FIGURE 3.

HP1α and HP1β, but not HP1γ, interact directly with MyoD in vitro via their chromoshadow domain and MyoD transactivating domain. A, HP1α and HP1β interact directly with MyoD in a GST pulldown assay. Equivalent amounts of bacterially produced untagged MyoD were incubated with GST beads (lane 4) or GST-HP1 beads (lanes 1–3). MyoD was detected by Western blotting using an anti-MyoD antibody (upper panel), and GST fusions using an anti-GST antibody (lower panel). IB, immunoblot. B, unlike MyoD, myogenin does not interact with HP1 proteins. Myogenin and MyoD were in vitro translated in the presence of radioactive [³⁵S] methionine (inputs on lanes 1 and 14, respectively). MyoD or myogenin were incubated with equivalent amounts of either GST or GST-HP1 proteins, as indicated. GST pulldown was then conducted as described under “Experimental Procedures,” except that at the end of the experiment, the radiolabeled proteins were detected by autoradiography. Lanes 1–8, GST pulldown experiment with [³⁵S]myogenin; lanes 9–14, with [³⁵S]MyoD. C, the chromoshadow domain of HP1α is required for interaction with MyoD. Equivalent amounts of bacterially produced MyoD were incubated with GST-HP1α deletion mutants on beads (lanes 2–5) or GST-HP1α wild type (lane 1). MyoD was detected by Western blotting using an anti-MyoD antibody (upper panel) and GST fusions using an anti-GST antibody (lower panel). D, HP1β interacts with MyoD wild type and with MyoD deletion mutants containing the C-terminal domain. Top panel, schematic diagram of MyoD functional domains. TAD, transactivation domain. Lower panels, wild type MyoD was detected using anti-FLAG antibody, and its deletion mutants were detected using anti-HA antibody (upper panels). GST and GST-HP1β were detected using anti-GST antibody (lower panel). E, Western blotting using anti-FLAG and anti-HA to verify the expression level of MyoD and its deletion mutants in HeLa cells used in D*, specific bands.