REV-ERBα regulates Fgf21 expression in the liver via hepatic nuclear factor 6

Abstract

The circadian clock contributes to the timing of many body functions including metabolism and reproduction. The hepatokine fibroblast growth factor 21 (FGF21) is a critical metabolic regulator involved in modulation of fertility. Here we show that lack of the clock component REV-ERBα elevates FGF21 levels in liver and plasma. At the molecular level, REV-ERBα modulates the expression of FGF21 via the liver-specific hepatic nuclear factor 6 (HNF6). We conclude that REV-ERBα regulates metabolism and reproduction, at least in part, via regulation of Fgf21.

Keywords: Circadian clock; Physiology; Transcription.

Fig. 1.

Female Rev-erbα^−/− mice display signs of reduced fertility. (A) Rev-erbα^−/− (red bar) mating pairs produce significantly less pups per litter compared to Rev-erbα^+/− (black bar) pairs. Unpaired two-tailed t-test, ***P<0.0001, n=18 for Rev-erbα^+/− and n=8 for Rev-erbα^−/−, F-test reveals no difference in variance, F=2.41, DFn=17, Dfd=7. (B) Number of litters per mating period is reduced in Rev-erbα^−/− (red bar) mating pairs compared to Rev-erbα^+/− (black bar) pairs. Unpaired two-tailed t-test, ***P<0.0001, n=13 for Rev-erbα^+/− and n=8 for Rev-erbα^−/−, F-test reveals difference in variance, F=51.38, DFn=12, Dfd=7. (C) Inter-litter period is significantly longer in Rev-erbα^−/− (red bar) compared to Rev-erbα^+/− (black bar) mating pairs. Unpaired two-tailed t-test, ***P<0.0001, n=16 for Rev-erbα^+/− and n=6 for Rev-erbα^−/−, F-test reveals difference in variance, F=8.793, DFn=5, Dfd=15. (D) Hepatic Fgf21 mRNA in the liver is increased in Rev-erbα^−/− (red line) compared to Rev-erbα^+/+ mice (black line). Two-way ANOVA with Bonferroni post-test. The two curves are significantly different, P=0.0008, n=4. (E) Plasma FGF21 protein levels are significantly increased in Rev-erbα^−/− (red line) compared to Rev-erbα^+/+ animals (black line). Two-way ANOVA with Bonferroni post-test. The two curves are significantly different, P=0.0003, n=4-6. All values are mean±s.e.m.

Fig. 2.

Regulation of the Fgf21 promoter by clock components. (A) Dose-dependent activation of the Per1::luc promoter by BMAL1 and CLOCK (black bars) and no BMAL1/CLOCK activation of the Fgf21::luc reporter (white bars) in NIH3T3 cells (n=3). (B) Dose dependent repression by REV-ERBα on the Bmal1::luc reporter (black bars) and the Fgf21::luc reporter (white bars) in NIH3T3 cells (n=4). (C) Mutation analysis of the Fgf21 promoter. Top left: schematic diagram showing the positions of the four ROREs (R1-R4), the PPAR element (blue) and the HNF6 binding site (green). Top right: diagram of the mutations in R1 and R4, respectively. Bottom: panels of fold change of the various constructs. Black bars: reference value for the Bmal1::luc and Fgf21::luc reporters, respectively. White bars: relative repression by Rev-erbα. Hatched bars: reduced repression by Rev-erbα on the mutated Fgf21::luc reporter (n=4). (D) Dose-dependent activating potential of RORα on the Bmal1::luc reporter (black bars) but not on the Fgf21::luc reporter (white bars) in NIH3T3 cells (n=3). (E) Repression of the Bmal1::luc (white bars) and Fgf21::luc (blue bars) reporters in Hepa-1c1c7 cells involves Hnf6. Sc shRNA, scrambled shRNA; Hnf6 shRNA, knockdown of Hnf6 (n=3). (F) Repressive potential of REV-ERBα in Hepa-1c1c7 cells on the Bmal1::luc, the Fgf21::luc and the Hnf6 site mutated Fgf21::luc (mut Fgf21::luc) reporters (n=3). (G) Fold induction of the Fgf21::luc reporter by Pparα, Rxrα and Per2 in NIH3T3 cells (n=4). All panels: one-way ANOVA with Bonferroni post-test. *P<0.05, **P<0.01, ***P<0.001, values are means±s.d.

Fig. 3.

Chromatin immunoprecipitation of REV-ERBα, HNF6, PPARα and PER2 on the Fgf21 promoter of liver chromatin. (A) Binding of REV-ERBα to its own promoter and to the Bmal1 and Fgf21 promoters at Zeitgeber time (ZT) 10 (black bars) and ZT22 (white bars), respectively. (B) Binding of HNF6 to the R1-R4 elements on the Fgf21 promoter at ZT10 and ZT22. (C) Binding of PPARα on the Bmal1 and Fgf21 promoters in the liver of wild-type (black bars) and Rev-erbα^−/− (red bars) animals at ZT8 and ZT20. (D) Binding of PER2 on the Bmal1 and Fgf21 promoters in the liver of wild-type (black bars) and Rev-erbα^−/− (red bars) animals at ZT8 and ZT20. All panels: two-way ANOVA with Bonferroni post-test, n=3, *P<0.05, ***P<0.001; values are means±s.e.m.

Fig. 4.

Schematic representing clock contribution to Fgf21 regulation in the liver. REV-ERBα (R orange circle) represses the Fgf21 promoter involving HNF6 (olive trapezoid). Furthermore, PPARα (green oval) with its heterodimerizing partner RXR (light blue oval) activates the Fgf21 promoter. PER2 (P2, red circle) most likely modulates one or both of these regulations due to its capacity to bind to PPARα and REV-ERBα. Our data indicate that BMAL1 (B blue circle) and CLOCK (C/N purple oval) are not significantly involved in the regulation of Fgf21.