C/EBPα and C/EBPϵ directly regulate the Per2 promoter. (A) Reporter assays were performed with NIH 3T3 cells cotransfected with murine Per2 reporter vector (pGL3-Per2) and either C/EBPα, C/EBPϵ, or DBP expression vectors. Control transfections indicated that the expression vectors had little to no effect on the pGL3 empty reporter vector. All transfections included the pRL-TK vector that served as an internal control for transfection efficiency. Results represent the mean ± SD of triplicate transfections. (B) EMSA was done using 10 μg nuclear extract proteins from NIH 3T3 cells either untransfected (UT) or transfected with a C/EBPα expression vector. Extracts were incubated with ³²P-labeled oligonucleotides containing the C/EBP site from the Per2 promoter. Unlabeled competitor oligonucleotides (× 100) or C/EBPα antibody was added as indicated. The asterisk indicates the position of the supershifted band. (C) Chromatin immunoprecipitation was performed from murine bone marrow cells using either C/EBPα or C/EBPϵ antibodies, preimmune serum, or no antibody. The samples were analyzed by PCR using primers specific for the C/EBP site in the murine Per2 promoter. Primers for the murine β-actin 5′ untranslated region were used as negative control. The input chromatin was included as a positive control. Images are inverted.