DEC2 modulates orexin expression and regulates sleep

Results

To explore the mechanism underlying the behavioral phenotype of decreased sleep time in DEC2 mutation carriers, we examined the expression of genes regulating sleep homeostasis in a mouse model of the human DEC2 mutation. We found that prepro-orexin (Hcrt) gene expression was up-regulated in the hypothalamus of hDEC2-P384R transgenic (Tg) mice compared with hDEC2-WT Tg mice (Fig. 1A). Mammals have two receptors for orexin peptides, orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) (13). In hDEC2 mutant mice, the expression of OX2R was also slightly increased (Fig. 1A). To confirm the up-regulation of orexin at the protein level, we analyzed its expression in the lateral hypothalamus at zeitgeber time 1 (ZT1) by immunohistochemistry using an antibody recognizing orexin precursor protein and the orexin A peptide. The number of orexin-positive cells was increased by the P384R mutation (Fig. 1B). We also generated FLAG-tagged hDEC2 BAC Tg mice harboring the Y362H mutation and found that these mice showed increased prepro-orexin levels (Fig. S1A). Tg mice expressing either Y362H-DEC2 or P384R-DEC2 exhibited increased activity and decreased rest behavior (Fig. S1B), similar to what has been shown previously in the P384R mutant mice (5).

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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.

Deletion of orexin neurons or knockout of prepro-orexin or orexin receptor genes results in narcolepsy in mammals, indicating that orexin signaling plays a critical role in maintaining arousal and consolidation of sleep (13). Furthermore, administration of orexin A or selective activation of the orexin neurons increases arousal and decreased sleep duration (13). Therefore, modulating orexin expression is a key step in sleep/wake regulation. Since the expression levels of prepro-orexin were increased in the mutant mice, we explored the possibility that DEC2 directly modulates the expression of orexin. Previous studies indicated that the 3.2-kb promoter region of prepro-orexin is sufficient for endogenous expression, which was confirmed by generating prepro-orexin promoter-driven LacZ Tg mice (14). Within this 3.2-kb region, two elements—orexin regulatory elements 1 and 2 (OE1 and OE2)—were recognized because of a high similarity between mice and humans. Of these, OE1 plays a more important role in regulating orexin expression (Fig. 2A) (15). To examine the role of DEC2 in orexin expression, we cloned promoter regions of the prepro-orexin gene including only OE1 or both OE1 and OE2. A luciferase assay using prepro-orexin promoter luciferase (ore-luc) constructs showed that DEC2 protein decreased ore-luc activity, especially with the 0.6-kb region of the promoter containing OE1 (Fig. 2B). We then examined the occupancy of endogenous mouse DEC2 in the promoter region of the prepro-orexin gene in brain and observed a chromatin immunoprecipitation (ChIP) signal in the OE1 region (0.6 kb; primer sets 1 and 2) but not outside of the 3.2-kb region (primer set 3) (Fig. 2C).

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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.

DEC1 and DEC2 directly or indirectly (through interaction with other transcription factors) bind to DNA and modulate the transactivation of CLOCK, BMAL1, MYC, MyoD1, and E12/47, all of which are E-box–binding transcription factors (8, 9, 16⇓–18). DEC1/2 proteins form homodimers and directly bind to class B E-boxes (CACGTG), which is the CLOCK-BMAL1 consensus sequence (8, 19). DEC1/2 also form complexes with other transcription factors, such as MyoD1 and its binding partner E12/47, to bind to another type of E-box sequence (CAGCTG) (18, 20) found in promoter region of orexin (Fig. 2A). Thus, we introduced mutations in putative E-boxes in the 0.6-kb region to examine the effect on DEC2 repressor activity (Fig. 2A). The mutations at E-boxes 1 and 2 attenuated DEC2 activity, suggesting that DEC2 represses ore-luc activity via these two elements (Fig. 2D). We then examined which binding partner of DEC2 is involved in orexin regulation mediated by these E-box sequences. Among E-box–binding transcription factors, MyoD1 dramatically activated ore-luc (Fig. 2E). In contrast, the transactivation of ore-luc by MYC and CLOCK/BMAL1 was much less than that by MyoD1.

Although MyoD1 is known for its role in regulating muscle cell differentiation and muscle regeneration, it is nonetheless widely expressed in many tissues, including brain. In the regulation of muscle differentiation, MyoD1 cooperates with E12 and E47 proteins, which are splice variants encoded by the TCF3 gene (16, 17, 20, 21). MyoD1/E47 heterodimer activates creatine kinase, M-type (Ckm) gene expression (a physiological target of MyoD1) (20), and DEC2 serves as repressor for MyoD1/E47 activity on Ckm expression. Thus, we asked whether E12/E47 also influences MyoD1 activity on ore-luc. In contrast to the case for Ckm, we found that overexpression of E12 and E47 inhibited MyoD1 activation of ore-luc. We then knocked down MYOD1 and its homolog (MYF5), which has a redundant role in muscle differentiation (22). Double knockdown of MYOD1 and MYF5 by shRNAs resulted in decreased ore-luc activity, while additional knockdown of TCF3 eliminated the reduction almost completely (Fig. 2F), consistent with E12 and E47 serving as repressors of MyoD1 for the prepro-orexin promoter. Taken together, these results suggest that MyoD1, E12, and E47 participate in the regulation of prepro-orexin expression, with E12 and E47 acting as repressors of MyoD1.

Interestingly, although DEC2 strongly inhibited ore-luc activation by MyoD1 (Fig. 3A), DEC2 repressor activity was completely abolished by the expression of E12 or E47 (Fig. 3A), with no effect on DEC2 expression levels (Fig. S2). It is possible that in the formation of functional complexes of transcription factors (MyoD1, E12/47, and DEC2), excessive E12/47 can interfere with proper DEC2 interaction and abolish DEC2 function. To confirm that E-box1 is the target of MyoD1, we used the mutated E-box sequences described above (Fig. 2 A and D). The mutations in E-box1 reduced MyoD1 transactivation and attenuated the DEC2 repressor activity of MyoD1 (Fig. 3B). Consistent with this, E-box1 is identical to the consensus sequence for MyoD1 rather than other DEC2-binding proteins, such as CLOCK and BMAL1. Moreover, sequence analysis revealed that only E-box1 is conserved in humans and mice prepro-orexin promoters, implying that E-box1 is more likely to be functional in vivo. However, mutations spanning all E-boxes within the 0.6-kb region (E-box1, E-box2, and E-box3) did not completely abolish MyoD1 transactivation (Fig. S3), suggesting that additional cis elements for MyoD1 exist in this region. We then examined the effect of the P384R mutation on DEC2 activity for ore-luc. We and another group have previously shown that the P384R mutation decreased the DEC2 repressor activity of CLOCK-BMAL1–mediated Per1-luc and Per2-luc transactivation (5, 10). Similarly, the P384R mutation reduced DEC2 repression of MyoD1 activity on promoter elements in both 0.6- and 3.2-kb ore-luc constructs (Fig. 3C). These data indicate that DEC2 and its binding partner MyoD1 inhibit and activate orexin expression, respectively. More importantly, the human mutation of DEC2 resulted in reduced DEC2 repressive activity and thus increased ore-luc activity. We also confirmed these findings in another cell line, SH-SY5Y, a neuroblastoma-derived cell line (Fig. 3D), because responses of the orexin promoter can vary among different cell lines (23). Taken together, our in vitro data are congruent with the finding that orexin expression is up-regulated in the mouse model of the human mutations (Fig. 1A).

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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.

We next asked how the mutation reduces DEC2 activity. We found that the protein levels of DEC2-WT, DEC2-P384R, and DEC2-Y362H were comparable, as has been reported previously (10). The degradation rate of DEC2 was not affected by either mutation (Fig. S4). A previous study demonstrated that the interaction with MyoD1 in vitro is reduced by deletion of the Gly-Ala rich domain of DEC2 (8, 20), in which the two mutations reside. Thus, the mutations may affect the interaction of DEC2 with other transcription factors. A coimmunoprecipitation experiment showed that the interaction of DEC2 with E12 was greatly strengthened by the expression of MyoD1 (Fig. 4A), implying that DEC2, MyoD1, and E12 form a complex, and that DEC2 binds to a MyoD1/E12 complex. We found that the complex formation of DEC2, MyoD1, and E12 was weakened by the P384R mutation, but not by the Y362H mutation, in cell culture (Fig. 4 A and B). It is possible that the reduced interaction of DEC2-P384R with E12/47 could impact the occupancy of DEC2 on the prepro-orexin promoter. To compare the DNA-binding affinity of WT and mutant DEC2 in brain, we used H11 locus-specific DEC2 Tg mice, in which a conditional allele of the gene was integrated into the H11 locus, so that we can exclude an effect of copy number and expression level of transgenes (Fig. S5A). After crossing these mice with Nestin-Cre Tg mice, the expression of Myc-tagged DEC2 proteins was driven by a constitutive promoter—the cytomegalovirus (CMV) early enhanced/chicken beta actin (CAG) promoter—and comparable amounts of DEC2 proteins were detected in mouse brain (Fig. S5B). We confirmed that in the H11 mice, DEC2-Myc binds to the E-box–containing promoters of prepro-orexin and Dbp genes, as expected (Fig. S5C); however, weakened binding of mutant DEC2 to the prepro-orexin promoter was observed in H11 P384R-DEC2 Tg mice (Fig. 4C). In contrast, there was no obvious effect of the Y362H mutation on the interaction of DEC2 with E12 in cell culture (Fig. 4 A and B), suggesting that a different molecular mechanism is responsible for the reduced repressor activity of DEC2-Y362H.

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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.

To further investigate the dynamics of the interaction of E12/47 with DEC2, we examined the effect of TCF3 knockdown on DEC2 repressor activity. TCF3 knockdown by siRNA decreased DEC2 repressive activity in a small, but statistically significant way (Fig. 4D). These results suggested that E12/47 helps to recruit DEC2 to the complex and to E-boxes. To test whether orexin mediates the sleep phenotype altered in the DEC2 mutant mice, we orally administrated orexin receptor antagonist to the Tg mice and recorded their wake/rest behavior. As shown previously (Fig. S1B), hDEC2-P384R mice have decreased total rest time compared with hDEC2-WT Tg mice, especially during the first half of the dark phase (Fig. 4E). Interestingly, the administration of MK-6096, a nonselective orexin receptor antagonist, partially cancelled the phenotype of decreased sleep time observed in hDEC2-P384R mice (Fig. 4E).

Taken together, the results presented here indicate that MyoD1 activates prepro-orexin gene expression, and that DEC2 is a repressor of this activation (Fig. 4F). Prepro-orexin expression and orexin levels show daily rhythms in hypothalamus and cerebrospinal fluid (24, 25). DEC2 expression oscillates in a circadian manner (9, 26) and thus may contribute to the expression rhythms of prepro-orexin. Our results suggest that E12/47, MyoD1, and DEC2 form a complex to regulate prepro-orexin expression. This complex formation is reduced by the P384R mutation, leading to orexin up-regulation. Overexpression of E12 or E47 attenuates DEC2 repressor activity, possibly because the interaction of DEC2 and E12 depends on MyoD1 (Fig. 4A), and excessive accumulation of E12/47 may prevent the DEC2–MyoD1 interaction (Fig. 4F, Bottom).