Fig. 5.

Setdb1 maintains endogenous MyoD expression without interfering transactivation by exogenous MyoD. (A, B) Setdb1 shRNA inhibits MyoD-luciferase reporter in C2C12 cells but not in C3H 10T1/2 cells. C2C12 myoblast cells (A) or C3H 10T1/2 mesenchymal cells (B) were transiently transfected for 24 h with plasmid expressing myc-MyoD and/or Setdb1 shRNA together with a MyoD-luciferase reporter. (C–D) Exogenous expression of Flag-Setdb1 had little effect on MyoD-luciferase reporter in both C2C12 and C3H 10T1/2 cells. C2C12 cells (C) or C3H 10T1/2 cells (D) were transfected with plasmid expressing myc-MyoD and/or Flag-Setdb1. For all reporter analysis, empty vector (pcDNA3) was added to adjust the total amount of transfected DNA to 1.0 μg. Data are presented as relative luciferase activity to the control (empty vector); Shown are representative data of three independent experiments performed in triplicate, and error bars indicate standard deviation. (E) Setdb1 did not bind to MyoD or myogenin promoter. Chromatin Immunoprecipitation (ChIP) was performed in C2C12 cells infected with retroviruses expressing Flag-Setdb1. The empty vector (pLZRS- IRES-GFP) was used as a control. Immunoprecipitated DNA was analyzed by PCR with specific primer sets described in Materials and methods followed by real-time qRT-PCR and agarose gel electrophoresis. Shown are representative data of three independent experiments. Nnat is a known Setdb1 target and was used as a positive control for Setdb1 binding, whereas Actin was used as a negative control.