Figure 7. CHOP associates with MyoD regulatory sequences and affects histone acetylation.

(A) A Chromatin IP experiment was performed on C2C12 cells expressing Flag-CHOP that were grown in DM for 24 hours. Immunoprecipitation of fragmented DNA was performed with anti-Flag antibodies. PCR amplification of fragments scattered throughout 6 Kb upstream region of the MyoD transcription unit and along 2 Kb upstream of the myogenin transcription unit was performed. PCR fragments were separated over agarose gels. Gels were scanned and values of band intensities are presented below. For each set of PCR primers, the value of the input was set to 1. (B) C2C12 cells expressing CHOP:ER chimera were grown in DM for 8 hours in the presence of ethanol or β estradiol (0.1 µM). Chromatin IP assay was performed on fragmented DNA with anti-acetylated histone H4 antibody. PCR amplification of fragments scattered along 6 Kb upstream of the MyoD transcription unit was performed. Gels were scanned and values of band intensities are presented below. For each set of PCR primers, the value of the input was set to 1 (C) 293T cells were transfected with expression plasmids as indicated. Cells were lysed under mild conditions and extracted proteins were analyzed (left) or immunoprecipitated with anti-Myc or anti-HA epitope antibodies as indicated (right). Proteins were analyzed by Western blotting as indicated.