Transcriptional feedback loops in the ovine circadian clock.

In mammals circadian time measurement depends on interlocked feedback loops involving clock genes and their protein products. The model of the mammalian circadian clock mostly rests on findings in the mouse. In comparison, little information is available in diurnal non-rodent species. In this respect, the sheep constitutes an excellent animal model. We cloned ovine clock components and proximal gene promoters and tested in-vitro, in NIH3T3 and COS7 cells, salient molecular characteristics of the circadian clock. We show that transcriptional features of the ovine circadian clock recapitulate those described for the mouse. These include (1) coordinated phasing of expression of Rev-erb alpha, Per1, Cry1 and Bmal1 as assessed by real-time luciferase assays, (2) CLOCK/BMAL1 transactivation at the Per1 and Rev-erb alpha promoters, (3) repression of CLOCK/BMAL1 by CRY1-2 and CIPC, (4) a role for REV-ERB alpha in inhibiting Bmal1 and Rev-erb alpha transcription. DEC1 has bidirectional transcriptional effects, repressor or activator, according to the promoter. We further show that some phosphorylation events affecting clock proteins appear conserved within the ovine clock. Taken together, these data are consistent with a broad conservation of transcriptional and post-translational mechanisms in the circadian clock of diurnal and nocturnal mammals.