FIGURE 4.

Endogenous clock genes are modulated in a target-specific manner by increased levels of CLOCK/BMAL1. A, MEFs were harvested at the indicated times after GFP or CLOCK/BMAL1 (CLK+BM) adenoviral infection. mRNA levels were measured by quantitative real-time PCR as described previously (15). Levels of Rev-erbα and Dbp were elevated (∼2- and ∼4-fold, respectively, above GFP MEFs), and rhythm amplitudes were significantly reduced in CLOCK/BMAL1-expressing MEFs. Amplitudes of Rev-erbα and Dbp mRNA rhythms in GFP MEFs were ∼2- and ∼3-fold, respectively. Relative values were calculated with the highest number in GFP MEFs set as 100 in each experiment. Results are shown as means ± S.E. of three experiments. B, PER2-LUC protein rhythms in GFP or CLOCK/BMAL1 MEFs. C, CLOCK binding to E-box motifs is elevated in Dbp, but it is not significantly increased in Per1 and Per2 when measured by ChIP. MEFs were infected with GFP or CLOCK/BMAL1 adenovirus (Adv), harvested 24 h later, and subjected to ChIP for CLOCK-bound chromatin as described previously (14, 38). Co-immunoprecipitated DNA levels were measured by real-time quantitative PCR, and relative values were calculated against the value in GFP MEFs. Results are shown as means ± S.E. of three experiments. NS, nonspecific immunoprecipitation. *, p < 0.05.