The domains of MyoD and Runx1 involved in their interaction.
A. The bHLH domain and the C-terminal transactivating domain of MyoD are involved in the interaction with Runx1 and CBFβ. Top panel, Schematic diagram of MyoD functional domains, TAD: transactivation domain, bHLH: basic Helix Loop Helix. Lower panel, Runx1 and CBFβ interact with MyoD wild-type and with MyoD deletion mutant containing the bHLH and the C-terminal domain. Lower panel, expression vectors for wild-type MyoD and its deletion mutants were transfected in HEK 293 cells using Calcium phosphate precipitation as described in Material and Methods section. 48 h post-transfection, cells were lysed and lysates were subjected to anti-HA immunoprecipitation. Precipitates were then analyzed by western blotting using with the indicated antibodies (WB Ab). *: specific bands. B. The transcription regulation domain located in C-terminal part of Runx1 is required for the interaction with MyoD. Top panel, Schematic diagram of Runx1 protein. The Runt, transactivation (TA), and transcription inhibition (ID) domains are indicated. Lower panel, Runx1 and its deletion mutants, or luciferase (Luc) were in vitro translated in the presence of 35S-Methionine (inputs shown on the right panel) and incubated with equivalent amounts of GST-MyoD or GST agarose beads (normalization is shown in the lower panel). GST pull-down was then conducted as described in the Material and Methods section, and the radiolabeled proteins were detected by autoradiography.