Fig. 4.

The Bmal2 promoter/enhancer is activated by BMAL1/CLOCK and BMAL1/NPAS2 heterodimers. Levels of Bmal1(A) and Bmal2 (B) mRNA levels in liver. Transcripts of Bmal1 and Bmal2 were quantified by real-time PCR and normalized with reference to Hprt mRNA levels as a control. The data were normalized by setting the maximum value of Bmal1 or Bmal2 mRNA levels to 1.0. Blue triangles show data from WT mice, while red squares show data from B1ko mice; lines connect the averages at each time point (n = 3 or 4 except for WT at 56 h, where n = 2). (C) Effect of CLOCK/NPAS2 and BMAL1/BMAL2 on expression of the mBmal2 promoter in 3T3 cells. The total amount of plasmid DNA that was transfected was kept constant among by addition of the empty vector DNA when appropriate. Data are mean±SD (n≥4) of firefly luciferase activity (P_mBmal2∷Fluc) normalized by the Renilla luciferase control (P_CMV∷Rluc). The activity in samples transfected with the reporter construct (i.e., “PmB2-Fluc” without plasmids expressing other proteins) was set as 1.0. Significant differences in pairwise comparisons at the p < 0.05 level are indicated by letters. (D) Control of circadian gene expression by Bmal1 and Bmal2. Left panel: in wild-type mice, E-box clock-controlled genes (CCGs) are activated by both BMAL1 and BMAL2 proteins in combination with CLOCK and NPAS2. Expression of the Bmal2 gene is regulated by BMAL1/CLOCK and/or BMAL1/NPAS2 heterodimers. Right panel: in the Bmal1ko/Bmal2Tg mouse, expression of BMAL2 from the transgene (and possibly from the endogenous Bmal2 gene that is also activated by BMAL2 from the transgene) regulates the expression of the CCGs, thereby rescuing clock and metabolic phenotypes that result from Bmal1/Bmal2 knock-out/down in the Bmal1ko mouse.