and Cx40 promoters by TBX5 and NKX2.5 were demonstrated (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, 20Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar). The promoters of ANF andCx40 harbor binding sites for both TBX5 and NKX2.5, and these sites were shown to be capable of binding to both transcriptional factors (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, 20Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar). Using an in vitro selection procedure with double-stranded random 26-mer oligonucleotides, a consensus sequence for TBX5 binding ((A/G)GGTGT(C/T/G)(A/G)) was identified (21Ghosh 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). This consensus sequence is part of a half-site from the Brachyury T-box target sequence (21Ghosh 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 can bind to either a single site or paired non-palindromic sites in vitro (21Ghosh 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). In the present study, we have used a biochemical system comprising of TBX5, NKX2.5, and the ANF promoter to characterize the TBX5 missense mutations identified in Holt-Oram syndrome patients. We demonstrate that the TBX5 missense mutations are functional mutations that cause a spectrum of defects in DNA binding, transcription activation, interaction with NKX2.5, synergistic transactivation with NKX2.5, and nuclear localization. These results also into of TBX5. The region from to from the transcription site of the ANF promoter was and into the in The was into the in the (20Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar). of were with and the other the as well as for TBX5. The expression for hemagglutinin and protein were as described from (20Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar). The missense mutations in Tbx5 were into the wild type by and by DNA The Tbx5 was also into the in the expression for TBX5. or were to in with and with and of DNA for the expression of DNA for the gene, and of control were and The of was by of cellular were by and to a The was with as the and the were used to the protein assay was a assay to the activity expressed from was used to the The were in are expressed as The site was by double-stranded with and and used in The site was with of and of TBX5 protein the system in of binding The was from DNA The were at for 40 and to For in vivo protein-protein were with expression for TBX5 and NKX2.5 for The were with in a and with I. cell was with The cell was with of and for at with the the proteins were with by and a The was with an and protein was by to the For in vitro protein-protein or proteins were in and on of and were with in TBX5 for at with proteins were from with glutathione and by with a and were on chamber at a of and at and were in in and with the in at The the TBX5 The a was and at for were with and were under a with The were with the for the of the protein in the nucleus cytoplasm. binding of TBX5 transcriptional factor to DNA is for activation of its target genes including the atrial natriuretic To the mechanism by which TBX5 missense mutations cause Holt-Oram syndrome, we the of mutant TBX5 proteins to bind to its target DNA-binding site Seven missense mutations in TBX5 were and they include and at the G80R, G169R, R237Q, and in the T-domain for DNA binding, and in the 1). The promoter region from the transcription contains a and this site was as of single The site was and used in with in TBX5 shown in the wild type TBX5 protein can bind to DNA and a specific with site dramatically reduced the and a DNA a sequence to the site to not These that the binding of TBX5 to the binding site is effect of missense mutations of TBX5 on DNA-binding The wild type and mutant TBX5 proteins were in vitro the The site is a double-stranded DNA to the region from to from the ANF transcriptional site no TBX5 wild type G80R, G169R, and S252I, TBX5 with missense a protein the of of wild type and mutant TBX5 proteins used in no TBX5 expression wild type Q49K, I54T, G80R, G169R, R237Q, R237W, and mutant TBX5, with of wild type TBX5 and mutants and the of the in was TBX5 The for specific binding of wild type TBX5 and mutants and is and mutations in the including G80R, R237Q, and R237W, dramatically reduced the binding of TBX5 to DNA The DNA-binding of wild type and mutant and TBX5 were determined of shown in and both mutant proteins have to DNA wild type TBX5. Mutations and moderate effect on DNA binding of TBX5. In was no in DNA binding between the wild type TBX5 and mutants and protein showed that both wild type and mutant TBX5 proteins were at results that a functional in DNA binding is the molecular mechanism for TBX5 mutations R237Q, and but not a mechanism for all TBX5 can transcription of its target but in the of NKX2.5, synergy in transcriptional activation by these two proteins was (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, 20Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar). We the functional effect of TBX5 mutations on transcriptional activation a gene in which promoter from to was to the luciferase The gene was with wild type or mutant TBX5 expression into activity was and expressed as luciferase shown in expression of wild type TBX5 transcription of mutations, including G80R, R237Q, and R237W, reduced transcription activation by TBX5, with that these mutations reduced DNA binding of TBX5. In TBX5 mutations Q49K, I54T, G169R, and effect on transcriptional activation by TBX5 showed that both wild type and all mutant TBX5 proteins were expressed in We whether the missense mutations in TBX5 the synergistic transactivation between TBX5 and NKX2.5. shown in either wild type TBX5 or NKX2.5 expression of the ANF but of TBX5 and NKX2.5 into showed synergistic activation of the ANF promoter as reported B.G. Logan M. Davis N. Levi T. Tabin C.J. Seidman J.G. Seidman C.E. Dev. Biol. 1999; 211: 100-108Google C.T. Huang T. Lin R.C. Bachinsky D.R. Weremowicz S. Vaglio A. Bruzzone R. Quadrelli R. Lerone M. Romeo G. Silengo M. Pereira A. Krieger J. Mesquita S.F. Kamisago M. Morton C.C. Pierpont M.E. Muller C.W. Seidman J.G. Seidman C.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2919-2924Google Scholar). However, all seven missense mutations of TBX5 not produce such synergistic effect with NKX2.5 all seven TBX5 missense mutations loss of synergistic activation of transcription by TBX5 and NKX2.5, we determined whether these mutations the interaction between these two transcriptional We used both in vivo and in vitro protein-protein interaction For the in vivo TBX5 and NKX2.5 with at the were in showed that both TBX5 and NKX2.5 were expressed in the The protein was with bind TBX5 or The protein or to was and by with an to the of NKX2.5. interaction between wild type TBX5 and NKX2.5 was was for TBX5 proteins with or were found for mutants I54T, G80R, R237Q, R237W, or These results that seven missense mutations studies here dramatically reduce the interaction between TBX5 and NKX2.5. We also used assay to the interaction of TBX5 mutants with NKX2.5. protein was to and TBX5. We TBX5 to and type TBX5, but of the mutant TBX5, showed interaction with NKX2.5 These results further the in vivo that missense mutations the interaction between TBX5 and NKX2.5. TBX5 is a transcription and is expected to be localized in the We that missense mutations in TBX5 may cause of the protein and in protein defects TBX5 from its function as a transcription To this we expressed wild type and mutant TBX5 proteins with a into and studied cellular localization of TBX5 by The of the nuclear and protein was and shown in B. wild type TBX5 was localized completely into the nucleus TBX5 a in with of the protein in the nucleus and in the the in TBX5 with of the in the and in the nucleus The of mutations G80R, G169R, R237W, and moderate effect on nuclear localization of TBX5 with found in both the nucleus and results were with or not This study that the seven TBX5 missense mutations identified in Holt-Oram syndrome patients are functional mutations with a spectrum of We the functional effect of these missense mutations on DNA binding and transcriptional activation by TBX5 as well as synergistic activation by the interaction between TBX5 and NKX2.5. Based on TBX5 missense mutations can be into two different functional The group of mutations G80R, R237W, and R237Q, which effect by the DNA-binding function of TBX5. DNA binding to reduction in transcription activation and loss of synergy between TBX5 and NKX2.5 in transactivation of target The second group of mutations Q49K, I54T, G169R, and S252I, which no or moderate effect on DNA This group of mutations not the transcriptional activation by TBX5, but they dramatic reduction in the interaction between TBX5 and NKX2.5 and the complete loss of synergy between TBX5 and NKX2.5 in transcriptional study the of all missense mutations identified in TBX5 to studies reported of a missense mutations in TBX5. (21Ghosh 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) reported of the effect of mutations, G80R, G169R, and S252I, on DNA binding to an in vitro with two sites a truncated of TBX5 (amino acids These not assay the effect of these mutations on transcription Mutations and were found to DNA binding of TBX5, mutations and DNA-binding activity (21Ghosh 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). DNA binding with the TBX5 and a site in the results on mutations G80R, G169R, R237Q, and (20Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar) the effect of mutations and on transcription activity of promoter but not effect on DNA reduction of transcriptional activation activity of TBX5 and loss of synergistic activation with NKX2.5. R237Q, which DNA binding, on transcriptional activation by TBX5 or synergistic activation by TBX5 and NKX2.5 (20Hiroi Y. Kudoh S. Monzen K. Ikeda Y. Yazaki Y. Nagai R. Komuro I. Nat. Genet. 2001; 28: 276-280Google Scholar) In study, results were for we found dramatic effect by R237Q, which is the DNA binding on this we the of seven missense mutations on the interaction and synergy in transactivation between TBX5 and NKX2.5. study further of molecular mechanisms of TBX5 mutant protein seven missense mutations the mutations in the G80R, R237W, and R237Q, dramatically reduced binding of TBX5 to the DNA further that the T-domain is the DNA-binding domain. It is that TBX5 with G169R, also at a highly conserved in the binding activity, suggesting that not all amino acid in the T-domain are for DNA Based on the from the T-domain of protein C.W. B.G. Nature. 1997; Scholar), both and are located to the critical amino acid for DNA binding, is to the and to the of the critical DNA-binding The different in to critical DNA-binding may the of binding activity to DNA for mutations at and from that for studies the of TBX5 and the N-terminal part of the T-domain (amino acids 1–90) as the binding site for NKX2.5 (21Ghosh 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). results that mutations Q49K, I54T, and the interaction and synergistic transcriptional activity between TBX5 and NKX2.5 further that the of TBX5 is in its interaction with NKX2.5. also other including and as critical in the interaction and synergistic transcription activation between TBX5 and NKX2.5. studies and studies with the TBX5 and NKX2.5 all critical in of with TBX5 showed that TBX5 proteins with the missense mutations were localized in both the nucleus and cytoplasm. The molecular mechanism for such defects is We that TBX5 missense mutations cause of TBX5, which the for TBX5 to be into the This of mutant TBX5 proteins to the of mutant TBX5 in both the nucleus and cytoplasm. of TBX5 into the these from transcriptional activation These results demonstrate nuclear localization of mutant TBX5 as a molecular mechanism for TBX5 missense protein defects have been reported for other including syndrome L. C. R. Schwartz P.J. Hum. Mol. Genet. 1999; Scholar) and syndrome G. S. C. M. 2000; Scholar). In we have shown that the missense mutations in TBX5 in this study are functional mutations that cause a spectrum of biochemical and cellular results indicate molecular mechanisms underlying the pathogenesis of Holt-Oram syndrome. TBX5 mutations cause a dramatic in its DNA-binding activity, which to reduced transcription activation by TBX5 and the loss of synergy between TBX5 and NKX2.5. TBX5 mutations not the DNA binding and transcriptional activation of TBX5 but cause the complete loss of synergy between TBX5 and NKX2.5 in transcription activation to dramatically reduced interaction between TBX5 and NKX2.5. nuclear localization is a by all missense mutations studied All these mechanisms are likely central to the pathogenesis of Holt-Oram syndrome. are with the hypothesis that the missense mutations in TBX5 by a We for and Komuro for the of expression