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Molecular and Cellular Biology, May 2008, p. 3070-3075, Vol. 28, No. 9
0270-7306/08/$08.00+0     doi:10.1128/MCB.01931-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Dipartimento di Biologia ed Evoluzione, Università di Ferrara, I-44100 Ferrara, Italy,¹ Circadian Rhythms and Sleep Disorders Program, Neuroscience Institute, Morehouse School of Medicine, Atlanta, Georgia 30310-1495,² Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390,³ Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, I-44100 Ferrara, Italy⁴

Received 26 October 2007/ Returned for modification 15 December 2007/ Accepted 24 February 2008

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^19/19 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.

Published ahead of print on 3 March 2008.