DEC2 modulates orexin expression and regulates sleep

Abstract

Adequate sleep is essential for physical and mental health. We previously identified a missense mutation in the human DEC2 gene (BHLHE41) leading to the familial natural short sleep behavioral trait. DEC2 is a transcription factor regulating the circadian clock in mammals, although its role in sleep regulation has been unclear. Here we report that prepro-orexin, also known as hypocretin (Hcrt), gene expression is increased in the mouse model expressing the mutant hDEC2 transgene (hDEC2-P384R). Prepro-orexin encodes a precursor protein of a neuropeptide producing orexin A and B (hcrt1 and hcrt2), which is enriched in the hypothalamus and regulates maintenance of arousal. In cell culture, DEC2 suppressed prepro-orexin promoter-luc (ore-luc) expression through cis-acting E-box elements. The mutant DEC2 has less repressor activity than WT-DEC2, resulting in increased orexin expression. DEC2-binding affinity for the prepro-orexin gene promoter is decreased by the P384R mutation, likely due to weakened interaction with other transcription factors. In vivo, the decreased immobility time of the mutant transgenic mice is attenuated by an orexin receptor antagonist. Our results suggested that DEC2 regulates sleep/wake duration, at least in part, by modulating the neuropeptide hormone orexin.

Keywords: behavior; genetics; mouse model; sleep.

Fig. 1.

Expression of orexinergic genes is up-regulated in the hDEC2-P384R mice. (A) mRNA levels of prepro-orexin, orexin receptor1 (OX1R), and orexin receptor2 (OX2R) in hypothalamus of hDEC2 BAC Tg mice at ZT1. mRNA levels were analyzed by real-time PCR using specific primers of indicated genes and normalized to Gapdh. Data are shown as mean ± SEM. n = 3. *P < 0.05, Student’s t test. (B) Immunohistochemistry of orexin A in the lateral hypothalamus of the BAC Tg mice at ZT1. Prepro-orexin and orexin A were immunostained with anti-orexin A antibody, and orexin A-positive cells were counted. Three representative images with the same experimental condition were shown. Data were collected from ∼30 slices and averaged for each mouse. Data are shown as mean ± SEM. n = 5. *P < 0.05, Student’s t test.

Fig. 2.

DEC2 inhibits E-box–mediated transcription of prepro-orexin. (A) Schematized model of the human prepro-orexin gene promoter. OE1–3 are the primer sets used for ChIP assays in C. (B) Luciferase assay in HEK293 cells. DEC2 expression inhibits ore-luc activity. Firefly luciferase activity was normalized to Renilla luciferase activity. (C) ChIP with mouse brain and anti-DEC2 antibody. Brains (including hypothalami) were collected at ZT8 and homogenized. ChIP signals were determined by real-time PCR using the primer sets shown in A. Signals were normalized to normal mouse IgG. Data are shown as mean ± SEM. n = 4. *P < 0.05, Tukey’s test. (D) Luciferase assay using E-box mutant constructs. Mutations are shown at the bottom of A. DEC2 repressor activity was not observed for E-box mutant ore-luc. Data are shown as mean ± SEM (n = 3). *P < 0.05, two-way ANOVA followed by post hoc test. (E) The effect of transcription factors known to bind E-boxes. FLAG-tagged transcription factors (TF) were expressed in HEK293 cells. C/B indicates FLAG-tagged CLOCK and BMAL1. Data are shown as mean ± SEM. n = 4. *P < 0.05, Tukey’s test. (F) The effect of MYOD1 and MYF5 knockdown by shRNA on ore-luc activity. shRNA of MYOD1/MYF5 (with/without TCF3 shRNAs) were transfected into HEK293 cells. Data are shown as mean ± SEM. n = 4. *P < 0.05, Tukey’s test.

Fig. 3.

The P384R mutation decreases DEC2 repressor activity. (A) Overexpression of E12 (Top) and E47 (Bottom) abrogated the effect of DEC2. HEK293 cells were transfected with the indicated plasmids. Data are shown as mean ± SEM. n = 4. *P < 0.05, Tukey’s test. (B) The mutation in E-box1 reduced MyoD1 activity and DEC2 repressive activity in both 0.6- and 3.2-kb ore-luc constructs. Data are shown as mean ± SEM. n = 4. *P < 0.05, Tukey’s test. (C) The effect of the P384R mutation on DEC2 activity in HEK293 cells. Data are shown as mean ± SEM. n = 4. *P < 0.05, Student’s t test for HEK293. (D) The effect of MyoD1 and DEC2 in SH-SY5Y cells. Data are shown as mean ± SEM. (Left) n = 4–8. *P < 0.05, Student’s t test. (Right) n = 4. *P < 0.05, Tukey’s test.

Fig. 4.

The P384R mutation decreases DEC2 binding to E12 and DNA. (A) Co-IP of E12 and DEC2 expressed in HEK293 cells. DEC2-Myc-His was immunoprecipitated with anti-Myc antibody. Results were obtained from three independent experiments. (B) The effect of the mutation on the interaction of DEC2 with E12 in HEK293 cells. (C) ChIP assay of DEC2 in mouse brain from H11 Tg mice. DEC2-Myc proteins were precipitated with anti-DEC2 antibody, and ChIP signals were determined using real-time PCR. Data are shown as mean ± SEM. n = 4. *P < 0.05, Student’s t test. (D) The effect of TCF3 knockdown on DEC2 repressor activity of ore-luc. Data are shown as mean ± SEM. n = 4. *P < 0.05, Student’s t test. (E) Locomotor activity of FLAG-hDEC2-P384R, and hDEC2-WT mice after injection of orexin receptor antagonist. MK-6096 (25 mg/kg) or PBS were administered p.o. at ZT11, and recording started from ZT12. Mouse movement was tracked by an infrared video camera from ZT12-24 in LD 12:12. The immobilization time was plotted every 60 min (Top) or every 6 h (Bottom). Data are shown as mean + SEM. n = 6. *P < 0.05, two-way ANOVA. (F) Proposed model for regulation of orexin expression by DEC2. DEC2 binds and competes with transcription factors (MyoD1, E12, and E47) and suppresses the transcriptional activity of orexin. Overexpressed E12 or E47 likely interfere with the binding of DEC2 to MyoD1/DNA. The P384R mutation in DEC2 decreased the interaction with E12 and DNA-binding affinity, leading to up-regulation of orexin.