MyoD occupies the Cdc6 promoter in myoblasts. (A) Schematic of the Cdc6 promoter. Closed ovals (E1 and E2) and squares identify the putative MyoD (E-boxes) and E2F sites within the promoter, respectively. The inverted arrows represent the primers used in the PCR amplification reactions. The transcription start site is depicted with an arrow. (B) Cross-linked chromatin from C2C12 myoblasts or primary mouse myoblasts cultured in GM was immunoprecipitated in parallel with anti-MyoD antibody (Ab) and then analyzed by semiquantitative PCR using primers surrounding the E1 and E2 sites in the Cdc6 promoter. A control reaction with the use of normal rabbit IgG (NR IgG) is shown along with input DNA (0.05%), which was amplified by the same set of primers. Black lines indicate that intervening lanes have been spliced out. (C) ChIP experiments were performed in parallel on chromatin from C2C12 myoblasts before and after differentiation using normal rabbit IgG or an antibody specific for MyoD. Precipitated DNA was then analyzed by PCR using the E1 primers, and afterward, the bands were quantified by ImageJ (version 1.36b; National Institutes of Health). Controls for PCR included the use of normal rabbit IgG and the titration of input DNA to ensure all amplifications were within the linear range (Fig. S1 D). (D) Values in the histogram represent the ratio of chromatin-bound MyoD to input genomic DNA and are the mean of three independent experiments with standard deviation.