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

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.

Figure 1. Identification of Tbx1 interactors using affinity purification.

Mouse P19-Cl6 embryonic carcinoma cells were stably transfected with plasmid expressing TEV-protein-A alone or C-terminally fused to Tbx1 and induced to differentiate with 1% DMSO. a) Constructs used to generate stably transfected P19CL6 cell clones. b) Western blot analysis of P19-Tbx1-PA and P19-PA (control) cell extracts. Proteins were separated by gel electrophoresis on 10% SDS-PAGE gel and immunoblotted with human IgG F(c), which recognize protein A. c) Colloidal coomassie-stained 10% SDS-PAGE gel containing nuclear extracts from P19-Tbx1-PA (lane 1) and P19-PA (lane 3) cells affinity purified by binding to IgG-Sepharose and then enzymatically eluted by cleavage with TEV protease. Compared with non-purified nuclear extract from Tbx1-TEV-PA cells (lane 2).

Figure 2. Tbx1 interacts with Smad1.

a) Western blot analysis of affinity-purified nuclear extracts from P19-Tbx1-PA (lane 1) and P19-PA (lane 3) cells, compared non-purified nuclear extracts of P19-Tbx1-PA cells (lane 2), using an antibody anti-Phospho-SMAD1/5/8. b) Western blot analyses of reciprocal coimmunoprecipitation (IP) experiments using the antibodies indicated. NIH3T3 cells were co-transfected transiently with Tbx1-cmyc and Smad1-Flag expression vectors. c) Western blot analyses of nuclear extracts from wild type E9.5 mouse embryos immunoprecipitated with an anti-Tbx1 antibody (lanes 1 and 2) or with anti-rabbit IgG (lane 3) and revealed with an anti-SMAD1 antibody (lane 1 and 3) and an anti-Phospho-SMAD1/5/8 antibody (lane 2). d) Immunoblotting with anti-Smad1 antibody of nuclear extracts of wild type mouse embryos (E9.5) coimmunoprecipitated with anti-Tbx1 (NE-IP), total nuclear extracts (NE), total cytoplasmic extracts (CE), and cytoplasmic extracts coimminoprecipitated with anti-Tbx1 (CE-IP). e) Coimmunoprecipitation of WT TBX1, TBX1^G145R, TBX1^F148Y, TBX1^G310S and Flag-SMAD1 transiently transfected in NIH 3T3 cells. The TBX1^G145R and TBX1^F148Y mutants physically interact with Smad1, while TBX1^G310S is unable to bind Smad1 indicating that Glycine 310 is important for this interaction. f) Luciferase assay using Cos7 cells transfected with the SMAD-responsive reporter NTK-tetramer-luc. Transfected SMAD1 activates the reporter while increasing amounts (5 to 100 ng) of co-transfected TBX1 suppresses it.

Figure 3. 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 TBX1^G145R 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.

Figure 4. Ectopic expression of Tbx1 in mouse embryos suppresses the Smad1 pathway in vivo.

a–a') A Ap2a^IRESCre/+;Coet embryo (a') at E18.5 shows bilateral cleft lip, compared with a control littermate (a). b–b') Three-dimensional reconstruction from digital images of histological sections of E16.5 hearts from control (b) and Ap2a^IRESCre/+;Coet embryos. The cavities of the right (RV) and left (LV) ventricles, as well as the great arteries are shown in red. Note the large ventricular septal defect (VSD), and the common origine of the aorta and pulmonary arteries from the right ventricle, a condition known as double outlet right ventricle (DORV). (c–f') Whole-mount in situ hybridization analysis of Msx1 expression in WT (c, d, e, f) and Ap2a^IRESCre/+;Coet embryos (c', d', e', f') at E9.5 (c–d') and E10.5 (e–f'). Mutant embryos show reduced expression in the maxillary region (white arrows) and in the second pharyngeal arch (black arrows). (g) Cleft lip present in Ap2a^IRESCre/+;Coet embryos (compare with panel a) is rescued by the FSMAD1 transgene.