Figure 3. Cyclic expression of BMAL1 is not required for intracellular core clock function.

(A) Bioluminescence patterns of fibroblasts derived from WT and Bmal1^−/−:mPer2^Luc mice. Bmal1^−/− fibroblasts are completely arrhythmic, suggesting that Bmal1 is required for clock function in fibroblasts. Circadian time: days after explant medium change. (B) Bioluminescence patterns in wild-type fibroblasts transduced with a lentiviral dLuc reporter. Bmal1(WT): Bmal1 promoter containing WT RORE sequence. Bmal1(Mut): Bmal1 promoter containing mutated RORE sequences. UbC: Ubiquitin C promoter. Unlike the Bmal1(WT), the Bmal1(Mut) and UbC promoters do not confer rhythmic luciferase expression. Circadian time: days after explant medium change. (C) Bioluminescence patterns in wild-type fibroblasts transduced with a lentiviral Bmal1::Luc fusion reporter. Unlike the Bmal1(WT), the Bmal1(Mut) and UbC promoters do not confer rhythmic BMAL1::LUC fusion protein expression. These results suggest that BMAL1 protein does not cycle and that only promoters that contain functional circadian elements confer rhythmic fusion protein expression. Circadian time: days after explant medium change. (D) Representative records of mPer2^Luc rhythms in Bmal1^−/− fibroblasts restored through genetic complementation. Lentiviral expression vectors carrying Bmal1 cDNA under control of different promoters were introduced into Bmal1^−/−:mPer2^Luc fibroblasts. The three promoters gave rise to similar levels of BMAL1 protein expression as determined by Q-PCR and Western blotting (data not shown). Both cyclically and constitutively expressed BMAL1 restored circadian rhythmicity in Bmal1^−/− fibroblasts, suggesting that the rhythm of BMAL1 protein is not required for basic core clock function. Circadian time: days after explant medium change.