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(Delta19/Delta19) 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.

FIG. 1.

Temporal variations of liver FVII mRNA expression (A) and of plasma FVII activity (B) levels in mice. Mice (n = 6 for each time point) exposed to LD or DD conditions were sampled at the indicated ZT/CT. Values were normalized with respect to the maximum value (100%) measured for each condition. The mean ± the standard error of the mean (SEM) at each time point is shown. Statistical analysis was carried out using one-way and two-way ANOVAs. Gray areas indicate dark phases. LD, filled circles; DD, empty squares.

FIG. 2.

Liver FVII mRNA expression levels in Clock^−/−; Npas2^−/− mice. Clock^−/−; Npas2^−/− (empty squares) and wild-type (filled circles) mice (n = 14 to 16 for each genotype) were sampled at the indicated CT on the first day in DD. FVII expression levels were normalized with respect to the maximum value (100%) measured for each strain. The mean ± SEM at each time point is shown. Statistical analysis was carried out using one-way and two-way ANOVAs. The gray area indicates the dark phase.

FIG. 3.

Liver FVII mRNA expression levels in Npas2^−/− mice. Npas2^−/− (empty squares) and wild-type (filled circles) mice (n = 10 for each genotype) under LD (A) and DD (B) conditions were sampled at the indicated ZT/CT. FVII expression levels were normalized with respect to the maximum value (100%) measured for each genotype/condition. The mean ± SEM at each time point is shown. Statistical analysis was carried out using the Kruskal-Wallis one-way ANOVA. Gray areas indicate dark phases.

FIG. 4.

FVII activity levels in Clock^Δ19/Δ19 mice. Clock^Δ19/Δ19 (empty squares) and wild-type (filled circles) mice (n = 8 for each time point) exposed to LD (A) and DD (B) conditions were sampled at different ZT/CT. Each point represents the mean ± SEM of the logarithm of FVII activity expressed as RFU per second. FVII levels were normalized with respect to the maximum value (100%) measured for each genotype/condition. Statistical analysis was carried out using one-way and two-way ANOVAs. The gray areas indicate dark phases.

FIG. 5.

Transcriptional regulation of mouse FVII promoter by circadian factors. (A) Schematic representation of the reporter gene construct. E-box elements (E1 and E2) in the 5′ regulatory region (1,087 bp) of the mouse FVII gene are represented by ellipses. The numbering refers to the translation start site (arrow). (B) Relative luciferase activity in Hepa1-6 cells transfected with pFVII-Luc and different combinations of circadian transcription factors (BMAL1, CLOCK, NPAS2, PER2, and CRY1). The abscissa displays mean (±SEM) induction levels of luciferase activity over that of the negative control (pGL3basic).

FIG. 6.

Functional role of E-boxes in the transcriptional regulation of mouse FVII promoter. The relative luciferase activity in Hepa1-6 cells transfected with pFVII-Luc variants (E1, E2, or E1/E2 mutation [mut]) and different combinations of circadian transcription factors (CLOCK, BMAL1, and NPAS2). The abscissa displays mean (±SEM) induction levels of luciferase activity over that of pGL3basic.