Figure 7.

TGF-β and Smad3 repress the transcriptional activity of MyoD by limiting its access to the E2A class of bHLH protein partners. (A,B) Increased E12 expression counteracts Smad3-mediated repression of transcription by MyoD. 10T1/2 cells were transfected with the 4R-tk-Luc (A) or MCK-Luc (B) reporter plasmid and expression plasmids for MyoD and Smad3, as well as increasing amounts of an E12 expression plasmid. Luciferase expression levels in the absence or presence of TGF-β were quantitated as in Fig. 4. (C,D) The activity of a covalently tethered MyoD∼E47 dimer is resistant to repression by TGF-β/Smad3 signaling. 10T1/2 cells were transfected with the 4R-tk-Luc (C) or MCK-Luc (D) reporter, together with expression plasmids for MyoD∼E47 and Smad2 or Smad3. Luciferase activities were scored relative to the value of MyoD∼E47 alone in the absence of TGF-β. Cotransfection of equivalent quantities of Smad3 and MyoD expression plasmid resulted in a repression of the MyoD activity by > 80% (see Fig. 4). (E) Mammalian two-hybrid analyses of the association between MyoD and E12. 10T1/2 cells were transfected with the indicated expression plasmids and a Gal4-responsive reporter Gal-Luc in the presence of increasing amounts of Smad3 expression plasmids. The normalized luciferase activities, as a consequence of the interaction between Gal4-E12 and MyoDbHLH-VP16 fusion proteins, are shown. (F) Evaluating the MyoD/E12 interaction by coimmunoprecipitation. HA-tagged MyoD and Myc-tagged E12 were expressed in 10T1/2 cells in the presence or absence of 2ng/mL TGF-β and/or cotransfected Smad3. Cell lysates were subjected to anti-HA immunoprecipitation, and MyoD and coprecipitated E12 in the protein complexes were detected by immunoblotting, using anti-HA (top) or anti-Myc antibodies (middle), respectively. The expression of E12 in the cell lysates was monitored by immunoblotting using anti-Myc antibody (bottom).