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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nat Neurosci. Author manuscript; available in PMC 2009 November 25.
Published in final edited form as:
Published online 2007 April 8. doi:  10.1038/nn1884
PMCID: PMC2782643
NIHMSID: NIHMS110495

CLOCK and NPAS2 have overlapping roles in the suprachiasmatic circadian clock

Abstract

Heterodimers of CLOCK and BMAL1, bHLH-PAS transcription factors, are believed to be the major transcriptional regulators of the circadian clock mechanism in mammals. However, a recent study shows that CLOCK-deficient mice continue to exhibit robust behavioral and molecular rhythms. Here we report that the transcription factor NPAS2 (MOP4) is able to functionally substitute for CLOCK in the master brain clock in mice to regulate circadian rhythmicity.

Circadian rhythms regulate biological events like the timing of the sleep-wake cycle and are generated by a hierarchy of circadian clocks1,2. Atop this hierarchy is a master clock that resides in the hypothalamic suprachiasmatic nuclei (SCN). The SCN clock is entrained by the daily light-dark cycle to the 24-h period by retina to SCN pathways, and it synchronizes the phase of circadian clocks in peripheral tissues that drive rhythmic changes in local physiology.

The intracellular circadian clock mechanism in the mouse is regulated by transcriptional feedback loops that drive the self-sustaining clock mechanism in both the SCN and peripheral tissues1,2. The critical molecular mechanism is thought to involve CLOCK:BMAL1 heterodimers that drive the rhythmic expression of three Period genes (mPer1–3) and two Cryptochrome genes (mCry1 and mCry2). The resulting proteins form PER:CRY complexes that translocate back into the nucleus to inhibit their own transcription, creating a negative feedback loop. A modulatory, interlocking positive transcriptional feedback loop involves the rhythmic regulation of Bmal1 transcription, through the coordinated actions of Rev-erbα (repressor) and Rora (activator), whose mRNA oscillations are antiphase to the mPer and mCry mRNA rhythms.

Recent genetic evidence has shown that CLOCK is not essential for the circadian rhythm in locomotor activity in mice (Fig. 1a, middle panel)3. However, compared with wild-type controls, CLOCK-deficient mice do have a slightly shortened circadian period in constant darkness and show altered circadian responses to light3. Without CLOCK, molecular and biochemical rhythms are also altered, but most persist3. Because BMAL1 is essential for the expression of circadian behavioral rhythms4 and homodimers are not transcriptionally active5, we sought an alternative dimerization partner for BMAL1.

Figure 1
Locomotor activity rhythms are abolished in double knockout mice. (a–d) Representative activity rhythms of (a) wild-type (left), CLOCK-deficient (Clock−/−; middle) and NPAS2-deficient mice (Npas2−/−; right), (b ...

NPAS2 (also called MOP4) is a paralog of CLOCK6,7 that can dimerize with BMAL1 and appears to function in a clockwork mechanism in mouse forebrain8. Its function in the SCN has been questioned, however, as previous studies were unable to detect Npas2 expression in the SCN8,9. Homozygous Npas2-mutant mice (Npas2−/−), which do not express functional NPAS210, display robust circadian rhythms in locomotor behavior11 (Fig. 1a, right panel). Like CLOCK-deficient mice, Npas2−/− mice also have a slightly shortened circadian period and an altered response to perturbations in the light-dark cycle11. These circadian phenotypes have been proposed to be a result of disrupted crosstalk between forebrain and SCN clocks, and not a result of NPAS2 deficiency in the SCN11,12.

To examine whether NPAS2 is the missing BMAL1 partner, we generated CLOCK-deficient mice carrying either one or no functional Npas2 alleles by interbreeding CLOCK-deficient mice with a previously generated null allele of Npas2 (ref. 10, Fig. 1b–d and Supplementary Methods online). Our animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the UMass Medical School. CLOCK-deficient animals carrying only one normal allele of Npas2 (Clock−/−;Npas2+/−) had substantially shorter circadian periods in constant darkness (22.7 ± 0.2 h) compared with wild-type mice (23.8 ± 0.1 h), with progressive rhythm instability (Fig. 1b,e,f). CLOCK-deficient mice with no functional NPAS2 (Clock−/−;Npas2−/−) exhibited arrhythmic locomotor behavior immediately on placement in constant darkness (Fig. 1c,e,f). These findings suggest that NPAS2 is the missing BMAL1 partner and that NPAS2 has a direct function in the SCN, the generator of rhythmic locomotor behavior. Notably, NPAS2 mutant mice carrying only one allele of Clock (Clock+/-;Npas2−/−) displayed behavioral rhythmicity in constant darkness that was similar to wild-type animals (Fig. 1d,e,f), suggesting that CLOCK may have a more prominent role than NPAS2 in the SCN clock.

We used in situ hybridization to evaluate the status of the molecular clock in the SCN of the double mutant (Clock−/−;Npas2−/−) mice. We found that the circadian rhythms of mPer1, mPer2, Rev-erbα and Bmal1 mRNA expressed in the SCN of wild-type mice were abolished in the double mutants (Fig. 2). Consistent with the idea that the transcriptional drive of both the negative and positive feedback loops are disrupted in the double mutants, mPer1, mPer2 and Rev-erbα mRNA levels were at constant low levels over the circadian cycle, whereas Bmal1 mRNA levels were at constant high values (Fig. 2), consistent with the idea that Bmal1 is repressed by Rev-erbα1,2.

Figure 2
Clock gene mRNAs are arrhythmic in the SCN of double knockout mice. (a) Brains from wild-type mice (black lines), NPAS2-deficient mice (Npas2−/−, green lines) and double-knockout mice (Clock−/−;Npas2−/− ...

Single knockout Npas2−/− mice displayed subtle alterations in the rhythmic expression of some genes in the SCN. For example, the mPer2 mRNA rhythm of Npas2−/− mice appeared to be slightly damped compared with wild-type mice (Fig. 2). In addition, Bmal1 levels, although rhythmic, were increased throughout the circadian day in Npas2−/− mice, compared with wild types (Fig. 2). The molecular defects in Npas2−/− SCN are more subtle than those observed in CLOCK-deficient mice3, suggesting that CLOCK normally has a more prominent role than NPAS2 in controlling circadian gene expression.

To further assess NPAS2 function in the SCN, we generated CLOCK-deficient mice carrying the mPer2Luciferase (mPer2Luc) allele. Mice carrying this allele at the mPer2 locus express a mPER2::LUC fusion protein, which allows real-time monitoring of circadian dynamics from tissue explants in culture (see Supplementary Methods)13. Using real-time reporting of bioluminescence from SCN explants, we found that isolated SCN from CLOCK-deficient mice expressing the fusion protein (Clock−/−; mPer2Luc) still maintained self-sustained molecular oscillations in culture that were similar to those from wild-type SCN expressing the fusion protein (Clock+/+; mPer2Luc, Fig. 3 and Supplementary Fig. 1 online). These data suggest that NPAS2 maintains the SCN clockwork without CLOCK, independent of a major influence from other brain regions.

Figure 3
SCN molecular rhythms persist in culture without CLOCK. Bioluminescence rhythms from cultures of the SCN of wild-type mice expressing mPeriod2::Luciferase (Clock+/+;mPer2Luc, top panel) and CLOCK-deficient mice expressing the fusion protein (Clock−/− ...

To verify that Npas2 is actually expressed in the SCN of wild-type and CLOCK-deficient mice, we used a quantitative real-time PCR approach. Our experiment clearly shows Npas2 expression in the SCN of both wild-type and Clock−/− mice (Supplementary Fig. 2 online).

We conclude that NPAS2 has a newly found, unexpected role in the SCN clock mechanism that controls circadian behavior. CLOCK and NPAS2 can independently heterodimerize with BMAL1 in the SCN to maintain molecular and behavioral rhythmicity. We cannot distinguish whether NPAS2 normally functions to regulate circadian gene expression in the SCN of wild-type mice, or whether NPAS2 only has functionally relevant effects on gene expression in the absence of CLOCK. Nevertheless, the results show that NPAS2 maintains circadian function in the absence of CLOCK. The differences in gene expression profiles between the Clock−/− and Npas2−/− single-knockouts suggests that different circadian promoters may have different affinities or requirements for CLOCK:BMAL1 versus NPAS2:BMAL1 heterodimers. Thus, NPAS2 may function coordinately with CLOCK in the SCN. These findings show a new level of transcriptional control in the SCN clockwork.

Supplementary Material

Supplementary Figure 1

Supplementary Figure 2

Supplementary Methods

ACKNOWLEDGMENTS

We thank C.M. Lambert for technical assistance, and S.L. McKnight and J.S. Takahashi for providing the mice we used to establish colonies of Npas2 mutant mice and mPer2::Luciferase reporter mice, respectively. This work was supported by US National Institutes of Health (NIH) grants R01 NS047141 (S.M.R.) and R01 NS056125 (D.R.W.). J.P.D. was supported in part by NIH National Research Service Award F32 GM074277.

Footnotes

Note: Supplementary information is available on the Nature Neuroscience website.

COMPETING INTERESTS STATEMENT

The authors declare no competing financial interests.

Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions

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