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Genes to Cells (2007) 12, 1225-1234. doi:10.1111/j.1365-2443.2007.01129.x
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PER2 controls circadian periods through nuclear localization in the suprachiasmatic nucleus

 Koyomi Miyazaki1, Miyuki Wakabayashi1, Sachiko Chikahisa2, Hiroyoshi Sei2 and Norio Ishida1,3,*

1 Clock Cell Biology Group, Institute for Biological Resource and Function, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
2 Department of Integrative Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan
3 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8572, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Molecular circadian clock regulation engages a negative feedback loop comprising components of the negative limb, PERs and CRYs. In addition to the rhythmic transcriptional regulation of clock genes, controlled subcellular localization might contribute to the molecular mechanism of the mammalian circadian clock. To address this issue, we generated transgenic (TG) mice lines harboring either rat PER2 (rPER2) with a deleted nuclear localizing domain [NLD(–)] or intact PER2. In comparison with wild-type (WT) control, the period of the circadian locomotor rhythm in TG mice over-expressing NLD(–) PER2 was longer, while that in TG mice over-expressing intact PER2 was shorter. The nuclear entry of endogenous PER2, CRY1 and CRY2 was delayed in the suprachiasmatic nucleus (SCN) of NLD(–) PER2 TG mice under constant darkness, whereas that of mouse PER2 (mPER2) is accelerated in the SCN of intact PER2 TG mice. Under constant light, the locomotor activity of NLD(–) PER2 TG mice became arrhythmic, whereas WT animals remained rhythmic. These data indicate that PER2 controls circadian periods through nuclear localization in the SCN. In addition, sleep architecture was also affected in intact PER2 TG mice, suggesting PER2 can modulate a sleep molecular mechanism.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
In mammals, circadian rhythms of physiology and behavior are coordinated by a master clock situated in the suprachiasmatic nucleus (SCN) of the basal hypothalamus (Weaver 1998). Molecular circadian regulation engages a transcription–translation feed back loop comprising clock proteins, CLOCK, BMAL1, PERs and CRYs (Dunlap 1999; Shearman et al. 2000; Ishida et al. 2001). In addition to the cyclic transcriptional regulation of clock genes, the controlled subcellular localization constitute important feature of circadian clocks. Transfection studies in COS7 cells have shown that exogenously expressed mouse PER2 (mPER2) can localize in the nucleus when co-expressed with mouse CRY (mCRY) proteins (Kume et al. 1999). We identified a nuclear localization sequence (NLS) in rat PER2 (rPER2), which was latent and activated after binding with CRY1 protein (Miyazaki et al. 2001). In addition, PER2 deleted around NLS, which is called as nuclear localization domain (NLD; residues 512–794), inhibited the nuclear entry of not only PER2 itself but also of CRY1, suggesting that the deleted domain is important for establishing nuclear accumulation of the PER2/CRY complex (Miyazaki et al. 2001). High levels of mCRY proteins are constantly expressed in the liver and they localize in nuclei when the abundance of PER protein increases (Lee et al. 2001). Considering these in vitro and in vivo results, mPER2 proteins seem to be rate limiting for mPER x mCRY interactions and for their nuclear translocation.

Circadian rhythmicity and sleep homeostasis interact to regulate sleep–wake cycle (Saper et al. 2005). Mutation in Per2 can affect rhythmicity by altering the fee-running period through changing PER2 protein stability and can result in circadian sleep disorders such as familial advanced sleep phase syndrome (FASPS) in humans (Toh et al. 2001). Period genes are considered to contribute individual differences in sleep timing by affecting circadian rhythmicity, but not sleep homeostasis (Kopp et al. 2002; Shiromani et al. 2004). But the genetic basis of clock regulation in sleep–wake cycle remains largely unknown (Tafti et al. 2005; Wisor & Kilduff 2005).

To determine whether nuclear localization of PER2 is important for generating circadian rhythm in vivo, we generate transgenic (TG) mice harboring either NLD-deleted rat PER2 [NLD(–) PER2] or full-length rPER2 (intact PER2). In comparison with wild-type (WT) control, the period of the circadian locomotor rhythm in TG mice over-expressing NLD(–) PER2 was longer, while that in TG mice over-expressing intact PER2 was shorter. The nuclear entry of endogenous PER2, CRY1 and CRY2 is monitored in SCN of NLD(–) PER2 TG under constant darkness. Under constant light, the circadian locomotor activity of NLD(–) PER2 TG mice was also analyzed. Furthermore, we report daily profile of core body temperature and sleep–wake cycle of NLD(–) PER2 TG and intact PER2 TG mice.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Inhibitory activity of NLD(–) PER2 on nuclear localization of mCRY proteins

In the previous report, we revealed that NLD(–) PER2 can inhibit the nuclear localization of not only NLD(–) PER2 itself but also of human CRY1 (hCRY1) (Miyazaki et al. 2001). Here, we tested whether NLD(–) PER2 inhibits the nuclear entry of mCRY1 and mCRY2. NLD(–) PER2 exogenously over-expressed with either mCRY1 or mCRY2 using COS1 cells, in which expression levels of endogenous clock genes, Per and Cry, are relatively lower than other cells. Both mCRY1 and mCRY2 were predominantly localized in nuclei (Supplementary Fig. S1F,H) when intact PER2 was co-transfected into COS1 cells (Supplementary Fig. S1B,D). When NLD(–) PER2 co-expressed with either mCRY1 or mCRY2, both mCRY1 and mCRY2 as well as NLD(–) PER2 were detected in the cytosol (Supplementary Fig. S1C,E,G and I). These data imply that NLD(–) PER2 also inhibits the nuclear entry of both of mCRY1 and mCRY2 (Kume et al. 1999; Miyazaki et al. 2001; Yagita et al. 2002), and that NLD(–) PER2 can function as a dominant negative form and inhibit the nuclear entry of both mCRY1 and mCRY2.

Generation of TG mice harboring NLD(–) or intact PER2 gene

To understand the role of the NLD of PER2 in circadian rhythm behavior, we generated NLD(–) PER2 or intact PER2 driven by a powerful promoter, namely the CMV-IE enhancer combined with the chicken ß-actin promoter (pCXN2 vector), in mice (Supplementary Fig. S2A). Linearized recombinant DNA was microinjected into fertilized eggs from C57BL6/SLC mice. Twelve (14%) TG lines harboring NLD(–) PER2 from 86 live offspring were identified by genomic PCR (Supplementary Fig. S2B) and seven of them expressed TG mRNA (Supplementary Fig. S2C). Ten (32%) TG lines harboring an intact PER2 gene were identified by PCR from 31 live offspring (Supplementary Fig. S2B), and RT-PCR confirmed that four of them expressed TG mRNA (Supplementary Fig. S2C). Expression levels of exogenous NLD(–) PER2 or intact PER2 were significantly higher than that of endogenous Per2 in brain, liver, kidney and muscle as shown in Supplementary Fig. S2D.

Circadian rhythm of locomotor behavior of TG mice harboring NLD(–) or intact PER2 genes

We postulated that either of over-expressed NLD(–) PER2 or intact PER2 can modulate the timing for the PER2/CRY complex to enter the nucleus and affect circadian rhythm generation in vivo. Over-expressed NLD(–) PER2 forms complexes with CRY and accumulates in the cytosol. Because of the decelerated nuclear entry of endogenous mCRYs, the nuclear entry of endogenous PER2/CRY complex becomes delayed, finally resulting in long periodicity. In contrast, the over-expression of intact PER2 might accelerate the nuclear entry of CRY proteins and advance the clock, finally inducing short periodicity. We tested this hypothesis by generating TG mice exogenously over-expressing either NLD(–) PER2 or intact PER2. To determine the effects of the Per2 transgene on the circadian system, we compared the circadian locomotor rhythms of the TG and control WT mice. The activities of animals entrained to a 12-h light, 12-h dark (LD) cycle did not significantly differ from those of WT controls, suggesting that the over-expression of either NLD(–) PER2 or intact PER2 does not affect the entrainment to external light cues. The free running rhythm of two of the TG lines expressing NLD(–) PER2, NLD(–) PER2-TG1 and PER2-TG2 under constant darkness was significantly longer (Fig. 1B,C and E) than that of control mice (Fig. 1A,E). Period length ({tau}) determined by analyzing {chi}2 periodgrams revealed an average circadian period length of 23.67 ± 0.03 h (mean ± SE; n = 10) for WT mice, 25.21 ± 0.15 h (n = 22) for TG1 and 24.86 ± 0.18 h (n = 12) for TG2 mice. In contrast, the period of a TG mice expressing intact PER2 was significantly shorter (22.95 ± 0.08 h; n = 24; Fig. 1D,E) than that of the WT control. The difference of period length between TG mice and WT controls were statistically significant (P > 0.00001). These data suggest that the per2 gene have a role for the period generation of locomotor rhythm in mice.


Figure 1
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  		Figure 1  Effect of NLD(–) and intact PER2 transgene on circadian locomotor activity rhythms. (A–D) Drinking activity of individual mice was double-plotted according to conventional methods. Animals were maintained on LD 12 : 12 cycles as indicated by bars above records for 10 days and then transferred to constant darkness at indicated by asterisks right of each record. Activity records of mice: WT (A), NLD(–) PER2 TG1 (B), NLD(–) PER2 TG2 (C), intact PER2 TG (D). (E) Effect of NLD(–) PER2 and intact PER2 transgene on circadian period. Free-running period was estimated by {chi}2 periodgrams from days 10 to 20 under constant dark (DD). Means and SEM of each genotype are illustrated. Sample sizes (N) are as follows: WT, n = 10; NLD(–) PER2 TG1, n = 22; NLD(–) PER2 TG2, n = 12; intact PER2 TG, n = 24. Asterisks indicate significant differences in period length of TG vs. WT mice by one-way ANOVA analysis, with P < 0.00001 as criterion for significance.

 
Retarded nuclear entry of endogenous PER2, CRY1 and CRY2 in the SCN of NLD(–) PER2 TG mice

We postulated that NLD(–) PER2 protein over-expression inhibits the nuclear entry of endogenous clock proteins, including mPER2 and mCRYs. To determine whether the nuclear localization of endogenous PER2 is affected in the SCN of TG mice, we assessed the nuclear localization of mPER2 protein by immunohistochemistry using an anti-mPER2 antibody, which recognizes endogenous PER2 but neither exogenous NLD(–) or intact rPER2 (Supplementary Fig. S3). Under constant darkness, nuclear staining has revealed robust circadian rhythms of mPER2 immunoreactivity in the mouse SCN (Shearman et al. 2000; Maywood et al. 2003). Robust circadian nuclear accumulation of mPER2 was maintained in both WT and TG mice (Fig. 2). But, mPER2 staining peaked later in nuclei from the SCN of NLD(–) PER2 TG mice at 62 and 66 h after placing under constant darkness, compared with WT animals at 58 and 62 h (Fig. 2). This is consistent with the differences in the length of the free-running period (Fig. 1). In contrast, mPER2 staining in nuclei of SCN in intact PER2 TG peaked at 54 and 58 h. These findings suggest that the timing of the nuclear entry of mPER2 was delayed in NLD(–) PER2 TG mice and accelerated in intact PER2 TG mice.


Figure 2
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  		Figure 2  Immunohistochemical analysis of mPER2 nuclear entry in the SCN of WT, NLD(–) PER2 TG and intact PER2 TG mice. Shown are representative micrographs of SCN sections immnostained anti-mPER2 antibody (ADI). NLD(–) PER2 TG1, TG2, WT control, and intact PER2 TG mice were sacrificed and fixed at indicated times after transition to constant darkness (four mice per time point). Coronal brain sections above the optic chiasma were reacted with anti-mPER2 antibody and visualized using Alexa488-anti-rabbit IgG antibody.

 
In vitro over-expression of NLD(–) PER2 inhibited the nuclear entry of either CRY1 or CRY2 (Supplementary Fig. S1). To determine whether the nuclear entry of CRY1 and CRY2 is affected even in vivo, we also examined the nuclear accumulation of CRY1 and CRY2 in the SCN of NLD(–) PER2 TG mice using mCRY1 and mCRY2 antibodies. The nuclear entry of mCRY1 (Supplementary Fig. S4) and mCRY2 (data not shown) in the SCN of TG mice was also delayed like that of PER2. These data suggested that NLD(–) PER2 decelerates the nuclear translocation of the PER2/CRY complex.

Analysis of circadian locomotor activity by NLD(–) PER2 TG mice under constant light

Recent reports showed that constant light (LL) conditions affect PER2 expression in the SCN (Steinlechner et al. 2002; Munoz et al. 2005). Thus, we assessed the circadian phenotype of NLD(–) PER2 TG mice exposed to constant light by monitoring the periodicity of locomotor activity. Under LL conditions, all of the WT animals displayed a circadian rhythmicity with a period length of over 24 h as reported (Fig. 3C) (Steinlechner et al. 2002). In contrast, 6 of 11 NLD(–) PER2 TG1 and 8 of 10 NLD(–) PER2 TG2 became arrhythmic under LL conditions (Fig. 3A, B and D). {chi}2 periodgrams analysis revealed that 5 of 11 NLD(–) PER2 TG1 and 2 of 10 NLD(–) PER2 TG2 exhibit obscure circadian rhythm than WT control (data not shown). These results suggest that PER2 also have a role for the proper rhythm generation under constant light. We do not know the mechanism for the arrhythmicity of NLD(–) PER2 TG, but the arrhythmicity of NLD(–) PER2 TG mice under LL conditions might affect cellular communication between the SCN cells, because one study has already shown that constant light disturbs circadian behavior rhythms by disrupting such cellular communication (Ohta et al. 2005). It is possible that interaction between endogenous clock components and exogenous NLD(–) PER2 in NLD(–) PER2 TG mice may have disrupted molecular clock mechanism under LL conditions, leading to behavioral arrhythmicity in the TG mice.


Figure 3
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  		Figure 3  Representative locomotor activity records of NLD(–) PER2 TG1 (A), TG2 (B) and WT control mice (C) under LL conditions. For the first 10 days, animals were housed under cycle of LD 12 : 12 as indicated by bars above each record. Animals were then transferred to LL as indicated by asterisks right of each record. (D) Arrhythmicity was determined by {chi}2 periodgram analysis (statistical significance at P < 0.05).

 
Daily profile of core body temperature and sleep–wake cycle of NLD(–) PER2 TG and intact PER2 TG mice

Recent experimental studies have demonstrated important links between sleep architecture and circadian rhythms (Mignot & Takahashi 2007). We compared the rhythm of core body temperature among NLD(–) PER2, intact PER2 TG and WT control mice (Fig. 4A,B). Fluctuations in the body temperature of intact PER2 TG mice significantly differed from those of NLD(–) PER2 TG and control mice. The temperature of intact PER2 TG mice started to increase earlier, at the end of light period. During the late dark period, body temperature remained high in intact PER2 TG mice, but started to decrease in NLD(–) PER2 TG and WT mice. The different profile of body temperature between intact PER2 TG and WT control was statistically significant by ANOVA followed by post-hoc Scheffe-test (P < 0.05) as shown in asterisks in Fig. 4A. Furthermore, the body temperature of intact PER2 TG mice was higher in overall day period (Fig. 4A).


Figure 4
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  		Figure 4  Daily profiles (mean ± SEM) of body temperature (A, B) and wake (C, D) under LD 12 : 12 cycle conditions in intact PER2 TG (A and C, red circle with red line; n = 4) NLD(–) PER2 TG1 (B and D, red circle with red line; n = 7) and WT control (A–D, open circle with black line; n = 6) mice. Following electrode implantation, sleep–wake EEG was recorded. Sleep was scored by a visual assessment of EEG and electromyograms for each 5 s epoch. Asterisks indicate significant differences in body temperature and wake time of TG vs. WT mice by ANOVA followed by post-hoc Scheffe-test, with P < 0.05 as criterion for significance.

 
Daily changes in wake, non-rapid and rapid eye movement (NREM and REM, respectively) sleep recordings in NLD(–)PER2 TG, intact PER2 TG and control mice are shown in Fig. 4C,D, and Supplementary Fig. S5. Corresponding to the daily changes in body temperature, intact PER2 TG mice significantly awakened earlier at the end of light period and did not sleep during the late dark period as shown in asterisks in Fig. 4A (P < 0.05). Furthermore, exogenously expressed intact PER2 significantly reduced total sleep duration (Supplementary Fig. S5A). Following sleep deprivation, NREM delta power in recovery sleep was smaller in intact PER2 TG mice (data not shown). The results indicated that PER2 over-expression changes the regulation of sleep architecture and preference for sleep. However, the daily profiles of sleep–wake cycles did not significantly differ between NLD(–) PER2 TG and WT control mice (Fig. 4D and Supplementary Fig. S5B,D).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The present study focused on the function of PER2 nuclear entry for rhythm generation in vivo. When intact PER2 was expressed with mCRY1 or mCRY2 in COS-1 cells, both PER2/mCRY1 and PER2/mCRY2 complex localized in nuclei (Supplementary Fig. S1), as similar to previous reports (Kume et al. 1999; Miyazaki et al. 2001; Yagita et al. 2002). The accumulations of these complexes were changed from nuclei to cytosol by expression of NLD(–) PER2 (Supplementary Fig. S1). These data suggested that NLD(–) PER2 can function as dominant negative form and inhibits nuclear entry of both mCRY1 and mCRY2.

As shown in Fig. 5, we hypothesized over-expressed either NLD(–) PER2 or intact PER2 can modulate a timing for nuclear entry of PER2/CRY complex and effect on circadian rhythm generation of period in vivo. Over-expressed NLD(–) PER2 binds with CRY and the complexes accumulate in the cytosol. As a result of decelerated nuclear entry of endogenous mCRYs, the nuclear entry of endogenous PER2/CRY complex becomes delayed, resulting in long periodicity of behavior. In contrast, the over-expression of intact PER2 might accelerate the nuclear entry of CRY proteins and advance the clock, finally inducing short periodicity. To test this hypothesis, we generated TG mice exogenously over-expressing either NLD(–) PER2 or intact PER2.


Figure 5
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  		Figure 5  Schematic representation of post-translational events induced by exogenously over-expressed NLD(–) PER2 or intact PER2 in vivo. Left panel: over-expressed NLD(–) PER2 binds with CRYs and traps the complex in cytosol. As a result, nuclear entry of endogenous PER2/CRY complex is decelerate, clock delays and long periodicity is induced. Right panel: over-expressed intact PER2 accelerate nuclear entry of CRY, clock advances and short periodicity is induced.

 
The over-expression of NLD(–) PER2 in TG mice significantly lengthened the circadian period in constant darkness. In contrast, TG mice over-expressing full-length PER2 exhibited shorter periodicity (Fig. 1). We also found the nuclear entry of endogenous PER2 in SCN was delayed in NLD(–) PER2 TG compared with WT controls. We thus concluded that the extended periodicity of NLD(–) PER2 TG is due to the decelerated nuclear entry of endogenous PER2 protein, which in turn induced a delay of the clock (Fig. 2). Furthermore, NLD(–) PER2 can inhibit the nuclear entry of CRY even in vivo (Supplementary Fig. S4) as similar to in vitro (Supplementary Fig. S1). In contrast, intact PER2 promotes the nuclear entry of endogenous PER2 and CRY in vivo. The data indicated that NLD(–) PER2 protein is an apparent rate limiting factor for the nuclear entry of CRY proteins in vivo as well as in vitro and that PER2 plays an important role in determining period length.

Mutations in the mPer2 gene, mPer2Brdm1 and mPer2Idc elicit a short period following by a loss of circadian rhythmicity (Zheng et al. 1999; Bae et al. 2001). In contrast, the locomotion of NLD(–) PER2 TG mice is rhythmic with long periodicity. The gene expression of several clock genes, including Per1, Cry1 and Bmal1 is suppressed in mPer2Brdm1 and mPer2Idc knockout mutants (Zheng et al. 2001; Oster et al. 2002). In contrast, NLD(–) PER2 TG did not affect the expression levels of endogenous PER2 protein (Fig. 2) and of other clock genes, including Per1, Bmal and Dbp (K. Miyazaki, unpublished data). These differences might explain the contrary effects on the circadian periodicity of each genotype. Exogenously expressed NLD(–) PER2 and intact PER2 can modulate the function of other endogenous clock components than CRY, such as PER1/3 and BMAL1 (Lee et al. 2001), and change the periodicity.

A circadian profile of the gene expression of bmal1, per1 and dbp, in NLD(–) PER2 gene was similar to that of WT control (K. Miyazaki, unpublished data). The expression of bmal1, per1 and dbp are controlled by RORE- and E box-dependent transcriptional regulation with ROR/Rev-Erb{alpha} or CLOCK/BMAL complex, respectively (Sato et al. 2006). Our result suggests that NLD(–) PER2 may not affect a timing of both E box- and RORE-dependent transcriptional regulation.

Under constant light conditions, most of NLD(–) PER2 TGs became behaviorally arrhythmic, whereas all of WT control mice were rhythmic. The remainder of NLD(–) PER2 TG displayed a weak rhythm with a significantly longer period than WT control animals. This indicated that NLD(–) PER2 disturbed the generation of circadian rhythmicity under constant light. A recent report has indicated that constant light disturbs circadian behavior rhythms by disrupting the cellular communication of the SCN clock cells (Ohta et al. 2005). The arrhythmicity of NLD(–) PER2 TG under constant light suggests that exogenously expressed NLD(–) PER2 may affects the cellular communication of the SCN clock. Long-term constant light induced constitutive elevated expression PER2 protein in the mouse SCN by inhibiting the degradation of PER2 and then modulate the circadian oscillator (Munoz et al. 2005). It is possible that interaction between endogenous clock components PER2 induced by constant light and exogenous NLD(–) PER2 in TG mice may explain the behavioral arrhythmicity under LL conditions in the TG mice.

The period length of Drosophila correlates with dPeriod RNA expression level, so period length of locomotor was shorter in accordance with a higher expression levels of Period mRNA (Baylies et al. 1987). The period of locomotor activities of TG mice over-expressing intact and NLD(–) PER2 were, respectively, short and long. The data may be explained by mRNA expression level of Per2, because NLD(–) PER2 have a dominant negative effect. Unfortunately we could not generate another TG line with different expression levels of NLD(–) PER2 or intact PER2 in brain.

Nucleocytoplasmic shuttling of PER/CRY complex regulates their own protein stability, such as ubiquitylation and degradation (Yagita et al. 2002; Miyazaki et al. 2003). In addition to modulation of nuclear entry timing, NLD(–) PER2 and intact PER2 may affect a CRY protein stability by modulating subcellular localization. Casein kinase I{varepsilon} phosphorylates and controls the destabilization of PER2 (Toh et al. 2001; Eide et al. 2005). Contrary, a phosphorylation at Ser 659, which is mutated in patients suffering from FASPS, was reported to result in nuclear localization and stabilization of mPER2 (Vanselow et al. 2006). NLD in rPER2 includes 10 of 21 serine or threonin residues phosphorylated endogenously. Another explanation for the long periodicity of NLD(–) PER2 TG is that exogenously expressed NLD(–) PER2 may affect the intact phosphorylation and degradation of PER2, because NLD of PER2 includes both casein kinase I{varepsilon} binding and phosphorylation domains.

Per genes are considered to contribute individual differences in sleep timing by affecting circadian rhythmicity (Toh et al. 2001), but not sleep homeostasis (Kopp et al. 2002; Shiromani et al. 2004). Intact PER2 TG mice were significantly affected on a rhythm of sleep and wakefulness distribution across the 24 h period with reducing total sleep time. Intact PER2 TG woke up earlier than WT control at the end of light regime due to short periodicity. In addition, a sleeping at the end of night regime was disappeared in intact PER2 TG mice. These results indicate that exogenously expressed intact PER2 affects not only sleep timing by affecting circadian rhythmicity but also sleep architecture. Deletion of Bmal1 attenuated rhythm of sleep and wakefulness distribution across the 24 h period, and increase in total sleep time (Laposky et al. 2005). Circadian clock components can function not only in circadian rhythm generation but also in sleep homeostasis.

Circadian periodicity was significantly affected in TG mice exogenously expressing intact PER2 or NLD(–) PER2. We concluded that PER2 is a key factor in the generation of a proper circadian period through the nuclear localization in SCN. Our findings also suggested that PER2 expression affects daily changes in body temperature and homeostatic regulation of sleep in mice. These TG mice will provide novel insights into the mechanism and function of PER2 in rhythms and sleep generation.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
TG mice and behavioral analysis

The Animal Care and Use Committee at AIST approved this study. PER and NLD(–) PER2 expression cassettes for transgenes were excised from the respective reported plasmids (Supplementary Fig. S2A and Miyazaki et al. 2001). Transgenic animals were generated according to standard protocols. Genotypes were determined by PCR analysis of genomic DNA extracted from mouse tails using the primers, 5'-actcaggctatgaagctcctaga and 5'-gtgcagagggacagctagac (Supplementary Fig. S2B). The expression of mRNA transcribed from the transgenes was examined using a one-step RT-PCR kit (Invitrogen, Carlsbad, CA) and the primer pair as described above or Northern blotting as reported (Sakamoto et al. 1998).

Behavior was analyzed as described (Oishi et al. 2002). Drinking activities were monitored using an online personal computer equipped with a Chronobiology Kit (Stanford Software Systems, Santa Cruz, CA). Mice were initially maintained for approximately 10 days under a 12 h light, 12 h dark (LD) regime and then transferred to constant dark (DD) or LL (approximately 300 lux) conditions. To determine period length, activity data for a 10-day interval upon release into DD and LL were analyzed using a {chi}2 periodgram. Differences between groups were evaluated by ANOVA and values were considered significantly different at P < 0.0001.

Immunostaining

We generated rabbit anti-mCRY1 and mCRY2 antiserum. Purified recombinant fragments of mCRY1 (amino acid residue numbers 438–606) and mCRY2 (amino acid residue numbers 440–592) were, respectively, used as immunogens.

Subcellular localization of over-expressed PER2 and CRYs in COS-1 cells were analyzed according to a previous report (Miyazaki et al. 2001).

For the analysis of nuclear entry of endogenous PER2, CRY1 and CRY2 in SCN, brains were perfusion-fixed at indicated time after transfer to constant dark condition (four mice at each time point) for immunohistochemistry as described (Sakamoto et al. 1998; Oishi et al. 2002). After cryopreservation overnight in 20% sucrose in PBS, 8 µm brain slices cut in a Leica cryostat were washed twice in Tris-buffered saline containing 0.3% Triton X-100 (TBS-T). The sections on slides were washed with TBS-T twice, blocked in TBS-T containing 10% horse serum, incubated with affinity purified anti-mPER2 antibody (ADI, San Antonio, TX) overnight at 4 °C. Species specificity of anti-mPER2 was examined by immunostaining of Cos-1 cells over-expressed PER2 or NLD(–) PER2 with this antibody. Exogenously expressed intact PER2 and NLD(–) PER2 could be detected by anti-rPER2 but not anti-mPER2 (Supplementary Fig. S3). Serial sections were also reacted with anti-mCRY1 antiserum diluted 1 : 1000. Primary antibodies were visualized using Alexa Fluor 488-conjugated anti-rabbit IgG (Invitrogen).

Telemetric recordings of body temperature and sleep

Body temperature and sleep rhythms were telemetrically recorded as described (Sei et al. 2001). Time course data during sleep and core body temperature were analyzed using analysis of variance (ANOVA) followed by post-hoc Scheffe-test.


   Acknowledgements
 
We thank K. Oishi and H. Ohta for helpful discussion, K. Suzuki, J. Narukawa, I. Kodomari, and Y. Hara for excellent technical assistance and I. Shibasaki for maintaining the mice. This study was supported by an Industrial Technology Research Grant from NEDO and an AIST Internal Grant.


   Footnotes
 
Communicated by: Masayuki Yamamoto (Tohoku University)

* Correspondence: E-mail: n.ishida{at}aist.go.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Bae, K., Jin, X., Maywood, E.S., Hastings, M.H., Reppert, S.M. & Weaver, D.R. (2001) Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock. Neuron 30, 525–536.[CrossRef][Medline]

Baylies, M.K., Bargiello, T.A., Jackson, F.R. & Young, M.W. (1987) Changes in abundance or structure of the per gene product can alter periodicity of the Drosophila clock. Nature 326, 390–392.[CrossRef][Medline]

Dunlap, J.C. (1999) Molecular bases for circadian clocks. Cell 96, 271–290.[CrossRef][Medline]

Eide, E.J., Woolf, M.F., Kang, H., Woolf, P., Hurst, W., Camacho, F., Vielhaber, E.L., Giovanni, A. & Virshup, D.M. (2005) Control of mammalian circadian rhythm by CKI{varepsilon}-regulated proteasome-mediated PER2 degradation. Mol. Cell. Biol. 25, 2795–2807.[Abstract/Free Full Text]

Ishida, N., Miyazaki, K. & Sakai, T. (2001) Circadian rhythm biochemistry: from protein degradation to sleep and mating. Biochem. Biophys. Res. Commun. 286, 1–5.[CrossRef][Medline]

Kopp, C., Albrecht, U., Zheng, B. & Tobler, I. (2002) Homeostatic sleep regulation is preserved in mPer1 and mPer2 mutant mice. Eur. J. Neurosci. 16, 1099–1106.[CrossRef][Medline]

Kume, K., Zylka, M.J., Sriram, S., Shearman, L.P., Weaver, D.R., Jin, X., Maywood, E.S., Hastings, M.H. & Reppert, S.M. (1999) mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98, 193–205.[CrossRef][Medline]

Laposky, A., Easton, A., Dugovic, C., Walisser, J., Bradfield, C. & Turek, F. (2005) Deletion of the mammalian circadian clock gene BMAL1/Mop3 alters baseline sleep architecture and the response to sleep deprivation. Sleep 28, 395–409.[Medline]

Lee, C., Etchegaray, J.P., Cagampang, F.R., Loudon, A.S. & Reppert, S.M. (2001) Posttranslational mechanisms regulate the mammalian circadian clock. Cell 107, 855–867.[CrossRef][Medline]

Maywood, E.S., O’Brien, J.A. & Hastings, M.H. (2003) Expression of mCLOCK and other circadian clock-relevant proteins in the mouse suprachiasmatic nuclei. J. Neuroendocrinol. 15, 329–334.[CrossRef][Medline]

Mignot, E. & Takahashi, J.S. (2007) A circadian sleep disorder reveals a complex clock. Cell 128, 22–23.[CrossRef][Medline]

Miyazaki, K., Mesaki, M. & Ishida, N. (2001) Nuclear entry mechanism of rat PER2 (rPER2): role of rPER2 in nuclear localization of CRY protein. Mol. Cell. Biol. 21, 6651–6659.[Abstract/Free Full Text]

Miyazaki, K., Mezaki, M. & Ishida, N. (2003) The role of phosphorylation and degradation of hPER protein oscillation in normal human fibroblasts. Novartis. Found. Symp. 253, 238–248; discussion 249.[Medline]

Munoz, M., Peirson, S.N., Hankins, M.W. & Foster, R.G. (2005) Long-term constant light induces constitutive elevated expression of mPER2 protein in the murine SCN: a molecular basis for Aschoff's rule? J. Biol. Rhythms 20, 3–14.[Abstract/Free Full Text]

Ohta, H., Yamazaki, S. & McMahon, D.G. (2005) Constant light desynchronizes mammalian clock neurons. Nat. Neurosci. 8, 267–269.[CrossRef][Medline]

Oishi, K., Miyazaki, K. & Ishida, N. (2002) Functional CLOCK is not involved in the entrainment of peripheral clocks to the restricted feeding: entrainable expression of mPer2 and BMAL1 mRNAs in the heart of Clock mutant mice on Jcl:ICR background. Biochem. Biophys. Res. Commun. 298, 198–202.[CrossRef][Medline]

Oster, H., Yasui, A., van der Horst, G.T. & Albrecht, U. (2002) Disruption of mCry2 restores circadian rhythmicity in mPer2 mutant mice. Genes Dev. 16, 2633–2638.[Abstract/Free Full Text]

Sakamoto, K., Nagase, T., Fukui, H., Horikawa, K., Okada, T., Tanaka, H., Sato, K., Miyake, Y., Ohara, O., Kako, K. & Ishida, N. (1998) Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain. J. Biol. Chem. 273, 27039–27042.[Abstract/Free Full Text]

Saper, C.B., Scammell, T.E. & Lu, J. (2005) Hypothalamic regulation of sleep and circadian rhythms. Nature 437, 1257–1263.[CrossRef][Medline]

Sato, T.K., Yamada, R.G., Ukai, H., Baggs, J.E., Miraglia, L.J., Kobayashi, T.J., Welsh, D.K., Kay, S.A., Ueda, H.R. & Hogenesch, J.B. (2006) Feedback repression is required for mammalian circadian clock function. Nat. Genet. 38, 312–319.[CrossRef][Medline]

Sei, H., Oishi, K., Morita, Y. & Ishida, N. (2001) Mouse model for morningness/eveningness. Neuroreport 12, 1461–1464.[CrossRef][Medline]

Shearman, L.P., Sriram, S., Weaver, D.R., Maywood, E.S., Chaves, I., Zheng, B., Kume, K., Lee, C.C., van der Horst, G.T., Hastings, M.H. & Reppert, S.M. (2000) Interacting molecular loops in the mammalian circadian clock. Science 288, 1013–1019.[Abstract/Free Full Text]

Shiromani, P.J., Xu, M., Winston, E.M., Shiromani, S.N., Gerashchenko, D. & Weaver, D.R. (2004) Sleep rhythmicity and homeostasis in mice with targeted disruption of mPeriod genes. Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R47–R57.[Abstract/Free Full Text]

Steinlechner, S., Jacobmeier, B., Scherbarth, F., Dernbach, H., Kruse, F. & Albrecht, U. (2002) Robust circadian rhythmicity of Per1 and Per2 mutant mice in constant light, and dynamics of Per1 and Per2 gene expression under long and short photoperiods. J. Biol. Rhythms 17, 202–209.[Abstract/Free Full Text]

Tafti, M., Maret, S. & Dauvilliers, Y. (2005) Genes for normal sleep and sleep disorders. Ann. Med. 37, 580–589.[CrossRef][Medline]

Toh, K.L., Jones, C.R., He, Y., Eide, E.J., Hinz, W.A., Virshup, D.M., Ptacek, L.J. & Fu, Y.H. (2001) An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome. Science 291, 1040–1043.[Abstract/Free Full Text]

Vanselow, K., Vanselow, J.T., Westermark, P.O., Reischl, S., Maier, B., Korte, T., Herrmann, A., Herzel, H., Schlosser, A. & Kramer, A. (2006) Differential effects of PER2 phosphorylation: molecular basis for the human familial advanced sleep phase syndrome (FASPS). Genes Dev. 20, 2660–2672.[Abstract/Free Full Text]

Weaver, D.R. (1998) The suprachiasmatic nucleus: a 25-year retrospective. J. Biol. Rhythms 13, 100–112.[Abstract/Free Full Text]

Wisor, J.P. & Kilduff, T.S. (2005) Molecular genetic advances in sleep research and their relevance to sleep medicine. Sleep 28, 357–367.[Medline]

Yagita, K., Tamanini, F., Yasuda, M., Hoeijmakers, J.H., van der Horst, G.T. & Okamura, H. (2002) Nucleocytoplasmic shuttling and mCRY-dependent inhibition of ubiquitylation of the mPER2 clock protein. EMBO J. 21, 1301–1314.[CrossRef][Medline]

Zheng, B., Albrecht, U., Kaasik, K., Sage, M., Lu, W., Vaishnav, S., Li, Q., Sun, Z.S., Eichele, G., Bradley, A. & Lee, C.C. (2001) Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock. Cell 105, 683–694.[CrossRef][Medline]

Zheng, B., Larkin, D.W., Albrecht, U., Sun, Z.S., Sage, M., Eichele, G., Lee, C.C. & Bradley, A. (1999) The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature 400, 169–173.[CrossRef][Medline]

Received: 2 May 2007
Accepted: 30 July 2007




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