PER1 Phosphorylation Specifies Feeding Rhythm in Mice

Please cite this article in press as: Liu et al., PER1 Phosphorylation Specifies Feeding Rhythm in Mice, Cell Reports (2014), http://dx.doi.org/10.1016/ j.celrep.2014.04.032

Cell Reports

Article PER1 Phosphorylation Specifies Feeding Rhythm in Mice Zhiwei Liu,1,5 Moli Huang,2,5 Xi Wu,1 Guangsen Shi,1 Lijuan Xing,1 Zhen Dong,1 Zhipeng Qu,1 Jie Yan,1,2 Ling Yang,2 Satchidananda Panda,3,* and Ying Xu1,2,4,* 1MOE

Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, Nanjing 210061, China Suda Genome Resource Center, Soochow University, Suzhou 215006, China 3Salk Institute for Biological Studies, La Jolla, CA 92037, USA 4Collaborative Innovation Center for Genetics and Development, Fudan University, Shanghai 200433, China 5Co-first author *Correspondence: [email protected] (S.P.), [email protected] (Y.X.) http://dx.doi.org/10.1016/j.celrep.2014.04.032 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). 2Cambridge

SUMMARY

Organization of circadian behavior, physiology, and metabolism is important for human health. An S662G mutation in hPER2 has been linked to familial advanced sleep-phase syndrome (FASPS). Although the paralogous phosphorylation site S714 in PER1 is conserved in mice, its specific function in circadian organization remains unknown. Here, we find that the PER1S714G mutation accelerates the molecular feedback loop. Furthermore, hPER1S714G mice, but not hPER2S662G mice, exhibit peak time of food intake that is several hours before daily energy expenditure peaks. Both the advanced feeding behavior and the accelerated clock disrupt the phase of expression of several key metabolic regulators in the liver and adipose tissue. Consequently, hPER1S714G mice rapidly develop obesity on a highfat diet. Our studies demonstrate that PER1 and PER2 are linked to different downstream pathways and that PER1 maintains coherence between the circadian clock and energy metabolism.

INTRODUCTION The circadian clock orchestrates the daily rhythms in physiology and behaviors that allow organisms to anticipate regular environmental cycles and increase their adaptive fitness. The current mammalian clock model is composed of a transcriptional-translational feedback network that includes the Per-Amt-Simdomain-containing helix-loop-helix transcription factors Clock and Bmal1, the Period genes (Per1, Per2, and Per3), and the cryptochrome genes (Cry1 and Cry2). The CLOCK:BMAL1 complex activates the transcription of the period and cryptochrome genes by binding to E-boxes in their promoters, whereas the PER:CRY complex closes the negative feedback loop by repressing the activity of CLOCK:BMAL1 (Lowrey and Takahashi, 2004; Reppert and Weaver, 2002; Schibler and Naef, 2005).

Bmal1 and Cry1 expression is also regulated by a secondary feedback loop comprised of the nuclear hormone receptors Rev-Erba/Rev-Erbb and the retinoid-related orphan receptors (Preitner et al., 2002; Sato et al., 2004; Ueda et al., 2005; UkaiTadenuma et al., 2011). Collectively, these complexes sustain the endogenous circadian oscillators. Although the basic function of the molecular clock is conserved, mammals employ multiple paralogous clock genes. Analysis of Per1/Per2, Cry1/Cry2, and Rev-erba/Rev-erbb double knockout mice indicated that these family genes exhibited a certain degree of redundancy in sustaining rhythms; however, important differences among family genes do exist (Bae et al., 2001; Cho et al., 2012; van der Horst et al., 1999; Vitaterna et al., 1999). Clock mutations significantly affect many physiological processes and have been linked to previously unexpected physiological function, even in the members of a gene family (Ko and Takahashi, 2006; Liu et al., 2008), suggesting that the expansion of the clock gene members has acquired additional functions in complex mammalian physiology rather than simple redundancy to confer robustness. Posttranslational modifications significantly contribute to the precision of the mammalian clock. However, the specific roles of these posttranslational modifications in shaping the period, amplitude, and phase of the oscillator and the consequent effects on wholeanimal physiology remain largely unexplored. Phosphorylation of clock components determines the rate of protein accumulation, nuclear-cytoplasmic distribution, and their degradation, thus setting the robustness, pace, and phase of the circadian oscillator, which ultimately translates to change in the overt circadian rhythms. Among the PER proteins, individuals carrying an S662G mutation in hPER2 exhibit familial advanced sleep-phase syndrome (FASPS) (Jones et al., 1999). The S662 position of the hPER2 protein is the first of five serines spaced three amino acids apart, and the S662G mutation impedes sequential phosphorylation by casein kinase and alters the protein stability (Toh et al., 2001; Xu et al., 2007). The SXXS motif is highly conserved in mammalian PER proteins (Figure S1A; Toh et al., 2001), and this serial phosphorylation of the PER2 protein shows striking temporal changes (Xu et al., 2007). We thus hypothesize that (1) this motif arose before duplications to retain the essential time-keeping function and similar Cell Reports 7, 1–12, June 12, 2014 ª2014 The Authors 1

Please cite this article in press as: Liu et al., PER1 Phosphorylation Specifies Feeding Rhythm in Mice, Cell Reports (2014), http://dx.doi.org/10.1016/ j.celrep.2014.04.032

A

PER1S714 L

τ = 23.73 PER1S714 H

C

τ = 23.67

1 Normalized bioluminescence intensity

0 LD DD day

30

τ = 23.36 PER1S714G H

PER1S714G L

τ = 22.67

0 LD DD

adipose

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spleen days 1

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-1 days

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PER1S714H PER1S714G H PER2S662G

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PER1S714G H;Per1-/-τ = 21.8 Per1 -/-

τ = 23.33

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PE R 1S 71 PE 4 R PE 1 S7 L 14 R PE 1 S7 H 14 R 1S PE 71 GL 4G R 1S 71 P H 4G er 1 -/ H ; Pe PE r1 R 2 S6 /62 G

24.0 23.5 23.0 22.5 22.0 21.5 21.0

p < 0.001 p < 0.001

Relative mRNA intensity

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SCN

adipose lung liver 35 35 12 30 30 10 25 25 8 20 20 6 15 15 4 10 10 5 2 5 0 0 0 spleen 2.0 80 Hypothalamus ZT 0 4 8 12162024 1.6 mPer1 60 1.2 mPer2 40 0.8 20 0.4 0 0 ZT 0 4 8 12162024 ZT 0 4 8 12162024

Figure 1. The PER1S714G Mutation Impairs Normal Circadian Oscillator Function (A) Representative actograms of locomotor activity in mice. The mice were entrained to a 12 hr light/12 hr dark (LD) cycle for 7–10 days and then maintained in constant darkness (DD). The red lines represent the phase of activity onset in constant darkness. The period length from days 8 and 21 in DD was calculated using ClockLab. (B) Average period length (±SD) quantification of the indicated mouse lines. The indicated p value was determined using Student’s t test. (C) Representative Per2Luc bioluminescence traces of explants from hPER1S714H (green), hPER1S714GH (red), and hPER2S662G (blue) mice. The explants were prepared within 1 hr before dark onset. The indicated time windows were used to determine the period over 3 consecutive days. (D) The period and phase in different tissues and genotypes are shown as mean ± SD. The indicated sample sizes (i.e., the number of rhythmic tissues) are from three mice for each genotype. One-way ANOVA indicated a significant difference between hPER1S714H and hPER1S714GH or hPER2S662G mice. Black stars

(legend continued on next page)

2 Cell Reports 7, 1–12, June 12, 2014 ª2014 The Authors

Please cite this article in press as: Liu et al., PER1 Phosphorylation Specifies Feeding Rhythm in Mice, Cell Reports (2014), http://dx.doi.org/10.1016/ j.celrep.2014.04.032

selective pressure constraints on this motif and (2) PER1 phosphorylation may be critical for maintaining a physiologic process that is properly attuned to the environmental light-dark cycle. Therefore, mutational phenotype may be observed through an amino acid change in the first serine of this motif in the hPER1 protein by applying the general principles learned from PER2 regulation. Here, we generated bacterial artificial chromosome (BAC) transgenic mice carrying an S714G mutation in the hPER1 and found that S714 in hPER1 is a key site that drives behavioral rhythms in food intake and plays a critical role in physiological optimization for feeding behavior and energy expenditure. RESULTS Generation and Characterization of hPER1 Mutant Mice Constitutive expression from nonnative promoters is known to dampen circadian oscillation (Chen et al., 2009; Numano et al., 2006), thus precluding the ability to distinguish the effects of posttranslational modifications from transcriptional control. Therefore, to explore the specific role of PER1 phosphorylation, we generated BAC transgenic mice in C57BL/6J background carrying either an S714G mutation or the wild-type followed by a C-terminal Myc-tag (Figure S1B). We used a human transgene because it is highly homologous to the mouse gene (99% identity) and can be identified easily. The resultant BAC construct included the promoter and a large flanking region (which extended 85 kb upstream and 50 kb downstream) that contained known cis-acting regulatory elements to recapitulate the expression pattern of the endogenous loci (Maston et al., 2006). We obtained 17 founders for wild-type and mutant lines. After identifying copy numbers with six probes covering whole hPER1 gene using quantitative PCR, we selected two wildtype lines (L:1-2 copy and H:6-8 copy) and five S714G mutant lines (L1:2 copy; H:3-4 copy; L2:1-2 copy; L3:1 copy; and L4:1 copy) for further characterization. The copy numbers of seven lines were further confirmed using Southern blot analysis (Figure S1C). One low-copy line from S714G mutation did not go germline. Other three low-copy mutant lines show similar phenotypes referred as hPERS714GL and high copy referred as hPER1S714GH corresponding to wild-type hPER1S714L and hPER1S714H, respectively. Similar phenotype among low- or high-copy-number lines implied negligible effect of site of integration on the phenotype. We analyzed their circadian wheel-running activity rhythms. As expected from the known dosage effect of Per genes on overt rhythms (Zheng et al., 1999, 2001), hPER2 transgenic mice exhibited a dose-dependent period elongation under constant darkness (Gu et al., 2012). However, hPER1 transgenic mice carrying low- or high-copy numbers of the hPER1 gene exhibited no differences in activity rhythms (hPER1S714L: t = 23.73 ± 0.16; hPER1S714H: t = 23.72 ± 0.14; Figures 1A and 1B), suggesting that the copy number of the wild-type (WT) PER1 allele and its

genomic location does not discernibly affect the pace of the oscillator. hPER1S714G mice exhibited a dose-dependent shortening of their activity rhythm (hPERS714GL: t = 23.36 ± 0.26; hPER1S714GH: t = 22.67 ± 0.16; Figures 1A and 1B), suggesting that posttranslational regulation plays a dominant role in the kinetics of PER1 oscillation, which in turn alters the periodicity of overt rhythms. Notably, the hPER1S714G mutation (either low or high copy) resulted in a milder locomotor period defect than the hPER2S662G mutant mice (one to two copies; Xu et al., 2007; Figure 1B). hPER1S714GH; Per1 / mice exhibited a shorter period than Per1 / mice (Zheng et al., 2001; Figure 1A), which further indicated the dominant role of phosphorylation in setting the periodicity of the clock. Given the prominent role of Per1 in sustaining robust rhythms in tissue-autonomous oscillators (Liu et al., 2007), we bred hPER1S714H, hPER1S714GH, or hPER2S662G mice with Per2Luc knockin reporter mice (Yoo et al., 2004). Longitudinal monitoring of luciferase activity from suprachiasmatic nuclei (SCN), lung, liver, adipose, and spleen tissues revealed marked shortening of the period and advanced phase by hPER1S714GH (red; Figures 1C and 1D). The short-period phenotype was distinct from the less persistent rhythms found in Per1 / adipose tissues, further verifying that both the whole-animal and tissue-level phenotypes of hPER1S714GH did not result from the loss of Per1 function (Figure S1D). Although the direction of period defect is consistent between behavioral period and SCN in hPER1S714GH and hPER2S662G mice, PER1S714G demonstrates a more-profound effect on the period and phase of peripheral tissues than does PER2S662G (Figures 1C and 1D) and PER2S662G exhibits a more-severe effect on the period of behavioral rhythm than does PER1S714G (Figures 1A and 1B). The discrepancy between locomotor activity (Figures 1A and 1B) and SCN explants (Figures 1C and 1D) may reflect the difference between the central clock and its behavior output downstream or studied in different context (in vitro versus in vivo; Brown et al., 2005; Liu et al., 2007). We also analyzed the relative expression levels of mPer1 and mPer2 for the potentially different roles related to tissue-specific behavior. The relative enrichments of mPer1 and mPer2 in liver, lung, adipose, hypothalamus, and spleen tissues vary (Figure 1E), reflecting the diversity of their functions across different organs. Accordingly, the most-profound effect of PER1 misregulation was detected in adipose, lung, and liver tissues, in which the Per2Luc oscillations are nearly antiphasic between hPER1S714H and hPER1S714GH during the first cycle (Figure 1C). Such severe tissue-specific clock perturbations indicated an internal misalignment in hPER1S714G mice. In summary, the whole-animal and tissue-level phenotypes indicated that PER1 phosphorylation plays a key role in setting the oscillator pace and that severe clock perturbations in adipose, liver, and in the lungs may lead to metabolic perturbations in hPER1S714G mice. Subsequently, we characterized the molecular changes in the cell autonomous clock and the consequences of these alterations on whole-animal metabolism.

represent a significant difference between hPER1S714H and hPER1S714GH. Purple stars represent a significant difference between hPER1S714GH and hPER2S662G mice (one star = p < 0.05, two stars = p < 0.01, and three stars = p < 0.001). (E) Enrichment of mPer1 and mPer2 mRNA in the indicated tissues from wild-type mice. All mRNA levels were normalized to Gapdh or 36B4 mRNA levels, and the mean ± SD was generated from three independent experiments from three independent mice.

Cell Reports 7, 1–12, June 12, 2014 ª2014 The Authors 3

Please cite this article in press as: Liu et al., PER1 Phosphorylation Specifies Feeding Rhythm in Mice, Cell Reports (2014), http://dx.doi.org/10.1016/ j.celrep.2014.04.032

B

A ZT 16

ZT 0 04 08 12 16 20 24 0 04 08 12 16 20 24 S714G

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PER1 PPase + -

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ACTIN

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S714G

PER1 PER1 nuclear PER1 nuclear CRY1 1.1 cytoplasmic CRY1 1.3 1.1 1.0 1.1 0.9 0.9 0.9 0.8 0.7 0.7 0.7 0.5 0.5 0.6 0.3 0.3 0.5 0.1 0.1 0.4 0 4 8 121620 0 4 8 121620 0 4 8 121620

Figure 2. The PER1S714G Mutation Affects PER Stability and Its Nuclear Entry (A) Nuclear hPER1 abundance and phosphorylation in hPER1S714H and hPER1S714GH liver extracts. The relative protein abundance was expressed as a percentage of the maximal value obtained from each experiment after normalization to ACTIN. Error bars represent the range from two independent experiments. The right panel showed that liver extracts from hPER1S714H and hPER1S714GH at ZT 16 were treated or not treated by lambda phosphatase (PPase). The values below western blot showed the normalized data, where labeled square density was normalized to ACTIN, showing that mobility shift was changed after PPase treatment. (B) The S714G mutation leads to a destabilization of the hPER1 protein. The MEFs (third passage) from hPER1S714H and hPER1S714GH transgenic embryos were treated with the protein translation inhibitor CHX and harvested at the indicated times. The amount of nuclear hPER1 protein was detected with anti-Myc using western blot analysis and was normalized to ACTIN. The quantification of the hPER1 protein is from three independent experiments and is expressed as the mean ± SD. The treatment of the cells with solvent did not result in PER1 degradation (data not shown). (C) Western blot analysis of nuclear hPER1 with anti-Myc and of both nuclear and cytoplasmic mCRY1 proteins with anti-CRY1 (Shi et al., 2013) in the synchronized MEFs. DEX washed and MEFs were transferred to normal medium at time zero. Error bars represent the range from two independent experiments. (D) After MEFs were treated with CHX for 16 hr to clear residual protein, DEX was added in the last 2 hr, MEFs were transferred to normal medium at time zero, and nuclear hPER1 and both nuclear and cytoplasmic mCRY1 proteins were detected at indicated time points. Error bars represent the range from two independent experiments.

PER1S714G Mutation Accelerates the Speed of the Feedback Loop We investigated the effect of the S714G mutation on the phosphorylation of the hPER1 protein. hPER2 phosphorylation of S662 affects both its mobility shift and abundance as detected on protein gels. Liver and lung nuclear extracts were prepared for western blot analysis because the cytoplasmic hPER1 is minimally detected. The wild-type hPER1 from hPER1S714H mice ex4 Cell Reports 7, 1–12, June 12, 2014 ª2014 The Authors

hibited prominent temporal changes in both abundance and mobility shift over the circadian cycle (Figures 2A, liver tissues, and S2, lung tissues). In contrast, the hPER1S714G mutant protein from hPER1S714GH mice exhibited less abundance and a lower mobility shift during the night phase and higher abundance during the daytime in comparison to hPER1S714. l protein phosphatase treatment alters the electrophoretic mobility of PER1S714 wildtype protein and induces the more rapidly migrating band of

Please cite this article in press as: Liu et al., PER1 Phosphorylation Specifies Feeding Rhythm in Mice, Cell Reports (2014), http://dx.doi.org/10.1016/ j.celrep.2014.04.032

A

B

C

PER1S714G mutant protein in liver tissue, suggesting multiple phosphorylation modifications occur in PER1 protein (Figure 2A, right panel).The reduced mobility shift likely reflects reduced phosphorylation of the mutant protein. We further estimated the stability of the nuclear hPER1 using cultured mouse embryo fibroblast cells (MEFs) from hPER1S714H and hPER1S714GH mice. In a cyclohexamide (CHX) chase analysis, we found that nuclear hPER1 carrying the S714G mutation accelerates the degradation of the hPER1 protein (Figure 2B). The accelerated degradation of the phosphorylation-deficient PER1 is consistent with the observation that the hPER1S714G protein is remarkably less abundant than hPER1S714 during the night phase (Figure 2A). To determine the consequences of the accelerated degradation and reduced levels of PER1 proteins on circadian rhythms, we examined the temporal expression pattern of two repressors, i.e., the PER1 and CRY1 proteins, in the synchronized MEFs from hPER1S714H and hPER1S714GH mice. Both hPER1S714 and hPER1S714G proteins in nuclear fraction exhibited circadian changes in abundance. However, the peak of the nuclear hPER1S714G protein appears several hours earlier in comparison to the hPER1S714 protein, and the mutant protein is minimally detected after 16–20 hr. Correspondingly, the CRY1 protein in hPER1S714GH fibroblasts exhibits earlier peaks in both the nucleus and the cytoplasm in comparison to the wild-type (Figure 2C), suggesting a shorter cycle in the hPER1S714GH MEFs. To determine the role of S714 on the nuclear transport, MEFs were treated with CHX for 14 hr to clear clock proteins from the cells and were then synchronized by dexamethasone (DEX). Nuclear PER1 and CRY1 were minimally detected at 0 hr after the removal of the CHX and DEX, suggesting the effectiveness of the CHX treatment and an equivalent initiation of protein syn-

Figure 3. The PER1S714G Mutation Alters Metabolic Homeostasis (A) The profiles of food intake, VO2, and activity for hPER1S714H and hPER1S714GH mice under a 12 hr light/12 hr dark cycle. Rhythms were plotted over a 72 hr time frame. The results were normalized and were then plotted as the mean ± SEM (n = 16 per genotype). The whole-body energy expenditure was measured by the volume of O2 consumed in the CLAMS chambers. D1 indicates the phase difference between the peaks of food intake in hPER1S714H and hPER1S714GH mice. D2 represents the phase difference between the peak of food intake and oxygen consumption in hPER1S714GH mice. (B) The acrophases of food intake and oxygen consumption in the indicated mice are shown as mean ± SEM. The numbers (n) represent the evaluated mice for each genotype. (C) The diurnal rhythms of food intake are shown. The results consist of the average food intake ± SD during the light and dark phases over a 3-day continuous monitoring period (n = 16 per genotype).

thesis in both cell lines (Figure 2D). Appreciable PER1S714G protein levels were detected at 4 hr following DEX and CHX removal followed by early accumulation of nuclear CRY1 at 4–8 hr in PER1S714G MEFs. In contrast, appreciable PER1S714 protein levels were detected at 8 hr after DEX and CHX removal followed by a peak of nuclear CRY1 at 12 hr in PER1S714 MEFs (Figure 2D). The advanced accumulation of PER1 and CRY1 proteins in the nucleus of hPER1S714GH MEFs reflects a more-rapid nuclear import at the molecular level. Previous studies indicated that coexpression of CK1ε or CK1d with mPER1 prevents mPER1 nuclear entry (Vielhaber et al., 2000), suggesting that unphosphorylated PER1 may alter the import kinetics of PER1 and CRY1. However, the enhanced degradation of PER1 proteins by CK1εtau during selective night phases is also suggested to cause short period and altered phase (Dey et al., 2005; Gallego et al., 2006; Meng et al., 2008). Thus, it is likely that impairment of posttranslational modification of the S714 site of PER1 accelerates nuclear import in the day phase and PER1 degradation in the night phase, both of which contribute to accelerate clocks. hPER1S714G Mice Exhibit Advanced Feeding Rhythms and Food Intake that Is Uncoupled from Energy Expenditure Liver, adipose tissue, and the lungs play important roles in whole-body energy metabolism, hunger, and oxygen use. The distortion of the phase relationship of individual clocks in these organs in hPER1S714G mice prompted us to measure foraging behavior and energy expenditure parameters using a Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments). A nonlinear, multiple-regression, cosine-fit analysis revealed that the food intake rhythms fit a 24 hr rhythm (p < 0.05 for all of the examined mice), and the circadian acrophases of food intake were calculated on 3 consecutive days (Figure 3A). Cell Reports 7, 1–12, June 12, 2014 ª2014 The Authors 5

Please cite this article in press as: Liu et al., PER1 Phosphorylation Specifies Feeding Rhythm in Mice, Cell Reports (2014), http://dx.doi.org/10.1016/ j.celrep.2014.04.032

25 PER1S714H PER1S714GH 20

E 3 2 1 0

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8 10 12 14 16 Age in weeks (Lid)

Total Feeding on HFD P=0.7030 N = 10 N = 14 P=0.0030

P=0.0056 PER1S714H PER1S714GH

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40 35 30 PER1S714H PER1S714GH

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P=0.6936

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P=0.1608 lean fat

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P=0.3619 25

Weight(g)

P = 0.96

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Figure 4. Average Body Weight Gain under NC and HFD (A) The BW of hPER1S714H and hPER1S714GH mice fed with NC over 16 weeks (hereafter, all BW values were shown as mean ± SEM; NC diet: 10% kcal/fat; n = 16 per genotype; p = 0.96). Two-way ANOVA was employed to analyze statistical significance thereafter. (B) BW of hPER1S714H and hPER1S714GH mice fed with a HFD over 8 weeks starting from 8 weeks old (60% kcal/fat; n = 15 per genotype; p = 0.033). (C) The body composition (mean ± SD) was measured using DEXA under NC at 2 months of age. p values are labeled at the corresponding positions; n = 16 per genotype. (D) The body composition (mean ± SD) as measured using DEXA under a HFD for 5 weeks; n = 14 per genotype. (E) The diurnal rhythms of food intake are shown. The results consist of the average food intake ± SD during the light and dark phases over a 3-day continuous monitoring period under ad libitum HFD. (F) BW of hPER1S714H and hPER1S714GH mice over an 8-week period in which food availability was limited at night with 2 weeks of entrainment under NC followed by a HFD. This protocol was initiated for all mice at 6 weeks of age (n = 10 per genotype; p = 0.59).

The peak of food intake was at zeitgeber time (ZT) 17.88 ± 0.42 and 15.44 ± 0.29 for hPER1S714L and hPER1S714H mice, respectively. The peak of food intake was advanced to ZT 13.48 ± 0.61 and 10.00 ± 0.54 for hPER1S714GL and hPER1S714GH mice, respectively (p < 0.0001; one-way ANOVA; Figures 3A and 3B). All transgenic lines from hPER1S714G exhibit significant phase advance of feeding rhythm than from hPER1S714 wild-type transgene. In addition, Per1 knockout mice exhibited a subtle advanced feeding phase (ZT 14.71 ± 0.40; Figure 3B). These results indicated that impaired PER1 leads to feeding behavior changes. We then compared the feeding behaviors in hPER2S662G and hPER1S714G mice. The peak of food intake was significantly earlier in hPER1S714G mice (hPER1S714GL: ZT 13.48 ± 0.61 and hPER1S714GH: 10.00 ± 0.54) than in hPER2S662G mice (ZT 14.68 ± 0.20; p < 0.0001; one-way ANOVA; Figure 3B). Furthermore, the D phase (feeding phase oxygen consumption phase) was 3.35 ± 0.45 in hPER1S714GH mice compared with 1.02 ± 0.38 in hPER1S714H mice (Figures 3A and 3B), suggesting that oxygen consumption is significantly misaligned with the feeding rhythms (p < 0.0001; one-way ANOVA). The D phase in hPER2S662G mice was not markedly different from that of wild6 Cell Reports 7, 1–12, June 12, 2014 ª2014 The Authors

type mice ( 1.37 ± 0.2; p > 0.05 compared with the wild-type mice). Altogether, these results suggest that PER1 phosphorylation at S714 site has a distinct effect on feeding rhythm and mutation at this site affects the normal phase relation between energy intake and expenditure. Consistent with this phenotype, mice carrying the hPER1S714GH transgene consumed more food during the light phase in comparison to the hPER1S714H controls (56.9% ± 9.3% versus 36.3% ± 5.7%, respectively; p < 0.0001; one-way ANOVA; Figure 3C). To test the biological significance to the misalignment between food intake and energy expenditure, we analyzed the body weight gain of hPER1S714H and hPER1S714GH mice fed a normal chow (NC). A comparison of the body weight (BW) using two-way ANOVA (genotype 3 time of day) did not reveal any genotype-specific differences in mice fed NC over 16 weeks (Figure 4A; p = 0.96). As the metabolic defects in several genotypes became more apparent when challenged with a high-fat diet (HFD), we subjected the mice to a HFD ad libitum. The hPER1S714GH mice gained BW more rapidly compared with the hPER1S714H control on a HFD (Figure 4B; p = 0.033; genotype 3 time; two-way ANOVA). We also compared body weight among hPER1S714H, hPER2S662G, and Per1 / mice on

Please cite this article in press as: Liu et al., PER1 Phosphorylation Specifies Feeding Rhythm in Mice, Cell Reports (2014), http://dx.doi.org/10.1016/ j.celrep.2014.04.032

A

B

Figure 5. The Profiles of Food Intake and VO2 for hPER1S714 and hPER1S714G Mice (A) Fed with an ad libitum HFD. (B) Night-only feeding on HFD. Rhythms were plotted over a 72 hr time frame. The results were normalized and were then plotted as the mean ± SEM (hPER1S714: n = 10; hPER1S714G: n = 14). The acrophases of food intake and oxygen consumption in the indicated mice are shown as mean ± SEM in bottom panels.

a HFD. There was no obvious difference in body weight among the three genotypes (p > 0.05; Figure S3), indicating that shortterm high-fat-induced obesity is specific to the hPER1S714G mice. Dual-energy X-ray absorption (DEXA) analysis indicated that the body compositions of 8-week-old hPERS714H and hPERS714GH mice were identical prior to a HFD challenge (Figure 4C). However, upon feeding a HFD for 5 weeks, the total fat mass of the hPER1S714GH mice increased in comparison to that of the hPER1S714H mice, although the total lean mass remained identical in both genotypes (Figure 4D), suggesting that this gained BW is largely due to fat mass gain. No difference was noted in total activity (Figures S4A and S4B) or total food intake (Figures 3C and 4E) for a NC or a HFD, suggesting that this increased BW was not due to decreased activity or increased food intake. To test whether the advanced feeding behavior contributed to high-fat-induced obesity in hPER1S714G mice, we subjected the mice to time-restricted access to food at night time. Such timerestricted feeding (tRF) paradigm is known to consolidate and align feeding time with peak night-time activity and energy expenditure. We subjected 6-week-old male hPER1S714H and hPER1S714GH mice to tRF for 2 weeks with NC and subsequently with HFD. The excess body weight gain that was observed in the hPER1S714GH mice fed ad libitum was prevented in hPER1S714GH mice that were fed exclusively at night time, and their weight was not significantly different from the control hPERS714H mice that were fed at night (Figure 4F; p = 0.59).

Furthermore, to determine whether altered feeding regimens can correct the misalignment between feeding rhythms and oxygen consumption (i.e., the D phase) under a HFD, we recorded foraging behavior and energy expenditure parameters using the CLAMS under tRF compared with an ad libitum HFD. hPER1S714GH mice exhibited a clear advanced feeding phase and misalignment between feeding phase and oxygen consumption phase under an ad libitum HFD (Figure 5A) with comparable total quantitation in locomotor activity, food intake, and oxygen consumption (Figures S4B–S4D), as was observed under an ad libitum NC diet. However, the hPER1S714GH mice showed improved alignment between feeding phase and oxygen consumption phase when they were fed only at night time (Figure 5B). Again, hPER1S714H and hPER1S714GH exhibited comparable quantitation in locomotor activity, oxygen consumption, and food intake (Figures S4E–S4G), suggesting that the misalignment between feeding rhythms and oxygen consumption can be prevented by imposing a feeding regimen. The overall improvement of the alignment between feeding behavior and oxygen consumption corresponded with the improved body weight gain in hPER1S714G mice under timerestricted feeding paradigms. Altered Feeding Rhythms and Clock Defects Reciprocally Reinforce Internal Misalignment The altered circadian clock and feeding pattern in S714G mutant likely result in changes in diurnal expression of transcripts in metabolic tissues. We examined the liver and adipose transcriptomes at ZT 1 and ZT 13 (lights on at ZT 0; lights off at ZT 12) using RNA microarray analysis. Transcripts at ZT 1 (corresponding to the end of feeding) and ZT 13 (time of feeding) that exhibited a greater than 2-fold difference in the expression level were defined as rhythmic transcripts. The total number of identified circadian genes (false discovery rate of <0.05) was significantly reduced in liver tissues (WT: 3,121 versus S714G: 369) and in adipose tissues (WT: 3,124 versus S714G: 2,003), suggesting that 88% of the identified diurnal transcripts in hPER1S714H altered their phase or dampened their amplitude in hPER1S714GH liver tissue (Figure 6A). The peak or trough of Cell Reports 7, 1–12, June 12, 2014 ª2014 The Authors 7

Please cite this article in press as: Liu et al., PER1 Phosphorylation Specifies Feeding Rhythm in Mice, Cell Reports (2014), http://dx.doi.org/10.1016/ j.celrep.2014.04.032

SG

ZT1 ZT1 >2 < 0.5 ZT13 ZT13 29 FDR <0.05 FDR <0.05 1195 205

ZT1 ZT13 Bmal1 Pparδ Avpr1a Nfil3 Lrfn3

369

3121

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Abcg8 Syt1 Nrg4 Pcsk4 Gm9992 Slc2a5 Slc7a2 Tef Dnajb11

ZT1 ZT1 < 0.5 >2 ZT13 32 ZT13 FDR <0.05 FDR <0.05 1926 164

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Figure 6. The Reciprocal Relationship between Feeding Rhythms and the Circadian Clock (A) A summary of the transcripts that are substantially affected by the S714G mutation in the liver and adipose tissues. The number of 2-fold increases or 2-fold decreases between ZT 1 and ZT 13 are individually shown in circles. The total number of altered transcripts is labeled outside of each circle for each genotype. The red number represents the number of genes that demonstrate a significantly different (inverted) phase between hPER1S714H and hPER1S714GH mice. (B) Hierarchical clustering analysis of 61 inverted transcripts between ZT 1 and ZT 13 in the liver and 15 inverted transcripts in adipose tissues of hPER1S714H and hPER1S714GH mice. High levels are shown in red, and low levels are shown in green. WT represents hPER1S714H, and SG represents hPER1S714GH. (C) Representative expression profiles for genes demonstrating an inverted phase over a 24 hr period in hPER1S714H and hPER1S714GH mice. (D) A representative expression profile for unaltered genes, according to our standard 24 hr period. (E) A Venn diagram depicts the number of overlapping genes between the genes that were identified as inverted transcripts (61 genes) and the previously reported tRF-modulated transcripts (290 genes from database were identified according to the above protocol; Vollmers et al., 2009). (F) Representative expression profiles for genes demonstrating an inverted phase under ad libitum feeding in comparison to time-restricted feeding (compared with Figure 6C, note that improved phase shift in hPER1S714GH). Transcript levels were measured using quantitative RT-PCR and were normalized to Gapdh mRNA levels. The mean ± SD was obtained as previously described from three independent experiments.

61 genes in liver tissue and 15 genes in adipose tissue was inverted between hPER1S714H and hPER1S714GH mice (Figure 6B; Tables S1 and S2). This group of phase-inverted transcripts 8 Cell Reports 7, 1–12, June 12, 2014 ª2014 The Authors

included metabolic and circadian regulators such as Clock, Bmal1, Elovl3, Dbp, Pgc1a, Lpin1, Fasn, Nfil3, Pdk4, Ppard, and Tef (p < 0.05; Figures 6B and 6C). Gene-enrichment analysis

Please cite this article in press as: Liu et al., PER1 Phosphorylation Specifies Feeding Rhythm in Mice, Cell Reports (2014), http://dx.doi.org/10.1016/ j.celrep.2014.04.032

indicated that these inverted genes were significantly associated with molecular functions that were related to biological rhythms (p = 3.41 3 10 7) and transcriptional regulation (p = 0.001). Protein products of these genes have been shown to play key roles in mediating circadian regulation of nutrient and xenobiotic metabolism (Asher and Schibler, 2011). High-fat-diet-induced obesity in mice is associated with dampened or misexpression of these genes (Hatori et al., 2012; Kohsaka et al., 2007). Furthermore, we examined diurnal genes that were considered as less-affected genes when compared with those invertedphase transcripts. We found that these transcripts display a phase that was advanced by approximately 4 hr (Figure 6D), reflecting a circadian defect. The phase of the transcripts in peripheral organs is an integration of its endogenous period with in vivo inputs. Time of food intake is a dominant zeitgeber for circadian oscillators in several peripheral organs. We compared 61 phase-inverted genes in hPER1S714G liver tissues with 290 genes that were previously reported to be altered by tRF (Table S3; Hatori et al., 2012) and detected 34 common genes, which suggested that the temporal expression pattern of these genes can be changed by externally imposing a feeding regimen (Figure 6E). We tested whether the advanced feeding behavior reinforced the phase shifts of targeted gene expression in liver tissue. For this purpose, the hPER1S714H and hPER1S714GH mice were fed from ZT 16 to ZT 20 with NC for 2 weeks. As previously reported for nocturnal animals (Damiola et al., 2000), in hPER1S714 mice, restriction of the feeding time to the night phase does not significantly alter the phase angle of rhythmic gene expression (Figure 6F versus 6C). In contrast, we observed tRF prevented or attenuated phase advancement for the Pdk4, Ppard, Nfil3, and Tef mRNA in the hPER1S714G mice (Figure 6F versus 6C), suggesting that a disrupted circadian clock along with advanced feeding reinforce the phase shifts of clock gene transcripts in the hPER1S714G mice. Thus, the synergistic interaction between clock and metabolism is exemplified in the responsive rhythmic transcripts. DISCUSSION Although a number of mice carrying loss-of-function alleles of clock components have been used to study the effects of clock genes on cell autonomous rhythms and overt phenotypes, specific roles of these paralogs in diverse functions of the mammalian circadian system remain unclear. Previous studies found that the SXXS motif was highly conserved among vertebrate PERIOD homologs, suggesting that this motif is critical for the proper functioning of PERIOD. The importance of this motif was confirmed by the fact that S662 in hPER2 is necessary to trigger a phosphorylation cascade and signal amplification mechanism (Xu et al., 2007). Other proteins including the nuclear factor of the activated T cell protein family and antigen-presenting cell (Okamura et al., 2004; Price, 2006) have also retained the SXXS motif during evolution. This observation suggests that the emergence of the SXXS motif occurred by an evolutionary speciation and may enable these proteins to adapt to varying environments. We hypothesized that modifying these motifs could be a useful tool to dissect the specific function of the paralogous pro-

teins that has been largely ignored. In this study, we uncovered a critical role for the S714 site in PER1 in mediating the feeding phase that was not observed in Per1 / , hPER2S662G, or Per2 / mice. This finding suggests that duplicated genes diverge over time and undergo functional specialization but partially overlap with the progenitor gene. The need to rest, eat, and adjust to daily changes in the physical environment may have resulted in the coevolution and integration of the circadian clock, metabolism, and the restactivity cycle, particularly in higher organisms. Generally, the environmental light-dark cycle provides the principal entraining signal to the SCN for the regulation of behavior, including restactivity cycles and, thus, feeding cycles (Albrecht, 2012). However, feeding cycles can act as a synchronizer and produce dynamic shifts in some rhythmic processes. hPER2S662G mice have been suggested as an advanced sleep-phase syndrome model and exhibit a 4 hr phase advance of activity rhythms in light-dark (LD) cycles (Xu et al., 2007). If rest-activity cycles are highly associated with feeding rhythms, then the feeding rhythm phase should be advanced relative to the phase of rest-activity cycles in hPER2S662G mice. In contrast, we found that hPER1S714G mice exhibit a markedly advanced phase of feeding behavior in comparison to hPER2S662G mice, irrespective of their rest-activity cycles, suggesting that PER1 and PER2 function differently and that rest-activity and feeding cycles are at least partially separate. The observation that hPER1S714G, but not hPER2S662G, mice rapidly develop obesity on a HFD due to altered feeding times further supports their specific function. In humans, night-eating syndrome (NES) is characterized by latenight binge eating (i.e., a lack of appetite in the morning and evening or nocturnal hyperphagia; Stunkard et al., 1955) and affects between 1% and 2% of the population. Most people with NES are also obese (Allison et al., 2006). Although a mutation screening for NES patients is currently unavailable, a genome-wide analysis of human disease alleles demonstrate that sequence variants co-occur at aligned amino acid residue pairs more frequently than expected by chance, due to similar functional constraints on paralogous protein sequences (Yandell et al., 2008). A previous study identified an S662G mutation in PER2 for FASPS, and it is highly possible that there is an S714G mutation in PER1 in NES. The mechanism of the diverse functions of PER1 and PER2 on the advanced phases of feeding-fasting cycles and activity-rest cycles remains to be determined. A recent study demonstrated that PER2 rather than PER1 integration with various nuclear receptors provides an explanation for the functional differences between these two PER proteins (Schmutz et al., 2010). Herein as well, the targeted transcripts in the liver and adipose tissues that were induced by PER1S714G varied substantially, and Per1 and Per2 were differentially enriched, indicating tissue-specific effects of PER1 on target gene expression. The whole-animal genetic perturbation models we used here is insufficient to point to the tissue type(s) that predispose hPER1S714G mice to altered feeding pattern. Hypothalamic nuclei-regulating activity, feeding, satiety, adipose tissues producing leptin and other metabolic signals, and liver playing a central role in nutrient and sterol metabolism contribute to pattern of food intake. Therefore, circadian Cell Reports 7, 1–12, June 12, 2014 ª2014 The Authors 9

Please cite this article in press as: Liu et al., PER1 Phosphorylation Specifies Feeding Rhythm in Mice, Cell Reports (2014), http://dx.doi.org/10.1016/ j.celrep.2014.04.032

defects in one or more of these tissues can potentially lead to altered feeding rhythm. As metabolism and circadian oscillator reciprocally interact, altered feeding rhythms further affect the circadian oscillator and clock-controlled genes in multiple organs. Further studies will be required to identify proteins that regulate phosphorylation state, stability, and subcellular activities of PER1 proteins and shed light on their possible role in metabolism using tissue-specific genetic manipulation techniques. EXPERIMENTAL PROCEDURES Generation of PER1 BAC Transgenic Mice RP11-1D5 is a BAC clone from the human genomic library containing the entire PER1 locus on a 163 kb genomic insert with 85 kb upstream of the gene (Children’s Hospital Oakland Research Institute). We modified this BAC clone by homologous recombination as previously described (Lee et al., 2012). The c-Myc tag-encoding sequence was introduced to the region before the TAG stop codon of the hPER1 gene to allow for protein detection. Then, the mutation (S714G) was inserted into the above BAC clone as hPER1S714G mutant form by recombination again and the nonmutated clone was a control. Transgenic mice were generated using microinjecting engineered BAC clones. The transgenic founders were backcrossed to C57/BL6J for greater than five generations. We characterized these mice based on their copy number by Southern blot and selected two transgenic lines as low and high copy for each genotype to screen phenotype. Animal Care and Behavioral Analysis Measurements of the free-running period were performed as previously described (Wang et al., 2010). All animal studies were carried out in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited, specific-pathogen-free animal facility, and all animal protocols were approved by the Animal Care and Use Committee of the Model Animal Research Center, Nanjing University. Luminescence Recording The detailed methods for real-time measurement of luminescence from ex vivo tissues followed the Shin Yamazaki method (Savelyev et al., 2011; Yamazaki and Takahashi, 2005) and are described in the method summary of the Supplemental Information. After recovering the rhythm, we estimate the phase and period as described previously (Pendergast et al., 2012). Here, the circadian clock oscillations are assumed to be the cosine wave. A nonlinear least-squares minimization method evaluates the parameters of the cosine wave. The period, phase, and amplitude of the most-powerful spectral peak in the fast Fourier transforms initialize a nonlinear least-squares minimization method. Microarray Assay Microarray labeling and hybridization are typically performed by Capitalbio and China using Agilent Whole Mouse Genome Oligo Microarray (4X44K). GeneChip hybridizations were read using Agilent G2565CA Microarray Scanner. Feature extraction was used to convert into GeneChip probe result files. GeneSpring GX software analyzed the probe level data. The raw data of signal intensity in all arrays were log transformed (base 2) and normalized with R (Bolstad et al., 2003). Only transcripts that demonstrated an expression level above 2 3 SD (background) were considered reliably detected. For significance analysis of microarray analyses (Tusher et al., 2001), transcripts between hPER1S714G and hPER1S714 in the liver and adipose at specific time points (ZT 1 and ZT 13) demonstrating >2-fold change and a false discovery rate of 5%, were statistically significant. The hierarchical clustering analysis was conducted with MeV using an average linkage method (Saeed et al., 2003). Three independent replicates for each time point and each genotype were profiled. Hierarchical clustering indicated that the microarray data are highly reproducible.

10 Cell Reports 7, 1–12, June 12, 2014 ª2014 The Authors

Metabolic Rhythm Measurement and Analysis Mice were entrained to normal LD cycles (lights on at 8:00 a.m. and lights off at 8:00 p.m.) for 1 week. Following the acclimation period for 3 days, mice were continuously recorded for another 3 days in 30 min time bins with the following measurements: food intake and VO2 in the comprehensive animal monitoring system (Oxymax; Columbus Instruments). The sampling time was transformed with the equation. x = ([hr 3 3600 + min 3 60 + s]/3600 8) 3 2, which the first day at 8:00 a.m. is as 0 and the next day at 8:00 a.m. is recorded as 48. Cumulative food intake and VO2 for the 72 hr period were imported into MATLAB. Then, food intake and VO2 values were smoothed with a running average method with a factor of 5. Data were fitted with the equation y = a 3 cos (2 3 pi/48 3 x b) + c. Factor b represents the phase of each mouse food intake or VO2 phase. The phase was then transformed to ZT time with the equation ZT = 24 3 b/2 3 pi. D phase was generated from feeding phase minus VO2 phase. Each value was shown as mean ± SEM. Feeding Schedule and Body Weight Measurement Age-matched wild-type and mutant mice were grouped housed (five per cage) to 8 weeks old under NC (LabDiet). Next, they were fed NC or HFD (Research Diets; D12492 60% fat kcal % diet) to 16 weeks. For a restricted feeding schedule, age-matched wild-type and mutant mice were grown to 6 weeks under NC and were restricted food access from ZT 12 to ZT 24 with NC for 2 weeks before changing to HFD. Body weight was recorded daily at ZT 0. At the beginning and end of the experiment, body composition (fat and lean mass) was determined by DEXA (PIXImus; GE Lunar Corporation) under Avertin anesthesia. Statistics Statistical analysis of food intake phase, VO2 phase, tissue period, and body composition was performed with one-way ANOVA. Growth curves either NC or HFD under ad libitum or restricted condition were analyzed by two-way ANOVA. p < 0.05 was considered statistically significant. ACCESSION NUMBERS The GEO accession number for the microarray data sets reported in this paper is GSE56161. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, four figures, and three tables and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2014.04.032. AUTHOR CONTRIBUTIONS Z.L., M.H., Y.X., and S.P. are responsible for designing research; Z.L. performed research; X.W., G.S., L.X., Z.D., Z.Q., and Y.X. contributed mouse models; M.H., X.W., S.P., and Y.X. performed bioinformatics analysis; Z.L., X.W., G.S., L.X., J.Y., L.Y., S.P., and Y.X. analyzed data; and Z.L., S.P., and Y.X. wrote the paper. ACKNOWLEDGMENTS We thank Cheng Chi Lee lab for Per1 knockout mice and Fu and Ptacek lab for hPER2S662G mice. This work was supported by grants from the Ministry of Science and Technology of China (2010CB945100 and 2013CB945203 to Y.X.), the National Science Foundation of China (31171343 and 31230049 to Y.X.), and the NIH (DK091618 and EY016807 to S.P.). Received: December 12, 2013 Revised: March 25, 2014 Accepted: April 16, 2014 Published: May 22, 2014

Please cite this article in press as: Liu et al., PER1 Phosphorylation Specifies Feeding Rhythm in Mice, Cell Reports (2014), http://dx.doi.org/10.1016/ j.celrep.2014.04.032

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