WO2015054819A1

WO2015054819A1 - Method for identifying advanced feeding rhythm syndrome and application thereof

Info

Publication number: WO2015054819A1
Application number: PCT/CN2013/085189
Authority: WO
Inventors: Ying Xu; Zhiwei Liu
Original assignee: Nanjing University
Priority date: 2013-10-14
Filing date: 2013-10-14
Publication date: 2015-04-23
Also published as: CN105744948B; CN105744948A

Abstract

The present invention provides a method for screening mammalian for conditions associated with altered feeding cycle or potential to develop such conditions, comprising detecting the phosphorylation status of PERI in the mammalian. In addition, the present invention provides a method for treating or preventing conditions associated with altered feeding cycle in the mammalian subject, and a method for screening for agents capable of treating or preventing conditions associated with altered feeding cycle.

Description

Method for Identifying Advanced Feeding Rhythm Syndrome and

Application Thereof

FIELD OF THE INVENTION

The present invention relates to a protein PERI (period circadian protein 1) involved in the mammalian feeding cycle. Specifically, the present invention includes the phosphorylation of PERI for conditions associated with altered feeding cycle.

BACKGROUND

The circadian clock allows an organism to anticipate environmental cyclic changes, such as the light-dark cycle and food cycle, and thus provides adaptive advantage. The current mammalian clock model is composed of a transcriptional-translational feedback network that includes the PAS (Per-Arnt-Sim) domain-containing helix-loop-helix transcription factors Clock and Bmall , Period genes (Perl, Per2, and Per3), and Cryptochrome genes (Cryl 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:BMALl . Taken together, these complexes cause the endogenous circadian oscillations (P. L. Lowrey, et al., Annu Rev Genomics Hum Genet 2004, 5 : 407; S. M. Reppert, et al., Nature 2002, 418: 935; U. Schibler, et al., Curr Opin Cell Biol 2005, 17: 223).

Accumulating evidences have revealed an intimate relationship between circadian clock and metabolism. In the main metabolic organ liver, many transcripts exhibited diurnal fluctuation (Panda et al._s Nature 2002, 417: 329-35; Oishi et al., J Biol Chem 2003, 278: 41519-27). Many metabolic parameters also exhibited daily rhythm, like body temperature, glucose, INSULIN and LEPTIN levels (Sinha et al., J Clin Invest 1996, 97: 1344-7; Van Cauter et al., Endocr Rev 1997, 18: 716-38) .

Lots of effort has been made to understand the molecular link between circadian clock and metabolism. Nuclear receptors were found to fluctuate in several major metabolic organs, liver, white or brown adipose and muscle, and given the close relationship of NRs with metabolism, NRs were believed to serve as mediator between circadian clock and metabolism (Yang et al., Cell 2006, 126, 801-10). Recent findings on SIRT1 , a NAD+ dependent deactylase involved in many metabolic processes, in modulating circadian clock via PER2 and BMALl made SIRT1 another candidate. Besides these mediators, some circadian mouse models developed metabolic phenotype. Clock mutant was hyperphagic and obese, and developed a metabolic syndrome of hyperleptinemia, hyperlipidemia, hepatic steatosis, hyperglycemia and hypoinsulinemia (Turek et al., Science 2005, 308, 1043-5). Bmall was found to regulate adipogenesisand influence life span (Shimba et al., PNAS 2005, 102: 12071 -6; Kondratov et al., Genes Dev 2006, 20: 1868-73). Rev-erb a co-localized with HDAC3 to regulate lipid metabolism, deficiency in Rev-erb a lead to hepatic steatosis (Feng et al., Science 2011, 331 : 13 15-19).

In another aspect, metabolism could also regulate circadian clock, as food could serve as a strong signal to entrain circadian clock in metabolic organ without influencing central clock (Damiola et al., Genes Dev 2000,

14: 2950-2961). Further work pointed out that glucocorticoid signaling and ADP-ribosylation were involved in food entrainment (Asher et al., Cell 2010, 142: 943-953; Le Minh et al., Embo J 2001, 20: 7128-7136). Not only when to eat, but also what to eat could affect circadian clock. High fat diet had a strong effect on circadian clock in terms of inducing period lengthening and attenuation of rhythm ( ohsaka et al., Cell Metab 2007, 6: 414-421 ). More and more evidences appeared to support the idea that feeding at improper times could lead to metabolic syndromes. Mice fed high fat diets at normal rest time had accelerated rate of body weight gain compared to mice fed equal amounts of food at normal active phase (Salgado-Delgado et aL, PLoS One 2009, 8: e60052; Arble et al., Obesity (Silver Spring) 2009, 17: 2100-2102). Restricted feeding to the normal active phase could completely rescue the metabolic disorder (Hatori et al., Cell Metab 2012), again verified the importance of coincidence of circadian clock with metabolism. More importantly; the clinical data also supported the conclusion from mouse studies; people subjected to shift work were likely to become obese compared with those working at regular time (Tasali et al._s Proc Natl Acad Sci USA 2008, 105: 1044-49).

How the circadian time clues transmitted to feeding remains unknown. The present understanding of feeding control was mainly based on studies of neuropeptides and circulating hormones. It was believed that the hypothalamus was the main site participating in feeding control through the counter-balance of neuropeptides. Slowly people has been building the connection of circadian clock with these neuropeptides. Some of these neuropeptides were shown to fluctuate around the circadian clock, though may be not a direct target of circadian clock (Lu et al., Endocrinology 2002, 143 : 3905-15; Stutz et al., Obesity (Silver Spring) 2007, 15 : 607-15; Xu et al., Endocrinology 1999, 140: 2868-75). Another important modular of feeding is circulating hormones. One of these hormonesis Leptin, which is a main adipokine secreted from white adipose tissue under circadian clock control (Kalsbeek et al., Endocrinology 2001 , 142: 2677-85).

There exists a circadian disease called Night-Eating Syndrome (NES) characterized by evening hyperphagia. Patients with NES display delayed circadian pattern of feeding rhythms but normal sleep-wake cycles, representing a dissociation of feeding rhythm with sleep control (O'Reardon et al., Obes Res 2004, 12: 1789-96). Mechanism underlying this phenomenon remains largely unknown.

Although the basic function of the molecular clock is largely conserved, mammals employ multiple paralogous clock genes. These clock components emerged and expanded in mammalian circadian systems, resulting in a high level of functional divergence in physiological functions. Mapping specific function in each paralogous gene is likely to provide general insights into the understanding of how the balance between clock systems and physiological homeostasis are achieved after the evolution of clock components. However, knockout these genes often exhibit subtle phenotypes due to genetic redundancy.

In lower invertebrates like drosophila, there exists only one dPer. During evolution, dPer divided into three homologs in vertebrates; PERI, PER2 and PER3. Gene duplication often came along with functional differentiation, as happened in the PER family. Since this century, much effort has been made to distinguish three PER family members. Multiple mouse models examining overexpression or loss of PERI , PER2and PER3 alone or in combination have been generated. Generally speaking, single knockout exhibited mild circadian phenotype with alteration in period length and weak influence on molecular clock. Double knockout of Perl and Per2 turned out to be arrthymic, indicating important function of Perl and Per2 in maintaining circadian rhythm, while Per3 seemed to work downstream of the core circadian clock (Bae et al., Neuron 2001 , 30: 525-36). Due to gene redundancy, however, none of the models could tell us if they developed specific functions outside of circadian system. Some published data suggested that PERI , PER2 and PER3 were related to cell cycle, metabolism, sleep, and so on (Goel et al., PLoS One 2009, 4: e5874; Dallmann and Weaver, Chronobiol Int 2010, 27: 1317-28; Grimaldi et al., Cell Metab 2010, 12: 509-20; Gu et al., Cell Death Differ 2012, 19: 397-405).

One excellent work on specifying PER2 function was done by Xu et al, which brought a mutation found in FASPS (hPER2^S662G, Toh et al., Science 2001, 291 : 1040-43) to a mouse model and clearly linked hPER2 to sleep control (Xu et al., Cell 2007, 128: 59-70). Further study on this site revealed a serial phosphorylation site with five serines forming an SXXS like sequence (Serine-rich motif, SR motif), which was targeted by casein kinase isoform epsilon (CKIe) and casein kinase isoform delta (CKI δ ). Phosphorylation at the first serine site (S662) was recognized by CKIs and CKI8 then PER2 was phosphorylated at the following serines.

Sequence comparison showed this motif to be highly conserved; it was only found in vertebrates PERs. Similar motifs also appeared in PERI and PER3 with evolutional conservation. One paper working on the same site in mPerl (S714 site is homologus to S662 in hPER2) suggested possible influences of this site on core circadian clock (Abraham et al., J Neurosci

2005, 25 : 8620-26).

SUMMARY OF THE INVENTION

The present invention relates to a method of screening mammalian subject for conditions associated with altered feeding cycle or potential to develop such conditions, comprising detecting the phosphorylation status of PERI in the subject. In certain embodiments, said phosphorylation status is determined through detecting the phosporylation status of S714 of SEQ ID NO: 1 , and the detection of hypophosphorylation of S714 indicates a positive diagnosis of said conditions. In some particular embodiments, the detection of phosporylation status of S714 of SEQ ID NO: 1 comprising detecting whether the Serine at the position 714 is mutated into Glycine. In certain embodiments, said condition is the Night-Eating Syndrome.

Another aspect of the present invention relates to a method of treating or preventing conditions associated with altered feeding cycle in a mammalian subject, comprising regulation of the phosphorylation status of PERI of the subject. In certain embodiments, said regulation of phosphorylation status of SEQ ID NO: l of PERI is carried out by a kinase that can phosphorylates PERL In one embodiment, the kinase is casein kinase isoform ε. In certain embodiment, the condition is Night-Eating Syndrome. In certain embodiments, said regulation of phosphorylation status comprising regulating the phosporylation of S714 of SEQ ID NO: 1.

Yet another aspect of the present invention relates to a method of screening for agents capable of treating or preventing conditions associated with altered feeding cycle in a mammalian subject. The method involves: a) providing test cells or tissues taken from the subjec; b) providing a plurality of candidate agents; and c) contacting the test cells or tissues with the candidate agents under circumstances effective for regulating phosphorylation of PERI, and identifying the candidate agents that can alter the phosphorylation status within PERI, and as a result, such identified agents having potential capability of treating or preventing conditions associated with altered feeding cycle in the mammalian subject. In certain embodiments, said regulation of phosphorylation comprising regulating phosporylation of S714 in the amino acid sequence of SEQ ID NO: 1. In certain embodiment, the condition is Night-Eating Syndrome.

A further aspect of the present invention provides the use of an agent capable of regulating the phosphorylation status of PERI in the manufacture of medicines for treating or preventing conditions associated with altered feeding cycle in mammalian. In certain embodiment, said regulation of phosphorylation comprising regulating the phosporylation of S714 of the amino acid sequence of SEQ ID NO: l .The agent maybe a kinase, or in particular kinase isoform ε. In certain embodiment, the condition is Night-Eating Syndrome.

The present invention provides new methods of screening, treating or preventing conditions associated with altered feeding cycle in mammalian and further provides agents capable of treading or preventing conditions associated with altered feeding cycle in mammalian and the method of screening thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The hPERl^S714G mutation impairs clock oscillators, (a) The period length quantification of the indicated genetic mice. The period was calculated from eight to 21 days after constant darkness using ClockLab. The bars indicate the mean ± standard deviation (SD). The p value indicated was determined by GraphPad Prism 5. (b) Representative PER2::Luc bioluminescence traces of explants from hPERl ^S7U (green), hPERl ^S714G (red), and hPER2^S662G (blue) mice. The explants were prepared within 1 h before the light went off. The time windows shown were used to determine the period over three consecutive days, (c) The period in the indicated tissues and genotypes are shown. The mean period ± SD was determined by a self-step algorithm. The sample sizes shown (number of rhythmic tissues) are from three mice for each genotype. One-way ANOVA indicated a significant difference between hPERl ^S714 and hPERl^S714G or hPER2^S662G mice. Purple stars represent a significant difference between hPERl^S7,4G and hPER2^S662G mice (one star - p < 0.05, two stars = p < 0.01 , three stars = p < 0.001). (d) Enrichment of mPerl and mPer2 mRNA in indicated tissues. All rriRNA levels were normalised to Gapdh, and the mean ± SD was generated from three independent experiments from three independent mice.

Figure 2. The PERI SV 14G mutation changes metabolic homeostasis, (a) The profiles of food intake and VO₂ for hPERl^S714 and PER1 ^{S7 I4G} mice under a 12 h light/12 h dark cycle. Rhythms were plotted over a 72 h timeframe. The data were normalised and 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. Δ 1 indicates the phase difference between the peaks of food intake in hPERl ^S714 and

PERI mice. Δ 2 represents the phase difference between the peak of food intake and oxygen consumption in PERI mice, (b) The acrophases of food intake and oxygen consumption, and the corresponding locomotor period in the indicated mice are shown. The numbers (N) represent the examined mice for each genotype, (c) The diurnal rhythms of food intake are shown. The data consist of the average food intake ± SD during the light and dark phases over a three-day continuous monitoring period (N = 16 per genotype), (d) The BW of hPERl^S7'⁴ and PER1 ^S714G mice over the six-month study with NC (10% kcal/fat; N = 16 per genotype, p = 0.96). Two-way ANOVA was employed to analyze statistical significance, thereafter, (e) BW of hPERl^S714 and PER1^{S7 I G} mice with a HFD over an eight-week study (60% kcal/fat, N = 15 per genotype, p = 0.033). (f) BW of hPERl^S714 and PER1^S714G mice over an eight-week study where food availability was limited at night with two weeks of entrainment under NC and then under a HFD. This protocol was initiated for all mice at six weeks of age (N = 10 per genotype, p = 0.59). (g) The profiles of food intake and VO₂ for hPERl^S714 and PER1^{S7, 4G} mice at night only feeding under HFD. Rhythms were plotted over a 72 h timeframe. The data were normalised and then plotted as the mean ± SEM (N = 8 per genotype).

Figure 3. The PER1 ^S714G mutation accelerates molecular feedback loops, (a) Nuclear hPERl abundance and phosphorylation in hPERl^S7M and hPERl^S714G liver and lung (Figure 5) extracts. Relative protein abundances were expressed as a percentage of the maximal value obtained from each experiment after being normalised to ACTIN. Error bars represent the range from two independent experiments, (b) The S714G mutation leads to a destabilisation of the hPERl protein. The MEFs (third passage) from hPERl and hPERl transgenic embryos were treated with the protein translation inhibitor cycloheximide (CHX) and harvested at the indicated times. The amount of nuclear hPERl protein was detected with anti-MYC by WB and normalised to ACTIN. The quantification of the hPERl protein is from three independent experiments and is expressed as the mean ± SD. The treatment of the cells with solvent did not lead to the degradation of PERI (data not shown), (c) Western blots of nuclear hPERl , and both nuclear and cytoplasmic mCRYl proteins over the clock in the synchronized MEFs. (d) After MEFs with CHX treatment for 16 h, nuclear hPERl, and both nuclear and cytoplasmic mCRYl proteins were detected over the clock. Error bars represent the range from two independent experiments, (e) There was an altered BMAL:CRY1 :E-box ratio in the Per2 and Dbp promoters. hPERl^S714 or PER1^{S7, 4G} liver tissue was used in the ChIP experiment. The immunoprecipitation was performed with anti-BMALl or anti-CRYl and IgG as a control. The ChIP was analysed by quantitative PCR. Original CRY1 and BMAL1 ChIP data are presented in Figure 11. The data are expressed as a percentage of the highest value in each experiment and the mean ± SD. The ratio ± SD of BMAL l :CRYl :E-box was obtained by dividing BMAL 1 enrichment by CRY1 enrichment at each time point from three independent experiments. (f) We observed altered RNAPII enrichment at the E-box in the Per2 and Dbp promoters. The data are expressed as described above. Two-way ANOVA demonstrated a significant difference between hPERl ^S714 and _{pER 1} S7 i 4G φ ø ooj) (_g) _The expression profiles of Per2 and Dbp mRNA in liver tissue are shown. All mRNA levels were normalised to Gapdh, and the mean ± SD was generated from three independent experiments. See Figure 13 for other clock genes in liver and adipose tissue.

Figure 4. The reciprocal relationship between feeding rhythms and the circadian clock, (a) The improved phase shifts of clock gene mRNA were reported in PERI mice under time-restricted feeding. Transcript levels were measured by qRT-PCR and normalised to Gapdh mRNA levels. The mean ± SD was obtained as described above from three independent experiments, (b-c) Hierarchical clustering analysis of inverted transcripts between ZT1 and ZT13 in the liver (b) and adipose (c) of hPERl^S7 and

AC

PERI mice. High levels are shown in red, and low levels are shown in green. WT represents hPERl^S714, and SG represents PER1^S714G (d) A Venn diagram depicts common genes between the altered expression and PERI binding sites (38) in the liver tissue, (e) Common genes between inverted transcripts and tRF modulated transcripts.

Figure 5. The generation of PERI S714 mutant mice, (a) The alignment of hPERIOD homologues in the vicinity of Serine 662 (S622) of hPER2 is shown. Serine residues are boxed, (b) The construction of BAC transgenic mice. A MYC-tag was inserted just before the stop codon. Mutation of S714G was introduced into the BAC-containing hPERl with 85 kb of flanking sequence 5' of the ATG start site and 50 kb downstream of the stop codon TGA site. The BAC without mutation was used for the generation of wild-type PER1 S714 control mice. Sequencing was employed to confirm the correct mutation, (c) Southern blot analyses of mutant and wild-type mice with a standard copy as reference. DNA gel electrophoresis was used as a loading control.

Figure 6. The representative actograms of locomotor activity in mice across the circadian day. The mice were entrained in a 12 h light/12 h dark cycle for seven to 10 days and then kept in constant darkness. The red lines represent the phase of activity onset in constant darkness. The period was calculated between days 8 and 21 of constant darkness.

Figure 7. A representative PER2::Luc bioluminescence trace of adipose explants from Perl-/-, hPERl S714, hPERl S714G, and hPER2S662G mice during the first and second days.

Figure 8. The BW of Perl-/- and hPER2S662G mice with a HFD over an eight- week study (60% kcal/fat, N = 10). Two-way ANOVA demonstrates that there is no significant difference between Perl-/- and hPER2S662G or wild-type mice under a HFD.

Figure 9. The measurement of metabolic parameters, (a) The body composition was measured by DEXA under NC at two months of age. P values are labelled in their corresponding positions, N = 16 per genotype.

(b) The body composition as measured by DEXA under a HFD for five weeks, N = 14 per genotype. (c,d) The total activity in the horizontal plane was measured by infrared beam breaks in the CLAMS chambers with NC

(c) or HFD (d). (e) Mice were fed with a HFD for five weeks, and the food intake was measured during the 12 h light/12 h dark cycle over six days. The results shown are the average food intake (in grams) ± SD (N = 8).

Figure 10. Nuclear hPERl abundance and phosphorylation in hPERl S714 and hPERl S714G lung extracts. Figure 1 1. The binding of BMAL 1 and CRY1 to the E-box regions of Per2 and Dbp promoter is altered in the liver tissue. IgG served as an interna] control. Chromatin samples from the livers of hPERl S714 (blue) and hPERl S714G (red) were analysed by ChIP with anti-BMAl l (a) or anti-CRYl (b) antibodies. ChIP was analysed by quantitative PCR. The data are expressed as a percentage of the highest value in each experiment. The mean ± SD was obtained from three independent experiments. Two-way ANOVA demonstrated significant differences between the hPERl S714 (blue) and hPERl S714G (red) liver tissues for BMAL1 (p <

0.001) and CRY1 (p < 0.001) binding to the Per2 and Dbp promoter.

Figure 12. ChIP experiment on Perl-/- liver tissues as above Figure 3. The immunoprecipitation was performed with anti-BMAL l or anti-CRYl and IgG as a control. The ChIP was analysed by quantitative PCR. The data are expressed as a percentage of the highest value in each experiment and the mean ± SD. The ratio ± SD of BMALl :CRYl :E-box was obtained by dividing BMAL 1 enrichment by CRY1 enrichment at each time point from three independent experiments.

Figure 13. The mRNA levels of circadian clock components Bmall, Cryl , Per2, and Dbp in adipose (upper) and liver (bottom) tissue at different time points. Transcription levels were measured by qRT-PCR and normalised to Gapdh mRNA. The data are expressed as a percentage of the highest value in each experiment. The mean ± SD was obtained from three independent experiments.

DETAILED DESCRIPTION

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

Throughout this specification, unless the context requires otherwise, the word "comprise" , or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are

I

provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

As used above, and throughout the description of this invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings. If not defined otherwise herein, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "circadian clock" is a biochemical mechanism that oscillates with a period of 24 hours and is coordinated with the day-night cycle, and is the central mechanisms which drive circadian rhythms that are intended to mean the regular variation in physiologic and behavioral parameters that occur over the course of about 24 hours. Such activities include the sleep cycle and nourishment cycle, as well as others.

As used herein, the term "amino acid" refers to the meaning including either of optical isomers, i.e., an L-isomer and a D-isomer of naturally- occurring and non-naturally-occurring amino acids. Furthermore, the term "amino acid" includes only twenty naturally-occurring amino acid residues which constitute natural proteins, as well as other alpha-amino acids, beta.-, gamma-and delta-amino acids, and non-naturally-occurring amino acids, and the like. Thus, the protein PERI may be modified with one or more amino acid residues conservative amino acid residues, for example, one having a similar charge, polarity or other property of one of the alpha-amino acid residue which constitute natural proteins, as well as other alpha-amino acids residues, and beta-, gamma-and delta-amino acid residues, non-natural amino acid residues, and the like. Examples of suitable beta-, gamma-and delta-amino acids include beta-alanine, gamma-aminobutyric acid and ornithine. Examples of other amino acid residues other than those constituting natural proteins or the non-natural amino acids include 3,4-dihydroxyphenylalanine, phenylglycine, cyclohexylglycine, l ,2,3,4-tetrahydroisoquinolin-3-carboxylic acid or nipecotinic acid. As used herein, the term "PERI" includes full length protein of mammalian Period 1 protein, as well as alleles, derivatives and any length of fragments thereof. In particular, derivatives herein include alternation from naturally-occurring forms of the protein by one or more different amino acids, truncated proteins, and fusion proteins of the full length or truncated protein containing either 3' or 5 '-'tags', as well as naturally occurring and non-naturally-occurring mutant sequences provided in the literature cited above and submitted to public databases such as in GeneBank. Derivatives also include proteins which contain a leader, epitope or other protein sequence, such as a Myc-tagged, his-tagged, or a

Flag epitope tag sequence.

The present invention relates to all mammalian PERI proteins. Preferably, the present invention refers to human PERI protein, whose sequence is accessible under Gene Bank Accession NP_002607, as shown in SEQ ID NO: L

As used herein, the term "S714 of SEQ ID NO: l" refer to the amino acid Serine located at position 714 of the full length hPERl protein, as shown in SEQ ID NO: 1. This serine site is the first phosphorylation site in the so-called Serine-Rich motif (SR motif, a serial phosphorylation sites with five serines forming an SXXS like sequence) that has been found in PERs, which was targeted by casein kinase isoform epsilon (CKIs) and casein kinase isoform delta (CKI δ ). Sequence comparison showed this motif to be highly conserved; it was only found in vertebrates' PERs. It is understood that phosporylation of this first serine site would lead the phosphorylation of the following serine sites in the SR motif, and even other potential phosphorylation sites in the protein.

As used herein, the term "phosphorylation status" refers to the phosphorylation levels of the concerned protein or fragment thereof. A full length protein or its fragment may contain many potential phosphorylation sites, and these sites may be phosphorylated by different kinases at different timing and under different conditions. According to the present invention, to determine the phosphorylation status of PERI protein means to attain an understanding of the phosphorylation status of one or plural potential phosphorylation sites presented on the PERI, e.g. one or two or more sites selected from the group consisting of amino acid sites that are phosphorylated.

According to the present invention, change the phosphorylation status of the S714 of SEQ ID NO. 1 may be directly or indirectly associated with the food intake behavior of the subject, in particular, the feeding cycle of the subject. As already mentioned, such SR motif containing this serine phosphorylation site is highly conserved in vertebrates' PERs; consequently, the first serine of this conserved SR motif presented in any homologous PERI proteins could have similar function, i.e., associating with the food intake behavior and altering the feeding cycles of the subject. Such a serine can be found, for example, in S714 of mouse PERI, or S713 of rat PERI , etc.

It is further understood that, according to the present invention, the "phosphorylation status" also refers to a change of the potential of being phosphorylated. For example, a genetic mutation of the perl gene could change the PERl 's potential of being phosphorylated, and if so, such a mutation could be considered as causing change of "phosphorylation status." As shown in the examples, a mutation in hPERl , i.e., changing S714 of SEQ ID NO: 1 to Glycine, permanently changes the phosphorylation map of the protein. Such a mutation thus should also be considered as changing the "phosphorylation status" of the protein. As used herein, the term "kinase" refers to a protein which is evaluated by one skilled in the art to have protein kinase activity, e. g., a protein which is capable of phosphorylating proteins during screening. The screening may use the same, or substantially similar, conditions as set forth in any one of examples below. However, methods of setting up phosphorylation assays are also well known in the art.

As used herein, the term "screening" refers to a set of conditions or agents suitable to permit phosphorylation of PERI. Generally, a screening system contains a ready source of phosphate. A preferred source of phosphate is a ready source of ATP. The screening system may be cell-based or in vitro. Cell-based screening system includes the use of cells which express any PERL A method for screening may be either a cell or a cell-free system. Suitable cell systems include yeast cells, such as S. cerevisia, bacterial cells, such as E. coli, insect cells, such as those used in bacculoviral expression systems, nematode cells, mammalian cells such as COS cells, lymphocytes, fibroblasts (3Y1 cells, NIH/3T3 cells, Rati cells, Balb/3T3 cells, etc.), human embryonic kidney cells, such as 293T cells, CHO cells, blood cells, tumor cells, smooth muscle cells, cardiac muscle cells, brain cells. Preferred cell systems are suprachiasmatic nuclei cells, nerve cells, myelocytes, gliacytes and astrocytes. In a cell based system, if the cell system does not express the PERI, then the cell must be transfected or transformed to express the PERI . Alternatively, a cell-free system may be used. Partially purified or purified PERI may be obtained from recombinant sources which express PERI or whereby the underlying base sequence of the original mRNA encoding the protein is modified.

Recombinant expression of a PERI in a cell may be the result of transfection with one or more suitable expression vectors containing, for example, a promoter and cDNA encoding PERI . Cell-based screening systems also include the use of cells in which the PERI is transuded or transduced into the cell as a fusion protein with a transduction or transducing sequence such as TAT protein obtained from HIV, Antennepedia transduction fragment, or any other means of introducing exogenous protein into a cell. Preferred in vitro screening systems include aqueous compositions comprising a ready source of phosphate. Preferred in vitro screening systems comprise ATP.

Examples of methods for determining the level of phosphorylation of a PERI protein includes standard methods of detecting the amount of protein phosphorylation, such as use of radiolabeled phosphorous and autoradiography, or indirectly by comparing the amount of radiolabeled phosphorous added and the resulting amount of unbound phosphorous.

Alternatively, colormetric or other detection means may be used to determine the level of phosphorylation. Another suitable method for determining the level of phosphorylation of a Period protein includes a cell-free system using glutathione Sepharose beads where PERI is bound to a solid support such as to Sepharose beads, and PERI is added. In addition, numerous alternative methods for determining the amount of PERI protein after are available, and include the use of 35S-Iabeled PERI protein degradation, colormetric assays, elution of bound PERI protein and the like.

The screening methods disclosed herein are particularly useful in that they can be automated, which allows for high through-put screening of large number of agents, either randomly designed agents or rationally-designed agents, in order to identify those agents that effectively modulate or alter the level of phosphorylation and/or degradation of the PERI protein, and hence alter the circadian rhythm of a mammal.

As used herein, the term "mammal" refers to human, primate, canine, rat and other higher organisms that are characterized by having a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young. According to the invention, humans are more preferred. Further, the term "subject" is used throughout the description to describe a mammal and preferably a human, to whom treatment, including prophylactic treatment, with the method and agents provided by the present invention.

"Agent or agents" for use in the present invention include any biological products or small molecule chemical compounds, such as a simple or complex organic molecules, peptides, analogues of peptides, proteins, oligonucleotides, compounds obtained from microorganism culture, naturally- occurring or synthetic organic compounds, and/or naturally-occurring or synthetic inorganic compounds. The choice of testing chemical compounds or biological products to be screened is well within the skill of the art.

As used herein, the term "condition associated with altered feeding cycle" refers to disease or phenomenon that is associated with an altered, abnormal feeding cycle of the subject. Such a condition is especially prominent on that it exhibits an uncoupled food intake behavior with the energy expenditure cycle. Such a condition may result in obesity and other metabolism irregularities. One of the examples of such conditions is the "Night-Eating Syndrome." As used herein, the term "potential to develop a condition" refers to a subject that possesses one or more symptoms or features indicative of having or will have the concerned condition. For example, a human subject having S714G in the SEQ ID NO: 1 in the PERI should at least be considered as having the Night-Eating-Syndrome.

As used herein, the term "feeding cycle" refers to a circadian rhythm associated with the food intake behavior of animals or human being. Such cycle can vary in many aspects, such as the feeding times, the duration of each feeding behavior, the intervals between each feeding behavior, the initial phase of the feeding cycle, etc.. The present invention relates to a method of regulating feeding cycle of a mammalian subject, e.g., a human being. It is important to understand that such regulation of the feeding cycle could affect each aspect of the feeding cycle.

As used herein, "treating" or "treatment" refers to an approach for obtaining beneficial or desired results, including and preferably clinical results. Treatment can involve optionally either the amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition.

As used herein, unless the context makes clear otherwise, "prevention," and similar words such as "prevented," "preventing" etc., indicates an approach for preventing, inhibiting, or reducing the likelihood of, the onset or recurrence of a disease or condition. It also refers to preventing, inhibiting, or reducing the likelihood of, the occurrence or recurrence of the symptoms of a disease or condition, or optionally an approach for delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.

The method provided by the present invention is related to screen or detect the phosphorylation status of PERI , and further related to diagnose conditions associated with altered feeding cycle or potential to develop such conditions. Supported by the method, the present invention further provides a way to screen agents that can effectively treat or prevent conditions associated with altered feeding cycle. It is worth of emphasizing that such agents screened according to the above-described methods can be further used in manufacturing effective drugs for treatment and prevention of altered feeding cycle related diseases, e.g. Night-Eating Syndrome.

EXAMPLES

Generation and characterisation of hPERl mutant mice

To explore the specific function of PERI, BAC transgenic mice carrying an S714G mutation (hPERl S714G) and wild type (hPERl S714) with a Myc-tag before the stop codon (Figure lb) were generated. A human transgene was used because it is highly homologous to the mouse gene and can be used to distinguish the transgene from endogenous expression. The resulting BAC construct included the promoter and a large flanking region (extending 85 kb upstream and 50 kb downstream) and was expected to have a high probability of containing most regulatory elements and locus control regions ( G. A. Maston, et al.„ Annu Rev Genomics Hum Genet 2006, 7, 29). Copy numbers of all lines were assessed by Southern blot analysis (Figure 5c). All mice were on a C57BL/6J background.

Low- (L) and high-copy (H) transgenic lines for each genotype were selected and were analysed their locomotor activity. In contrast to the hPER2 transgene, which exhibited a dose-dependent period elongation under constant darkness ( X. Gu et al., Cell Death Differ, 201 1), transgenic mice with either L or H wild-type hPERl exhibited no differences in wheel-running activity (L: τ =23.73 ± 0.16, H: τ = 23.72 ± 0.14) (Figure l a, Figure 6). hPERl S714G mice exhibited a minor dose-dependent shortening of their wheel-running period (L: hPERl S714G τ = 23.36 ± 0.26; H: hPERlS714G τ = 22.67 ± 0.16) (Figure la, Figure 6). hPERlS714G;Perl -/- exhibited a shorter period than Perl-/- mice (Figure l a), indicative of the dominant role for this mutation. These data suggest that the hPERl mutation leads to an accelerated clock by locomotor assay but this effect is relatively weaker when compared with hPER2S662G mice (Figure la).

Given the prominent role of Perl in sustaining robust rhythms in tissue-autonomous oscillators (A. C. Liu et al., Cell 2007, 129, 605), the circadian oscillations in peripheral tissues were monitored by breeding hPERl S714, hPERl S714G, or hPER2S662G mice with mPER2::LUC knock-in reporter mice ( S. H. Yoo et al., Proc Natl Acad Sci U S A 2004, 101 , 5339). Unexpectedly, ex vivo SCN, lung, liver, adipose, and spleen tissue are markedly shortening of period by hPERl S714G (red) (Figure lb, c). Although deletion of Perl exhibited a less persistent oscillation in peripheral tissues, the mean period from the first to second cycle was longer than hPERl S714G or hPER2S662G, again indicating that the phenotype from hPERl S714G mice is not due to a loss of Perl function (Figure 7). Relative expression levels of mPerl and mPer2 for the potentially different roles related to tissue-specific behaviour were also analyzed. The relative enrichments of mPerl and mPer2 in liver, lung, adipose and spleen tissues are varied (Figure I d), reflecting the diversity of their functions across different organs. In addition, the individual clocks are almost anti-phasic between hPERl S714 and hPERl S714G during the first cycle in adipose and lung tissue (Figure lb), providing a possibility for internal misalignment in hPERl S714G mice. Our in vitro data show that hPERl S714G has globally impairments on tissue-autonomous oscillators although its effect is variable across tissues. hPERl S714G mice exhibit advanced feeding rhythms and uncoupled food intake with energy expenditure.

Distortion of the phase relationship of individual clocks in hPERl S714G tissues prompted us to measure foraging behaviour and energy expenditure parameters using a Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments) under a 12 h light/12 h dark (LD) cycle. A non-linear multiple-regression, cosine-fit analysis revealed that the food intake rhythms in both hPERl S714 wild-type and hPERl S714G mutant mice fit a 24 hour rhythm (P <0.05, for all the mice examined). The circadian acrophases of food intake from hPERl S714 and hPERl S714G mice were calculated on three consecutive days. The peak of food intake was at ZT17.88 ± 0.42 and 15.44 ± 0.29 for L and H hPERl S714 mice, respectively. The peak was advanced to ZT13.48 ± 0.61 and 10.00 ± 0.54 for L and H hPERl S714G mice, respectively (P < 0.0001, one-way analysis of variance (ANOVA)) (Figures 2 a, b).

To assess paralogue-specific roles for the regulation of the feeding phase, the feeding behaviours in hPER2S662G and hPERl S714G mice were compared. The peak of food intake was significantly earlier in hPERl S714G mice (ZT10.00 ± 0.54) than in hPER2S662G mice (ZT14.68 ± 0.20), (P < 0.0001, one-way ANOVA) (Figure 2b). The hPER2S662G mice exhibited a 2 h shorter locomotor period than the hPERl S717G mice, indicating that the advanced feeding phase in the hPERlS717G feeding phase is specific and not a consequence of short behaviour period (Figure 2b left vs. right). In addition, Perl knockout mice exhibited a subtle advanced feeding phase (ZT14.71 ± 0.40), indicating PER1 S714G as a dominant role on this function (Figure 2b). Furthermore, the Δ phase (feeding phase - oxygen consumption phase) was -3.35 ± 0.45 in hPERl S714G mice compared to 0.1 1 ± 0.35 and -1.02 ± 0.38 in L and H hPERl S714 mice (Figure 2b), suggesting that oxygen consumption is significantly misaligned with the feeding rhythms (P < 0.0001 , one-way ANOVA). A weak misalignment between feeding rhythms and oxygen consumption (Δ phase) was also observed in the hPER2S662G mice (-1.37 ± 0.2, p = 0.06, compared with the wild-type mice). The effect is less severe than in the hPERl S714G mice, suggesting a paralogue-specific role in the misalignment between the feeding phase and oxygen consumption phase for hPERl . Consistent with this phenotype, mice with the S714G transgene consume more food during the light phase when compared with the hPERl S714 control (56.9% ± 9.3% vs. 36.3% ± 5.7%, respectively, P < 0.0001 , one-way ANOVA) (Figure 2c).

To assign a biological significance to the misalignment between food intake and energy expenditure, the growth curve of hPERl S714 and hPERlS714G mice fed with normal chow (NC) or high-fat diet (HFD) were analysed. A comparison of body weight (BW) using two-way ANOVA (genotype x time of day) did not reveal any genotype-specific differences in mice fed NC over nine months (Figure 2d) (p = 0.96). However, the hPERl S714G mice gained body weight (BW) more rapidly compared to the hPERlS714 control on HFD (Figure 2e) (p = 0.033, genotype x time, two-way ANOVA). No similar effects were observed for Perl knockout and hPER2S662G mice (P > 0.05, Figure 7), indicating that short-term high- fat induced obesity is specific to the hPERl S714G mice. Furthermore, dual-energy X-ray absorption (DEXA) analysis showed that the body compositions (BCs) are identical for both genotypes before a HFD in eight-week-old mice (Figure 9a). However, there is an increase in the total fat mass of the hPERl S714G mice versus hPERlS714 mice after being kept on a HFD for five weeks although the total lean mass remained identical in both genotypes (Figure 9b), suggesting that this gained BW is associated with altered body composition (fat accumulation). No difference was noted in total activity (Figure 9c, d) or total food intake (Figure 2c and Figure 9e) for NC or HFD as estimated by the CLAMS, suggesting that this increased BW was not due to decreased activity or increased food intake.

To assess whether advanced feeding behaviour accelerates high-fat-induced obesity in hPERl S714G mice, eight-week-old male hPERl S714 and hPERl S714G mice with NC were entrained at a fixed night phase for two weeks, and then changed to a HFD exclusively during the night phase. The hPERl S714 and hPERlS714G mice exhibited comparable BW gain (Figure 2f, p = 0.59) and less BW gain for night-only HFD when compared with feeding a day (Figure 2f vs 2e), suggesting that irregular mealtime is a major contributor to obesity in hPERlS714G mice. Furthermore, to test whether altered feeding regimen can correct misalignment between feeding rhythms and oxygen consumption (Δ phase) under HFD, foraging behaviour and energy expenditure parameters were recorded by CLAMS under night phase only feeding. The hPERl S714 and hPERlS714G mice both exhibited comparable pattern in food intake and oxygen consumption (Figure 2g), suggesting that advanced feeding is responsible for gained BW in the hPERl S714G mice.

PER1 S714G mutation accelerates feedback loop speed

Altered individual clocks and thus altered feeding behaviour likely reflect impaired function in the circadian negative feedback loop. Firstly, the effect of the S714G mutation on the phosphorylation of the hPERl protein was investigated. The hPERl S714 and hPERl S714G mice were sacrificed every 4 hr, and liver and lung nuclear extracts were prepared for Western blot because the cytoplasmic hPERl is barely detectable (thereafter). The wild-type hPERl (hPERl S714) exhibited prominent temporal changes in both abundance and mobility shift over the circadian cycle by anti-MYC antibody. In contrast, mutant hPERl exhibited a less abundance and lower mobility shift during the night phase, but not for daytime as hPER2S662G does (Y. Xu et al., Cell, 2007 128, 59) (Figure 3a and Figure 10). Over the past years, the observation that faster degradation and nuclear/cytoplasmic distribution alteration by impairing sequential phosphorylation of SXXS in PER2 has been suggested to explain circadian phenotype of PER2 (K. Vanselow et al., Genes & development 2006, 20, 2660). The enhanced degradation of PERI proteins by CK1 tau at selective night phase were translated into the shortening period and altered phase (J. Dey et al., Journal of biological rhythms 2005, 20, 99; M. Gallego, et al., Proceedings of the National Academy of Sciences of the United States of America 2006, 103, 10618; Q. J. Meng et al., Neuron 2008, 58, 78). Here, the stability of the nuclear hPERl was further estimated using cultured mouse embryo Fibroblast cells (MEFs) from hPERl S714 and hPERl S714G mice and found that nuclear hPERl carrying the S714G mutation accelerates the degradation of the hPERl protein by a cycloheximide (CHX)-chase analysis (Figure 3b). This is consistent with the observation that the hPERl ^S7140 protein was obviously less than hPERl⁸⁷¹⁴ during the night phase (Figure 3a). To extend this result to the circadian cycle changes, the kinetic expression levels of two repressors, PERI and CRYl proteins were examined, in the synchronized MEFs from hPERl ^S714 and hPERl^S714G mice around the clock. The nuclear hPERl^{S7 i 4} protein exhibited temporal changes over the clock in hPERl^S714 MEFs. However, the peak of nuclear PERI S71 G protein appears more early when compared with hPERl ^S714 protein, and is barely detectable after 16-20 h. Correspondingly, the CRYl protein is equally reflected in earlier peak in both the nucleus and the cytoplasm when compared with wild-type (Figure 3c), in a PERI reliant manner, suggesting a shorter cycle in the hPERl^{S7 ,4G} MEFs. To test S714 site on nuclear shuttle, MEFs were treated with CHX for 14 h to clear clock proteins from the cells, and then were synchronized by dexamethasone (DEX) after removing CHX. Interestingly, appreciable PER1 ^S714G protein was detected at 4 h of CHX removal, followed by early accumulation of nuclear CRYl at 4 to 8 h in PERI MEFs. In contrast, obvious PER1 ^S714 protein was detected at 8 h of CHX removal, followed by a peak of nuclear CRYl at 12 h in PERI⁵⁷¹⁴ MEFs (Figure 3d). Importantly, nuclear PERI and CRYl were barely detectable at 0 h, suggesting an equal start (Figure. 3d). The advanced accumulation of PERI and CRYl proteins in the nucleus in the hPERl^S714G MEFs reflects a faster nuclear import in molecular levels. Because endogenous mPERl exist in both MEFs, it is scenario that the early import of mutant hPERl with its partner CRYl is more a result of shortening period than of an altered degradation mechanism.

Then, time-dependent effect of the S714 status of PERI on the feedback loop of the circadian oscillator was determined. The binding of BMAL1 and CRYl proteins to the E-box regions of Per2 and the albumin D site-binding protein (Dbp) gene (Figure 1 1), an important circadian output gene ( M. Stratmann, et al., Genes & development 2010, 24, 1317) was examined. The ratio changes of BMAL1 :CRY1 on the E-box showed a daily oscillating pattern, which was in the same phase as rhythmic pattern of PERI protein in hPERl^S714 mice (Figure 3e, Figure 1 1 for BMAL1 and CRYl binding on the E-box). As PERI was reported previously to interact with CRYl to repress the BMAL1 :CLOCK activation on the E-box, it is highly possible that the cyclic accumulation of PERI may represent part of a mechanism to regulate phase of E-box activity. Interestingly, the ratios of BMAL CRYl are advanced by 8-10 h in hPERl^{S7 I 4G} mice when compared with hPERl ^S714 mice, indicating that S714G mutation in PERI changes the phase of the E-box activity, in consistent with the idea that S714G mutation leads an earlier nuclear import. The effect for S714G mutation on nuclear import and protein stability may reflect a phase shift and short period. To distinguish the difference between PER1 ^{S7 I4G} and PERl ^nu", it was examined the binding BMAL 1 and CRY1 proteins to the E-box regions of Per2 and Dbp gene in Perl^"'^" liver tissue.

Finally, to determine whether the ratios of BMAL1 :CRY1 represent transcriptional activity, the binding of RNA polymerase II (RNAPII) to the Per2 and Dbp promoters were examined ( J. P. Etchegaray, et al., Nature 2003, 421, 177). The phases of RNAPII-binding signals almost coincided with the altered ratio of BMAL1 :CRY1 in the hPERl^S714 and hPERl^{S7 i4G} liver tissue (Figure 3f). Again, the profiles of clock gene mRNA in liver and adipose tissue were analysed. These expression profiles exhibited anti-phasic or nearly anti-phasic patterns in hPERl^S714G when compared with hPERl ^S714 in both tissues (Figure 3g, Figure 13), suggesting that the S714G mutation in hPERl changes the counterbalance between BMAL CLOC and PER: CRY, and plays a crucial role in phase shift in the negative feedback loop.

Altered feeding rhythms and clock defects reciprocally reinforce internal misalignment

The phase of transcripts in each tissue is an integration of its endogenous period with in vivo inputs. Food is a dominant Zeitgeber for circadian oscillators in several mouse tissues, including liver, kidney and heart. Whether the advanced feeding behaviour reinforces the phase shifts of targeted gene expressions in adipose and liver tissue was to be determined. To do so, the hPERl ^S714 and the hPERl^S7,4G mice were fed from ZT16 to ZT20 with NC for two weeks. As reported previously for nocturnal animals ( F. Damiola et al., Genes & development 2000, 14, 2950), in hPERl⁵⁷¹⁴ mice, restricting feeding time to the night phase does not significantly alter the phase angle of cycle gene expression (Figure 4a compared with Figure 3g, Figure 13). In contrast, an improved phase advance from approximately ZTO-4 to ZT4-8 h for the Per2 and Dbp

¾^*7 1 A ^*

mRNA in the hPERl mice was observed, suggesting that advanced feeding as dominant Zeitgeber reinforces the phase shifts of clock gene transcripts in the hPERl ^S714G mice.

To systematically identify transcripts that are affected by the S714G mutation, the liver and adipose trans criptomes at Zeitgeber time (ZT) 1 and ZT13 (lights on at ZTO, lights off at ZT12) are compared using Agilent RNA microarray services from Capitalbio. A two-fold change threshold criterion was set, and the results were considered statistically significant at the 5% level (P < 0.05). Hierarchical cluster analysis identified 1 19 transcripts in the liver tissue (Figure 4b) and 28 transcripts in adipose tissues (Figure 4c) that were significantly inverted at ZT1 and ZT13, suggesting that advanced feeding is a major cause of enhanced phase shift of rhythmic transcriptions. Then, 1 19 inverted transcripts in liver tissue with 240 transcripts changed by tRF from Panda group data were compared ( M. Hatori et al., Cell metabolism 2012, 15, 848), and detected 44 common to both groups, again reflecting feeding consolidation (Figure 4d). Finally, Sixty of the live transcripts among 1 19 inverted transcripts colocalised with PERI binding sites reported in databases ( N. Koike et al., Science 2012, 338, 349), or the RRE, D-box, E-box-driven rhythmic genes determined by the Ueda group (http://www.dbsb.org/), including Bmal l , Nrld2, Nfil3, Dbp, Hmgcr, and PPAR . (Figure 4e). These data suggested that most of genes are directly regulated by PERI or depend on the clock system. Thus, the synergistic interaction between clock and metabolism is exemplified in the rhythmic transcripts.

Although a variety of clock-gene knockout mice have been used to study the effects of clock genes, the manner in which the clock gene paralogues evolved to integrate and facilitate the diversification of the mammalian circadian system remains unclear. Previous studies found that the SXXS motif was highly conserved among vertebrate PERIOD homologues, implying that this motif is critical to the proper functioning of PERIOD. The importance of this motif was confirmed by the fact that S662 residue in hPER2 is necessary for triggering a phosphorylation cascade and signal amplification mechanism ( Y. Xu et al., Cell 2007, 128, 59). Other proteins including the nuclear factor of activated T-cell protein (NFAT) family and APC ( H. Okamura et al., Molecular and cellular biology 2004, 24, 4184; M. A. Price, Genes & development 2006, 20, 399) developed the SXXS motif during evolution. This observation suggests that the emergence of the SXXS motif occurred by an evolutionary speciation and might enable these proteins to adapt to varying environmental ways. It was hypothesised that modifying these motifs could be a useful tool to dissect the specific function of the paralogous proteins that is often ignored. In this study, It was uncovered a critical role for S714 site in PERI in mediating the feeding phase that could not be found in Perl^"'^" mice and in hPER2^S662G or Per2^{" _} mice. This finding suggests that duplicated genes diverge over time and undergo functional specialisation but partly overlap with the progenitor gene.

Indeed, 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 rest-activity cycle, especially in higher organisms. Generally, the environmental light-dark cycle provides the principal entraining signal to the SCN for the regulation of behaviour, including rest-activity cycles and thus feeding cycles ( U. Albrecht, Neuron 2012, 74, 246). However, feeding cycles can act as a synchroniser and produce dynamic shifts in some rhythmic processes. hPER2^S662G mice has been suggested as an advanced sleep-phase syndrome (ASPS) model and exhibit a 4 h phase advance of activity rhythms in LD cycles (Y. Xu et al., Cell 2007, 128, 59). 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 hPER2^S662G mice. In contrast, it was found that hPERl^S714G mice exhibit a markedly advanced phase of feeding behaviour when compared with hPER2^S662G mice, irrespective of their rest-activity cycles, suggesting that PERI and PER2 function differently, and rest-activity and feeding cycle are at least in part separately. The observation that hPERl ^S714G mice develop obesity quickly on a HFD due to altered feeding times but not for hPER2^S662G mice further supports their specific function. In humans, Night-Eating Syndrome (NES) is characterised by late-night binge eating (i.e., lack of appetite in the morning and evening or nocturnal hyperphagia) ( A. J. Stunkard et al., Am J Med 1955, 19, 78) and affects between 1-2% of the population. Most people with NES are also obese ( K. C. Allison et al., Obesity 2006, 14 Suppl 2, 77 S). Although a mutation screen in NES patients is unavailable, a genome-wide analysis of human disease alleles demonstrate that sequence variants co-occur at aligned amino acid pairs more frequently than expected by chance, due to similar functional constraints on paralogous protein sequences (M. Yandell et al., PLoS Comput Biol 2008, 4, el 000218). Previous study identified an S662G in PER2 for FASPS, and it is highly possible that there is an S714G mutation in PERI in NES.

The mechanism of diverse function of PERI and PER2 on advance phases of feeding-fasting cycles and activity-rest cycles remains to be determined. A recent study demonstrated that PER2 rather than PERI integrating with various nuclear receptors provides to explain the functional differences between the two PER proteins ( I. Schmutz, et al., Genes & development 2010, 24, 345). Here as well, the targeted transcripts in the liver and adipose tissue induced by PER1^{S7 I4G} varied substantially and, Perl and Per2 enriched differently, indicating tissue-specific effects of PERI on target gene expression. Evidence that connections between the circadian clock and most of physiological processes are bidirectional is emerging. Further studies will be required to unravel PERI binding proteins and shed light on their possible role in metabolism using tissue-specific floxed techniques.

The examples set forth below are for illustrative purposes only and are not intended to limit, in any way, the scope of the present invention.

Example 1

Generation of PERI BAC transgenic mice

RP1 1 -1D5 is a bacterial artificial chromosome (BAC) clone from the human genomic library containing the entire PERI locus on a 163-kb genomic insert with 85 kb upstream of the gene (Children's Hospital Oakland Research Institute). This BAC clone was modified by homologous recombination as previously described ( H. Y. Lee et al., The Journal of clinical investigation 2012, 122, 507). The c-Myc Tag encoding sequence was introduced to the region before the TAG stop codon of the hPERl gene to allow for protein detection. Then, the mutation (S714G) was inserted into the above BAC clone as hPERl ^s714G mutant form by recombination again and the non-mutated clone was a control. Transgenic mice were generated using microinjecting engineered-BAC clones. The transgenic founders were backcrossed to C57/BL6J for > 5 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. Example 2

Animal care and behavioral analysis

Measurements of the free-running period were performed as previously described in X. Wang et al., The EMBO journal 2010, 29, 1389. Mice were placed in individual cages equipped with running wheels in light-tight ventilation chambers with timer-controlled lighting. Wheel-running activities were monitored over a 12: 12 h LD cycle for seven days followed DD up to four weeks. ClockLab system analyzed circadian parameters, and the period was calculated from 8 to 21 days after constant darkness (Actimetrics, IL). All animal studies were carried out in an AAALAC International (Association for Assessment and Accreditation of Laboratory Animal Care) accredited SPF animal facility, and all animal protocols were approved by the Animal Care and Use Committee of the Model Animal Research Center, the host for the National Resource Center for Mutant Mice in China, Nanjing University.

Example 3

Luminescence Recording

The detailed methods for real-time measurement of luminescence from ex vivo tissues followed the Shin Yamazaki method and personnel communication with Yamazaki and Sergey A. Savelyev for SCN culture ( S. Yamazaki, et al., Methods Enzymol 2005, 393, 288; S. A. Savelyev, et al., J Vis Exp, 2010). Explants were briefly prepared within 1 hr before lights shut off and cooled in Hank's balanced salt solution supplied with 10 mM HEPES, 4.5 mM NaHCO3, 100 U/ml penicillin, and 100 U/ml streptomycin. Explants were then transferred to DMEM (Product No. D2902, Sigma) supplemented with B27 supplement (Product No. 17504-044, Gibco), 10 mM HEPES (pH 7.2), antibiotics (100 U/ml penicillin, 100 U/ml streptomycin, 0.1 mM luciferin (Promega), 4.5 g/1 glucose, and 4.2 mM NaHCO3. SCN was cultured in the Millicell (0.4 uM, 30 ram diameter, Millipore). Dishes were sealed with Optical Adhesive Film (Applied Biosystems). All assays were done in a lumiCycle, a 32-channel automated luminometer (Actimetrics Inc, IL) placed in a 37^°C incubator and monitored over 10-minute intervals.

A self-step algorithm developed by Mariko Lzumo ( M. Izumo, et al., PLoS Comput Biol 2006, 2, el 36) was used to recover the rhythm of the luciferase:

(1) From the first data point, the linear regression is applied to a sub-series data sequence with a 24-hr length to calculate the sub trend. After that, the trend values of the sub-series sequence are temporarily stored in memory. From the second raw data point, the previous step was repeated, then the third raw data point, and so forth. The process does not cease until the sub-series data exceeds beyond the last raw data point.;

(2) All values stored at every time point are then averaged to gain the final trend;

(3) The rhythmic components are extracted by removing the trend from raw data;

(4) To reduce the influence of damping, each detrended data is divided by the standard deviation of the sub-series data sequence (24 hr) and temporarily stored in memory;

(5) Next, the data obtained in step (4) was averaged to produce a detrended time series of constant unit variance.

After recovering the rhythm, the phase and period were estimated. 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. Example 4

Tissue Collection

Mice of the indicated genotypes were entrained to a 12-12-h light dark cycle for at least seven days before tissue collection. Tissues were taken at 4-hr intervals Zeitgeber times (ZT) 0, 4, 8, 12, 16, 20 and 24, where ZT12 corresponds to the onset of subjective night. Each time point had average three to four mice for each genotype.

Example 5

RNA Isolation, RT-PCR and mRNA Expression Analyses

RNA isolation and RT-PCR (including primers for mRNA profiling) were carried out essentially as previously described ( X. Wang et al._; The EMBO journal 2010, 29, 1389). The relative levels of each RNA were normalized to the corresponding Gapdh or 36B4 RNA levels. Each ZT value used for these calculations is the mean of at least two duplicates of the same reaction. Relative RNA levels were expressed as percentage of the maximal value obtained for each experiment. Each mean ± s.d. was obtained from three independent experiments (and thereafter for all mean ± s.d.).

Example 6

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 ( B. M. Bolstad, et al., Bioinformatics 2003, 19, 185). For Significance Analysis of Microarray (SAM) analyses ( V. G. Tusher, et al., Proceedings of the National Academy of Sciences of the United States of America 2010, 98, 51 16), transcripts between hPERl^S714G and hPERl^S714 in the liver and adipose at specific time points (2T1 and ZT13) demonstrating >2-fold change or a false discovery rate of 5%, were statistically significant. The hierarchical clustering analysis was conducted with MeV using an average linkage method ( A. I. Saeed et al., BioTechniques 2003, 34, 374).

Example 7

Antibodies, Western Blotting (WB), and Chromatin Immunoprecipitation (ChIP)

Fresh liver extracts from designated Zeitgeber times were prepared according to a nuclear extraction kit (Active Motif, 100505). Briefly, 100 mg of fresh liver was washed with 5 ml of ice cold PBS/Phosphatase Inhibitors and transferred to 1 ml of ice-cold 1 X hypotonic buffer supplemented with 2 ul 1M DTT and 2 ul detergent and homogenized. After centrifugation for 10 min at 850 X g, cells were gently re-suspended in a 150 ul 1 X-Hypotonic buffer for another 15 min and centrifuged for 1 min at 14,000 g to collect nuclear pellets. The nuclear pellet was re-suspended in a 100 ul complete lysis buffer with a protein inhibitor cocktail. Nuclear protein was extracted. Cytoplasmic and nuclear fractions were quantified with the Bradford assay and aliquoted in liquid nitrogen. Mouse embryonic fibroblast cells were prepared from 13.5 -day-old embryos. The cells from the third passage were grown to confluence in 100-mm dishes. Nuclear and cytoplasmic proteins were harvested at the indicated times after treatment with a protein biosynthesis inhibitor CHX at 100 ug/ML. Nuclear and cytoplasmic factions were analyzed by Western blotting. Proteins were separated by electrophoresis through sodium dodecyl sulfate (SDS)-6% polyacrylamide gels (acrylamide 29.6 g; bisacrylamide 0.4 g) and transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk in PBS and incubated with MYC antibody (Sigma) diluted at 1 :500 in PBS containing 0.05% Tween 20, according to the manufacturer's instruction. Immunoreactive bands were detected using goat-anti-rabbit IgG-HRP (Santa Cruz sc-2030) and ECL (Amersham). ACTIN staining served as the loading control.

Chromatin immunoprecipitation (ChIP) assays were performed as previously described with modification (M. Yandell et al.₅ PLoS Comput Biol 2008, 4, el 000218). A hypotonic buffer (Active Motif, 100505) was used to obtain the nuclear extract. Rabbit igGs from non-immunized rabbits were employed as the negative control. For ChIP on E-box sites (at the Cryl and Per2 promoter), the 1^st intron of Cryl was used as the control locus; for ChIP on RRE sites (at the Bmall and Cryl promoter/enhancer), the Cryl promoter was used as the control site as described previously ( G. Shi et ah, Proceedings of the National Academy of Sciences of the United States of America 2013). The primers for q-PCR are listed in the Supplementary Table.

Example 8

Metabolic rhythm measurement and Analysis

Mice were entrained to normal LD cycles (lights on at 8:00 am and lights off at 8:00 pm) for one 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 = ((hours*3600+minutes*60+seconds)/3600-8)*2, which the first day at 8:00 am is as 0, and the next day at 8:00 am is recorded as 48. Cumulative food intake and V02 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 was fitted with the equation (y=a*cos (2*pi/48*x-b) + c). Factor b represents the phase of each mouse food intake or V02 phase. The phase was then transformed to ZT time with the equation ZT=24*b/2*pi. Δ phase was generated from feeding phase minus VO2 phase. Each value was shown as mean ± SEM.

Example 9

Feeding Schedule and Growth Curve

Age-matched wild type and mutant mice were grouped housed (five per cage) to 8 weeks old under normal chow (NC) (LabDiet). Next, they were fed NC or HFD (research diets, D 12492 60% fat kcal% diet) to 9 months. 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 ZT0 to ZT12 with NC for 2 weeks before changing to HFD. Body weight was recorded daily at ZT0. At the beginning and end of the experiment, body composition (fat and lean mass) was determined by dual X-ray absorptiometry (DEXA, PIXImus, GE Lunar Corporation, Madison, WI, USA) under Avertin anesthesia.

Example 10

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. Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

1. A method of screening mammalian for conditions associated with altered feeding cycle or potential to develop such conditions, comprising detecting the phosphorylation status of PERI in the mammalian.

2. The method according to claim 1, wherein said phosphorylation status is determined through detecting the phosporylation status of S714 of SEQ ID NO: 1, and the detection of hypophosphorylation of S714 indicates a positive diagnosis of said conditions.

3. The method according to claim 2, wherein the detection of phosporylation status of S714 of SEQ ID NO: 1 comprising detecting whether the Serine at the position 714 is mutated into Glycine.

4. The method according to any one of claims 1-3, wherein the condition is Night-Eating Syndrome.

5. A method of treating or preventing conditions associated with altered feeding cycle in mammalian, comprising regulation of the phosphorylation status of PERI of the mammalian.

6. The method according to claim 5, wherein said regulation of phosphorylation status comprising regulating the phosporylation of S714 of SEQ ID NO: 1.

7. The method according to claim 5, wherein said regulation of phosphorylation status of SEQ ID NO: l of PERI is carried out by a kinase that can phosphorylates PERI .

8. The method according to claim 7, wherein the kinase is casein kinase isoform ε.

9. The method according to claim 5-7, wherein the condition is Night-Eating Syndrome.

10. A method of screening for agents capable of treating or preventing conditions associated with altered feeding cycle in mammalian, comprising:

a) providing test cells or tissues taken from the mammalian;

b) providing a plurality of candidate agents; and

c) contacting the test cells or tissues with the candidate agent under conditions effective for regulating phosphorylation of PERI, and identifying the candidate agent that alters the phosphorylation status within PERI, and as a result, such identified agents having potential capability of treating or preventing conditions associated with altered feeding cycle in mammalian.

1 1. The method according to claim 10 , said regulation of phosphorylation comprising regulating the phosporylation of S714 of the amino acid sequence of SEQ ID NO: 1.

12. The method according to claim 10-1 1, wherein the condition is Night-Eating Syndrome.

13. The use of an agent capable of regulating the phosphorylation within PERI in manufacture of medicines for treating or preventing conditions associated with altered feeding cycle in mammalian.

14. The method according to claim 13 , said regulation of phosphorylation comprising regulating the phosporylation of S714 of the amino acid sequence of SEQ ID NO: 1.

15. The method according to claim 13, wherein the kinase is casein kinase isoform ε.

16. The method according to claim 13-15, wherein the condition is Night-Eating Syndrome.