Cryptochromes mediate rhythmic repression of the glucocorticoid receptor

Abstract

Mammalian metabolism is highly circadian and major hormonal circuits involving nuclear hormone receptors display interlinked diurnal cycling. However, mechanisms that logically explain the coordination of nuclear hormone receptors and the clock are poorly understood. Here we show that two circadian co-regulators, cryptochromes 1 and 2, interact with the glucocorticoid receptor in a ligand-dependent fashion and globally alter the transcriptional response to glucocorticoids in mouse embryonic fibroblasts: cryptochrome deficiency vastly decreases gene repression and approximately doubles the number of dexamethasone-induced genes, suggesting that cryptochromes broadly oppose glucocorticoid receptor activation and promote repression. In mice, genetic loss of cryptochrome 1 and/or 2 results in glucose intolerance and constitutively high levels of circulating corticosterone, suggesting reduced suppression of the hypothalamic-pituitary-adrenal axis coupled with increased glucocorticoid transactivation in the liver. Genomically, cryptochromes 1 and 2 associate with a glucocorticoid response element in the phosphoenolpyruvate carboxykinase 1 promoter in a hormone-dependent manner, and dexamethasone-induced transcription of the phosphoenolpyruvate carboxykinase 1 gene was strikingly increased in cryptochrome-deficient livers. These results reveal a specific mechanism through which cryptochromes couple the activity of clock and receptor target genes to complex genomic circuits underpinning normal metabolic homeostasis.

Figure 1

Cryptochromes interact with GR. (a–e, h) Immunoblots showing recovery of the indicated proteins from 293T cells expressing the indicated plasmids following IP with the indicated antibodies. In (e), cells were treated with vehicle (−) or 1 μM dexamethasone (+) for 16 hours. (f) Immunoblots showing recovery of endogenous GR and PER2 from MEFs stably expressing empty vector (−) or FLAG-Cry1 (+) following FLAG IP. (g) Luciferase activity in CV-1 cells transfected as indicated. Data represent the mean ± s.e.m. of triplicate samples. ***P < 0.001. Data represent typical results of 2–6 independent experiments.

Figure 2

Cryptochromes modulate GR-dependent transcription. (a) Heat map: color denotes dexamethasone-induced change in expression of all transcripts significantly altered by genotype or glucocorticoid stimulation. Selected genes are indicated on the left. (b) Transcripts altered by dexamethasone treatment in control (gray circles) or DKO (blue circles) MEFs. (c) Expression of indicated transcripts in control or DKO MEFs following overnight treatment with vehicle (−) or dexamethasone (+). (d,e) Expression of sgk1 in MEFs following treatment with dexamethasone for 1–4 hours. In (c–e), data represent the mean ± s.e.m. of triplicate samples analyzed in triplicate. * P < 0.01, ** P < 0.001.

Figure 3

Cryptochromes interact with GR on chromatin to regulate pck1. (a) Top, pck1 in mouse livers. *, ** P < 0.05, 0.01 vs. saline; #, ## P < 0.05, 0.01 vs. ZT2. Bottom, Cry1 immunoblots. (b) Immunoblots of liver lysates or IPs. (c) Left, pck1 at ZT16. Right, recovery of Pck1 GRE. *, ** P < 0.05, 0.01 vs. saline; #, ## P < 0.05, 0.01 vs. wildtype. (d) Recovery of Pck1 GRE or Dbp promoter E-box at ZT16. Data represent the mean ± s.e.m. of three samples (a,c) or duplicate samples (d) analyzed in triplicate.

Figure 4

Genetic loss of cryptochromes alters physiology. (a) Serum corticosterone. #P < 0.05 vs. ZT3-4. *P < 0.05, **P < 0.01 vs. wildtype. (b) Fasted and refed blood glucose **P < 0.01 vs. wildtype. (c) Glucose tolerance test. * P < 0.05, ** P < 0.01, ***P < 0.001 vs. wildtype. (d) Insulin tolerance test. (e) Corticosterone in sera collected at ZT10–11. (f) Fasted blood glucose. **P < 0.01 vs. saline-treated animals. ##P < 0.01 vs. wildtype. (g) Glucose tolerance tests. **P < 0.01 vs. saline-treated animals. In (a–d, f–g) data represent the mean ± s.e.m. for 6–8 animals per group.