Myocardin and Tbx5 interact directly.

<p>(<b>A</b>) COS-7 cells were transfected with expression plasmids encoding Flag-tagged Tbx5 and Myc-tagged myocardin as indicated. Tbx5 was immunoprecipitated (IP) by anti-Flag antibodies, and anti-Myc antibodies were used to detect the presence of myocardin in the immunoprecipitates by Western blot (WB) analysis (upper panel). One-fifteenth of cell extracts were directly Western blotted (WB) to detect the presence of myocardin and Tbx5 proteins by anti-Myc or anti-Flag antibodies, respectively (middle and lower panels, respectively). (<b>B</b>) An expression plasmid encoding full-length myocardin (1-935) fused to GAL4 DNA binding domain (Gal4Myocd) and the pL8G5-luciferase reporter were transfected in the absence or presence of Tbx5 expression plasmid into COS-7 cells and luciferase activity was determined. pcDNA-Myocardin (Myocd) and pcDNA-Tbx5 (Tbx5) were used as controls. Luciferase activity was determined 48 hr after transfection and was presented as fold of activation in which the control was assigned a value of 1. Data represent the mean ± s.d. from at least three independent experiments in duplicate. *P<0.05.</p

Synergistic activation of cardiac but not smooth muscle genes by myocardin and Tbx5.

<p>Luciferase reporters directed by cardiac and smooth muscle gene promoters were co-transfected with myocardin and Tbx5 expression plasmids in COS-7 cells (A-D) or neonatal rat cardiomyocytes (E). (<b>A</b>) Synergistic activation of the ANF promoter by myocardin and Tbx5. (<b>B</b>) Synergistic activation of the α-MHC promoter by myocardin and Tbx5. (<b>C</b>) The effect of myocardin and Tbx5 on the SM22 promoter. Myocardin activates the promoter reporter but there is no synergy between myocardin and Tbx5. (<b>D</b>) The effect of myocardin and Tbx5 on the SM-MHC promoter. Myocardin activates the promoter reporter but there is no synergy between myocardin and Tbx5. (<b>E</b>) Myocardin and Tbx5 synergistically activate the ANF promoter in neonatal rat cardiomyocytes. In all experiments, the luciferase activity was determined 48 hr after transfection and was presented as fold of activation in which the control was assigned a value of 1. Data represent the mean ± s.d. from at least three independent experiments in duplicate. *P<0.05.</p

The Tbx5 G80R mutant abolished the synergy between Tbx5 and myocardin to activate cardiac gene expression.

<p>COS-7 cells were transfected with an ANF promoter luciferase reporter and expression plasmids for myocardin, Tbx5 or indicated Tbx5 mutants and luciferase activity measured. Luciferase activity was determined 48 hr after transfection and was presented as relative luciferase activity in which the control was assigned a value of 1. Data represent the mean ± s.d. from at least three independent experiments in duplicate. *P<0.01.</p

The TBEs are required for the synergistic activation of ANF promoter by myocardin and Tbx5.

<p>(<b>A</b>) COS-7 cells were transfected with myocardin and Tbx5 expression plasmids and the indicated ANF promoter luciferase reporters in which the CArG boxes and the T-box factor-Binding Elements (TBEs) were indicated, and luciferase activity measured. (<b>B</b>) COS-7 cells were transfected with an ANF promoter luciferase reporter (ANF115) or a mutant reporter in which the TBE was mutated (ANF155m) together with myocardin and Tbx5 expression plasmids and luciferase activity measured. (<b>C</b>) COS-7 cells were transfected with an ANF promoter luciferase reporter (ANF115) (left three lanes) or a mutant reporter in which the CArG box was mutated (right three lanes) together with myocardin and Tbx5 expression plasmids and luciferase activity measured. (<b>D</b>) COS-7 cells were transfected with an ANF promoter luciferase reporter (ANF638) (left three lanes) or a mutant reporter in which both CArG boxes were mutated (ANF638m) (right three lanes) together with myocardin and Tbx5 expression plasmids and luciferase activity measured. (<b>E</b>) A luciferase reporter controlled by four tandemly repeats of a consensus Tbx binding elements (TBE) was transfected into COS-7 cells with myocardin and/or Tbx5 expression plasmids and luciferase activity measured. In all the experiments, the luciferase activity was determined 48 hr after transfection and was presented as relative luciferase activity in which the control was assigned a value of 1. Data represent the mean ± s.d. from at least three independent experiments in duplicate. *P<0.05.</p

Tbx5 and myocardin do not form a stable ternary complex on CArG-box or TBE.

<p>Myc-tagged Tbx5 and Flag-tagged myocardin proteins were expressed by <i>in vitro</i> transcription and translation and incubated with radiolabeled probes. (<b>A</b>) Electrophoresis mobility shift assays were performed with a ³²P-labeled TBE probe for Myc-tagged Tbx5 in the presence and absence of increasing amount of Flag-myocardin. Anti-Myc antibodies were applied for supershift. The Tbx5/TBE complexes and the supershift complex were indicated. (<b>B</b>) Electrophoresis mobility shift assays were performed with a ³²P-labeled CArG probe for Flag-myocardin in the presence and absence of SRF, or in the presence and absence of Myc-Tbx5. Anti-SRF antibodies were applied for supershift. The SRF/CArG complexes (SRF), the Myocd/SRF/CArG ternary complexes (SRF+Myocd) and supershift complex (Supershift) were indicated.</p

Mapping of the myocardin and Tbx5 interaction domains.

<p>(<b>A</b>) COS-7 cells were transfected with expression plasmids encoding Myc-tagged Tbx5 and a collection of Flag-tagged myocardin deletion mutants. Tbx5 was immunoprecipitated (IP) by anti-Myc antibodies, and anti-Flay antibodies were used to detect the presence of myocardin in the immunoprecipitates by immunoblot analysis (IB) (upper panel). One-fifteenth of cell extracts were directly immunoblotted (IB) to detect the presence of myocardin and Tbx5 proteins by anti-Flag or anti-Myc antibodies, respectively (middle and lower panels, respectively). (<b>B</b>) A schematic summary of myocardin and Tbx5 protein interaction domains. Myocardin domains abbreviated as follows: B, basic domain; Q, a stretch of glutamine residues; SAP, <u>S</u>AF A/B, <u>A</u>cinus, <u>P</u>IAS domain; TAD, transactivation domain. (<b>C</b>) COS-7 cells were transfected with expression plasmids encoding Flag-tagged myocardin and a collection of Myc-tagged Tbx5 deletion mutants. Myocardin was immunoprecipitated (IP) by anti-Flag antibodies, and anti-Myc antibodies were used to detect the presence of Tbx5 in the immunoprecipitates by immunoblot (IB) analysis (upper panel). One-fifteenth of cell extracts were directly immunoblotted (IB) to detect the presence of Tbx5 and myocardin proteins by anti-Myc or anti-Flag antibodies, respectively (middle and lower panels, respectively). (<b>D</b>) A schematic summary of myocardin and Tbx5 protein interaction domains.</p