E1 and E2 sites contribute to circadian expression of C/ebpα.

(A) Schematic representation of mutant constructs of C/ebpα promoter region. Either or both E-boxes (sites E1 and E2) were mutated (E1, 5′-CACGTG-3′ to 5′-CACCAC-3′; E2, 5′-CACGTG-3′ to 5′-CACCAC-3′). (B) Real time reporter assay. NIH3T3 cells transfected with indicated mutant constructs were incubated with dexamethasone (100 nM) and then bioluminescence was measured. Plasmid pGL3-dLuc-Cebp-WT, wild type 1.4 kb upstream region of C/ebpα gene;, pGL3-dLuc-Cebp-Mut I, mutant E1 site; pGL3-dLuc-Cebp-Mut II, mutant E2 site; pGL3-dLuc-Cebp-Mut I-II, mutant E1 and E2 sites; mPer2-dLuc, Per2 promoter region (−798 to +331 relative to the cap site) cloned into pGL3-dLuc [23]. Each experiment was repeated 3-5 times and representative data were used for these graphs.