Evidence for an overlapping role of clock and npas2 transcription factors in liver circadian oscillators

The mechanisms underlying the circadian control of gene expression in peripheral tissues, and influencing many biological pathways, are poorly defined. Factor VII (FVII), the protease triggering blood coagulation, represents a valuable model to address this issue in liver since its plasma levels oscillate in a circadian manner and its promoter contains E-boxes, putative DNA-binding sites for CLOCK:BMAL1 and NPAS2:BMAL1 heterodimers and hallmarks of circadian regulation. Peaks of FVII mRNA levels in liver of wild type mice preceded those in plasma, to indicate a transcriptional regulation, and were abolished in Clock-/-;Npas2-/- mice, thus demonstrating a role for CLOCK and NPAS2 circadian transcription factors. The investigation in Npas2-/- and ClockD19/D19 mice, which express functionally defective heterodimers, revealed robust rhythms of FVII expression in both animal models, suggesting a redundant role for NPAS2 and CLOCK. The molecular bases of these observations were established through reporter gene assays. FVII transactivation activity of the NPAS2:BMAL1 and CLOCK:BMAL1 heterodimers resulted to be i) comparable (4-fold increase), ii) dampened by the negative circadian regulators PER2 and CRY1, and iii) abolished upon E-box mutagenesis. Our data provide the first evidence in peripheral oscillators for an overlapping role of CLOCK and NPAS2 in the regulation of circadian controlled genes.