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Tbx1 regulates the BMP-Smad1 pathway in a transcription independent manner.

Fulcoli FG, Huynh T, Scambler PJ, Baldini A - PLoS ONE (2009)

Bottom Line: Tbx1 interferes with Smad1 to Smad4 binding, and a mutation of Tbx1 that abolishes transactivation, does not affect Smad1 binding nor does affect the ability to suppress Smad1 activity.In addition, a disease-associated mutation of TBX1 that does not prevent transactivation, prevents the TBX1-SMAD1 interaction.Our data indicate that Tbx1 interferes with Bmp/Smad1 signaling and provide strong evidence that a T-box transcription factor has functions unrelated to transactivation.

View Article: PubMed Central - PubMed

Affiliation: Telethon Institute of Genetics and Medicine, Naples, Italy.

ABSTRACT
Tbx1 is a T-box transcription factor implicated in DiGeorge syndrome. The molecular function of Tbx1 is unclear although it can transactivate reporters with T-box binding elements. We discovered that Tbx1 binds Smad1 and suppresses the Bmp4/Smad1 signaling. Tbx1 interferes with Smad1 to Smad4 binding, and a mutation of Tbx1 that abolishes transactivation, does not affect Smad1 binding nor does affect the ability to suppress Smad1 activity. In addition, a disease-associated mutation of TBX1 that does not prevent transactivation, prevents the TBX1-SMAD1 interaction. Expression of Tbx1 in transgenic mice generates phenotypes similar to those associated with loss of a Bmp receptor. One phenotype could be rescued by transgenic Smad1 expression. Our data indicate that Tbx1 interferes with Bmp/Smad1 signaling and provide strong evidence that a T-box transcription factor has functions unrelated to transactivation.

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Transactivation ability of Tbx1 is not required for Smad pathway suppression; Tbx1 interferes with Smad1/Smad4 binding.a) A luciferase assay showing the inability of the TBX1G145R mutant to transactivate a T-box reporter construct in Jeg3 cells. Error bars indicate the standard error mean. b) A luciferase assay with a SMAD reporter showing that the mutant is capable of suppressing SMAD transactivation. c) Western blot analyses of nuclear extracts from C2C12 transfected with Tbx1 and SMAD1-flag expression vectors (as indicated). The top two rows are samples immunoprecipitated with an anti-flag antibody. The bottom two rows are non-immunoprecipitated nuclear extracts from the same samples. Note the strong reduction of Smad4 co-immunoprecipitated with Smad1 in the presence of transfected TBX1.
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pone-0006049-g003: Transactivation ability of Tbx1 is not required for Smad pathway suppression; Tbx1 interferes with Smad1/Smad4 binding.a) A luciferase assay showing the inability of the TBX1G145R mutant to transactivate a T-box reporter construct in Jeg3 cells. Error bars indicate the standard error mean. b) A luciferase assay with a SMAD reporter showing that the mutant is capable of suppressing SMAD transactivation. c) Western blot analyses of nuclear extracts from C2C12 transfected with Tbx1 and SMAD1-flag expression vectors (as indicated). The top two rows are samples immunoprecipitated with an anti-flag antibody. The bottom two rows are non-immunoprecipitated nuclear extracts from the same samples. Note the strong reduction of Smad4 co-immunoprecipitated with Smad1 in the presence of transfected TBX1.

Mentions: To determine whether the interaction between Tbx1 and Smad1 has functional consequences, we overexpressed Tbx1 and assessed the transactivation ability of Smad1. We carried out a luciferase assay in Cos-7 and C2C12 cells with the Smad-responsive reporter NTK-tetramer-luc, which contains four copies of a Smad consensus-binding element [21]. The reporter was activated by transfection of a SMAD1 expression vector in Cos7 cells or by adding BMP4 to the culture media of C2C12 cells (Figure 2f and Figure S3, respectively). In both cases we observed that increasing amounts of transiently transfected TBX1 is capable of suppressing Smad1- or BMP4-induced activation of the reporter (Figure 2f and Figure S3a). TBX1 expression did not affect the level of P-Smad1/5/8, Smad1 or the inhibitory Smad6 (Figure S4). To assess the role of transcriptional activity for the Bmp-Smad suppression activity, we expressed a mutant isoform of TBX1 (G145R) that carries a T-box mutation, which prevents DNA binding [20]. As shown in Fig. 3a, TBX1G145R was unable to transactivate a T-box reporter, but it was still able to bind Smad1 (Figure 2e) and to suppress the Smad1 signaling in the luciferase assay, indicating that the anti-Smad activity of TBX1 is independent from transcriptional activity. We obtained the same results by activating the Smad reporter with BMP4 (Figure S3b).


Tbx1 regulates the BMP-Smad1 pathway in a transcription independent manner.

Fulcoli FG, Huynh T, Scambler PJ, Baldini A - PLoS ONE (2009)

Transactivation ability of Tbx1 is not required for Smad pathway suppression; Tbx1 interferes with Smad1/Smad4 binding.a) A luciferase assay showing the inability of the TBX1G145R mutant to transactivate a T-box reporter construct in Jeg3 cells. Error bars indicate the standard error mean. b) A luciferase assay with a SMAD reporter showing that the mutant is capable of suppressing SMAD transactivation. c) Western blot analyses of nuclear extracts from C2C12 transfected with Tbx1 and SMAD1-flag expression vectors (as indicated). The top two rows are samples immunoprecipitated with an anti-flag antibody. The bottom two rows are non-immunoprecipitated nuclear extracts from the same samples. Note the strong reduction of Smad4 co-immunoprecipitated with Smad1 in the presence of transfected TBX1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2698216&req=5

pone-0006049-g003: Transactivation ability of Tbx1 is not required for Smad pathway suppression; Tbx1 interferes with Smad1/Smad4 binding.a) A luciferase assay showing the inability of the TBX1G145R mutant to transactivate a T-box reporter construct in Jeg3 cells. Error bars indicate the standard error mean. b) A luciferase assay with a SMAD reporter showing that the mutant is capable of suppressing SMAD transactivation. c) Western blot analyses of nuclear extracts from C2C12 transfected with Tbx1 and SMAD1-flag expression vectors (as indicated). The top two rows are samples immunoprecipitated with an anti-flag antibody. The bottom two rows are non-immunoprecipitated nuclear extracts from the same samples. Note the strong reduction of Smad4 co-immunoprecipitated with Smad1 in the presence of transfected TBX1.
Mentions: To determine whether the interaction between Tbx1 and Smad1 has functional consequences, we overexpressed Tbx1 and assessed the transactivation ability of Smad1. We carried out a luciferase assay in Cos-7 and C2C12 cells with the Smad-responsive reporter NTK-tetramer-luc, which contains four copies of a Smad consensus-binding element [21]. The reporter was activated by transfection of a SMAD1 expression vector in Cos7 cells or by adding BMP4 to the culture media of C2C12 cells (Figure 2f and Figure S3, respectively). In both cases we observed that increasing amounts of transiently transfected TBX1 is capable of suppressing Smad1- or BMP4-induced activation of the reporter (Figure 2f and Figure S3a). TBX1 expression did not affect the level of P-Smad1/5/8, Smad1 or the inhibitory Smad6 (Figure S4). To assess the role of transcriptional activity for the Bmp-Smad suppression activity, we expressed a mutant isoform of TBX1 (G145R) that carries a T-box mutation, which prevents DNA binding [20]. As shown in Fig. 3a, TBX1G145R was unable to transactivate a T-box reporter, but it was still able to bind Smad1 (Figure 2e) and to suppress the Smad1 signaling in the luciferase assay, indicating that the anti-Smad activity of TBX1 is independent from transcriptional activity. We obtained the same results by activating the Smad reporter with BMP4 (Figure S3b).

Bottom Line: Tbx1 interferes with Smad1 to Smad4 binding, and a mutation of Tbx1 that abolishes transactivation, does not affect Smad1 binding nor does affect the ability to suppress Smad1 activity.In addition, a disease-associated mutation of TBX1 that does not prevent transactivation, prevents the TBX1-SMAD1 interaction.Our data indicate that Tbx1 interferes with Bmp/Smad1 signaling and provide strong evidence that a T-box transcription factor has functions unrelated to transactivation.

View Article: PubMed Central - PubMed

Affiliation: Telethon Institute of Genetics and Medicine, Naples, Italy.

ABSTRACT
Tbx1 is a T-box transcription factor implicated in DiGeorge syndrome. The molecular function of Tbx1 is unclear although it can transactivate reporters with T-box binding elements. We discovered that Tbx1 binds Smad1 and suppresses the Bmp4/Smad1 signaling. Tbx1 interferes with Smad1 to Smad4 binding, and a mutation of Tbx1 that abolishes transactivation, does not affect Smad1 binding nor does affect the ability to suppress Smad1 activity. In addition, a disease-associated mutation of TBX1 that does not prevent transactivation, prevents the TBX1-SMAD1 interaction. Expression of Tbx1 in transgenic mice generates phenotypes similar to those associated with loss of a Bmp receptor. One phenotype could be rescued by transgenic Smad1 expression. Our data indicate that Tbx1 interferes with Bmp/Smad1 signaling and provide strong evidence that a T-box transcription factor has functions unrelated to transactivation.

Show MeSH
Related in: MedlinePlus