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. 2016 May 13;291(20):10867-75.
doi: 10.1074/jbc.M116.719120. Epub 2016 Mar 21.

The Nuclear Receptor Rev-erbα Regulates Adipose Tissue-specific FGF21 Signaling

Jennifer Jager  1 , Fenfen Wang  1 , Bin Fang  1 , Hee-Woong Lim  1 , Lindsey C Peed  1 , David J Steger  1 , Kyoung-Jae Won  1 , Alexei Kharitonenkov  2 , Andrew C Adams  3 , Mitchell A Lazar  4
Affiliations

The Nuclear Receptor Rev-erbα Regulates Adipose Tissue-specific FGF21 Signaling

Jennifer Jager et al. J Biol Chem. .

Abstract

FGF21 is an atypical member of the FGF family that functions as a hormone to regulate carbohydrate and lipid metabolism. Here we demonstrate that the actions of FGF21 in mouse adipose tissue, but not in liver, are modulated by the nuclear receptor Rev-erbα, a potent transcriptional repressor. Interrogation of genes induced in the absence of Rev-erbα for Rev-erbα-binding sites identified βKlotho, an essential coreceptor for FGF21, as a direct target gene of Rev-erbα in white adipose tissue but not liver. Rev-erbα ablation led to the robust elevated expression of βKlotho. Consequently, the effects of FGF21 were markedly enhanced in the white adipose tissue of mice lacking Rev-erbα. A major Rev-erbα-controlled enhancer at the Klb locus was also bound by the adipocytic transcription factor peroxisome proliferator-activated receptor (PPAR) γ, which regulates its activity in the opposite direction. These findings establish Rev-erbα as a specific modulator of FGF21 signaling in adipose tissue.

Keywords: DNA binding protein; Rev-ErbAα (NR1D1); adipose tissue metabolism; clock gene; metabolic regulation; microarray; mouse; nuclear receptor; transcription enhancer.

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Figures

FIGURE 1.
FIGURE 1.
Identification of klb as a direct target gene of Rev-erbα. Rev-erbα direct target genes in EWAT were identified by combining Rev-erbα ChIP-seq data and a microarray performed on EWAT of WT and Rev-erbα KO mice at ZT 10 (5 p.m.). Of the direct target genes identified, Klb was the top candidate.
FIGURE 2.
FIGURE 2.
βKlotho mRNA and protein levels are increased in white adipose tissue of Rev-erbα KO mice. A, relative mRNA level of Klb in the EWAT, IWAT, liver, hypothalamus (Hypo), and BAT of WT and Rev-erbα KO mice at ZT 10. Data are expressed as the mean ± S.E. and normalized to the WT (Student's t test; *, p < 0.01; **, p < 0.001 versus WT; n = 4–6). B and C, Western blotting analysis of KLB and HSP90 (loading control) proteins levels in EWAT, IWAT, and liver of WT and Rev-erbα KO mice at ZT 10. Representative immunoblots are shown (B), and KLB protein amounts were quantified by densitometry scanning analysis, expressed as the mean ± S.E., normalized to the WT (Student's t test; *, p < 0.01 versus WT; n = 4).
FIGURE 3.
FIGURE 3.
Rev-erbα controls enhancer RNA expression at the Klb locus specifically in mouse white adipose tissue. A, ChIP-seq profiles of Rev-erbα binding at the Klb locus (green tracks) in EWAT, liver (39), and BAT (18) of WT mice at ZT 10. Rev-erbα peaks at the Klb locus are highlighted in yellow boxes. Also shown are ChIP-seq profiles of H3K27ac at the Klb locus (blue track) in EWAT (43). GRO-seq was performed on EWAT, IWAT, liver, and BAT of WT and Rev-erbα KO mice at ZT 10. Genome browser views of nascent transcripts at the Klb locus are shown. GRO-seq signals on the + and − strand are illustrated in blue and red, respectively. Intragenic nascent eRNA at the Klb locus is highlighted in yellow boxes. The y axis scale refers to the normalized tag count per ten million reads. B, magnification of the major Rev-erbα peak at the Klb locus in EWAT, liver, and BAT. The center of eRNA in each tissue is highlighted in blue. C, RT-qPCR validation of transcription of intragenic eRNA at the Klb locus in EWAT, IWAT, liver, and BAT of WT and Rev-erbα KO mice. Tissues were harvested at ZT 10. Data are expressed as the mean ± S.E. and normalized to the WT (Student's t test; *, p < 0.01 versus WT; n = 4).
FIGURE 4.
FIGURE 4.
Rev-erbα controls the circadian transcription of Klb in WAT. A and B, relative mRNA levels of Rev-erbα (A) and Klb (B) in EWAT, IWAT, and liver of WT and Rev-erbα KO mice throughout 24 h. Values are the mean ± S.E. and normalized to the WT at ZT 10 (n = 4–6/time point). C, RT-qPCR of eRNA at the Klb locus in EWAT, IWAT, and liver of WT and Rev-erbα KO mice throughout 24 h. Data are expressed as the mean ± S.E. (n = 4–6/time point) and normalized to the WT at ZT 10. D, Western blotting analysis of KLB, Rev-erbα (ns, nonspecific band), and HSP90 (loading control) protein levels in IWAT of WT and Rev-erbα KO mice throughout 24 h (n = 2/time point). Representative immunoblots are presented.
FIGURE 5.
FIGURE 5.
PPARγ binds and modulates the Rev-erbα-regulated enhancer at the Klb locus. A, ChIP-seq profiles of Rev-erbα (green tracks, also shown in Fig. 3A) (39, 48) and PPARγ (purple track) (41) or PPARα (orange track) (42) binding at the Klb locus in, respectively, EWAT or liver of WT mice. The major Rev-erbα peak at the Klb locus is highlighted in pink. The y axis scale refers to the normalized tag count per million reads. B, ChIP-seq profiles of PPARγ binding at the Klb locus in 3T3-L1 adipocytes (40). The y axis scale refers to the normalized tag count per million reads. C, GRO-seq was performed in 3T3-L1 adipocytes treated with rosiglitazone (Rosi) for 10 min or left untreated (46). Genome browser views of nascent transcripts at the Klb locus are shown. GRO-seq signals on the + and − strand are illustrated in blue and red, respectively. The y axis scale refers to the normalized tag count per million reads. D, Klb gene expression in 3T3-L1 adipocytes treated with siRNA against scrambled (siSCR) or PPARγ (siPPARγ) mRNA (35). Data are expressed as the mean ± S.E. and normalized to the WT (Student's t test; *, p < 0.001 versus the WT; n = 3).
FIGURE 6.
FIGURE 6.
The FGF21 response is enhanced in white adipose tissue of Rev-erbα KO mice. A–F, relative mRNA levels of the FGF21 target genes cFos, Egr1, and Glut1 in EWAT (A–C) and IWAT (D--F) of WT and Rev-erbα KO mice injected with either vehicle or 0.6 mg/kg FGF21. Tissues were harvested at ZT 10, 2 h after the injection. Values are expressed as the mean ± S.E. and normalized to WT vehicle (two-way analysis of variance and Tukey's post hoc test; *, p < 0.01; **, p < 0.001; n = 8). G and H, body weight (G) and EWAT weight (H) of WT and Rev-erbα KO mice. Data are expressed as the mean ± S.E. (Student's t test; *, p < 0.05 versus WT; n = 5). I, blood glucose level of WT and Rev-erbα KO mice injected with either vehicle or 0.6 mg/kg of FGF21. Blood was collected at ZT 10, 2 h after the injection. Data are expressed as the mean ± S.E. (two-way analysis of variance; *, p < 0.05 versus WT vehicle; n = 6).
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