Evidence for an Overlapping Role of CLOCK and NPAS2 Transcription Factors in Liver Circadian Oscillators

Abstract

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, which are putative DNA-binding sites for CLOCK-BMAL1 and NPAS2-BMAL1 heterodimers and hallmarks of circadian regulation. The peaks of FVII mRNA levels in livers of wild-type mice preceded those in plasma, indicating a transcriptional regulation, and were abolished in Clock^−/−; Npas2^−/− mice, thus demonstrating a role for CLOCK and NPAS2 circadian transcription factors. The investigation of Npas2^−/− and Clock^Δ19/Δ19 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 activities of the NPAS2-BMAL1 and CLOCK-BMAL1 heterodimers were (i) comparable (a fourfold 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 circadianly controlled genes.

ACKNOWLEDGMENTS

The work was supported by grants from the University of Ferrara (M.P., C.B., A.F., and F.B.), the Fondazione Cassa di Risparmio di Cento (I.C., A.F., and C.B.), Telethon (GP05214; M.P. and F.B.), and the National Institutes of Health NS-43459 (G.T.).

We are grateful to Urs Albrecht (University of Fribourg, Switzerland), Pablo Sdrenko (Max Planck Institute for Molecular Endocrinology, Hannover, Germany), Steven L. McKnight (University of Texas Southwestern Medical Center, Dallas, TX), Steven M. Reppert (University of Massachusetts Medical School, Worcester, MA), and Michael H. Hastings (Medical Research Council, Cambridge, United Kingdom), who kindly provided expression vectors for the mouse circadian transcription factors. We also thank Steven L. McKnight (University of Texas Southwestern Medical Center) and Jason P. DeBruyne (University of Massachusetts Medical School) for providing us with samples from Npas2^−/− and Clock^−/−; Npas2^−/− mutant mice, respectively.