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(The FASEB Journal. 2001;15:2613-2622.)
© 2001 FASEB

Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain

DMITRI CHILOV, THOMAS HOFER, CHRISTIAN BAUER, ROLAND H. WENGER1 and MAX GASSMANN2

Institutes of Physiology and Veterinary Physiology, University of Zürich, CH-8057 Zürich, Switzerland

2Correspondence: Physiologisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. E-mail: maxg{at}access.unizh.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The key elements of circadian clockwork and oxygen homeostasis are the PAS protein family members PER and CLOCK and hypoxia-inducible factor 1{alpha} (HIF-1{alpha}). The PAS domain serves as an interface for protein–protein interactions. We asked whether a cross-talk exists between the PAS components of hypoxic and circadian pathways. We found several isoforms of PER1 protein that exhibit tissue-specific size differences. In the mouse brain, a predominantly nuclear 48 kDa isoform that followed a daily rhythm was observed. The 48 kDa form was found in the nuclear fractions derived from mouse liver, Swiss3T3 fibroblasts, and N2A neuroblastoma cells. In mouse kidney and human 293 kidney cells, a 55 kDa PER1 form was detected. CLOCK was observed as a predicted 100 kDa protein in rat-1 cells and in all analyzed mouse tissues including brain, liver, kidney, and spleen. In contrast to PER1, CLOCK protein expression was not rhythmic. Exposure to hypoxia led to increased PER1 and CLOCK protein levels in mice. Based on coimmunoprecipitation experiments that showed protein–protein interaction between PER1 and the {alpha} subunit of HIF-1, we suggest that these hypoxic effects may be modulated by HIF-1{alpha}.—Chilov, D., Hofer, T., Bauer, C., Wenger, R. H., Gassmann, M. Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain.


Key Words: circadian clock • hypoxia • HIF • PAS proteins


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CIRCADIAN CLOCK, A mechanism conserved among species ranging from bacteria to eukaryotes, governs daily rhythmicity of molecular, physiological, and behavioral processes (1 2 3 4) . In mammals, a master clock generating circadian rhythms is located in the suprachiasmatic nucleus (SCN) of the hypothalamus (5 , 6) . The SCN is an autonomous pacemaker (7 , 8) that can be entrained by environmental cues, thus synchronizing the SCN-driven output rhythms to 24 h (9 10 11 12) . Autonomous oscillators are also found in several peripheral tissues of mammals (13 14 15 16 17) . Recently, several components of the mammalian circadian clock have been identified. These include the PAS (Per-ARNT-Sim) proteins PER1 (18 , 19) , PER2 (15 , 20 , 21) , and PER3 (22) . Two other PAS proteins, CLOCK and BMAL1/MOP3/ARNT3, were cloned (23 24 25 26 27 28 29) and characterized as positive transcription factors that bind to the E-box elements present in the promoter of the mouse per1 gene (30 , 31) . The three mPER proteins are each able to inhibit CLOCK:BMAL1-induced transcription in NIH3T3 cells (9 , 32) . In addition, the mouse Cryptochrome genes mCry1 and mCry2 have been implicated in regulation of the mammalian clock. A molecular mechanism regulating rhythmic output from SCN has been described and involves a positive regulation of vasopressin mRNA by CLOCK:BMAL1 heterodimers that is inhibited by mPER1 and mTIM proteins (9) .

An array of pharmacological and genetic approaches used to study circadian rhythms in mammals resulted in the observations that homozygous Clock-deficient mice are arrhythmic in constant darkness (DD) (25 , 33) and show reduced mRNA levels of mPer1, mPer2, mPer3, mCry1, and mCry2 mRNA (9 , 34 , 35) . Moreover, homozygous Per2 mutant mice (termed mPer2Brdm1) lose circadian rhythmicity and show reduced oscillation of mPer1 and mPer2 mRNAs in DD (36) . Mice deficient in both mPER1 and mPER2 do not express circadian rhythms (37) . Analysis of Clock/Clock, mPer2Brdm1 mutant, and Cryptochrome-deficient mice provided evidence for the mammalian clock model in which CLOCK-BMAL1 drives transcription of the Per and Cry genes, whereas PER2 acts as a positive regulator of the Bmal1 and CRYs as negative regulators of the Per and Cry (38) . mPER1 was shown to influence rhythmicity primarily through interaction with other clock proteins (39) .

Oxygen is essential to all higher organisms because it serves as the terminal electron acceptor in mitochondrial oxidative phosphorylation and because several enzymatic processes require molecular oxygen as a substrate (reviewed in ref 40 ). In response to reduced oxygenation, activation of the hypoxia-inducible factor 1 (HIF-1) regulates transcription of several genes involved in oxygen homeostasis (reviewed in refs 41 42 43 ). HIF-1 is a heterodimeric complex composed of the two basic helix-loop-helix (bHLH) PAS subunits, HIF-1{alpha} and HIF-1ß/ARNT. HIF-1{alpha} is efficiently translated under normoxic and hypoxic conditions (44) and is activated by a redox-dependent proteolytic stabilization. Hypoxic induction of HIF-1{alpha} is instantaneous (45) and results from blocking of the von Hippel-Lindau factor-mediated ubiquitinylation (46 , 47) and rapid degradation of HIF-1{alpha} in proteasomes (48 , 49) . It was recently shown that the interaction between pVHL and HIF-1{alpha} is regulated through hydroxylation of a proline residue that requires molecular O2 and Fe2+ (50 , 51) . Mice homozygous for a targeted deletion in the gene encoding HIF-1{alpha} are not viable and die around midgestation, mainly due to defective vascularization, heart malformations, and failure in neuronal tube closure (52 53 54) . Similar to HIF-1{alpha}, ARNT deficiency is also embryonic lethal (55 , 56) , indicating that HIF-1 is a nonredundant master regulator of oxygen homeostasis.

HIF-1{alpha} was shown to interact and form functional DNA binding complexes with BMAL1/MOP3 in vitro (30) , indicating that HIF-1{alpha} may interfere with the circadian clockwork. To test whether hypoxia affects circadian gene expression, we analyzed mPER1 mRNA and the mPER1 and CLOCK protein levels in mice subjected to hypoxia and assayed a putative PER1:HIF-1{alpha} protein–protein interaction. We found that PER1 protein is observed as several isoforms. A shorter nuclear isoform of PER1 was predominantly nuclear and exhibited daily rhythmicity in mouse brains.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
The Swiss3T3 fibroblasts, N2A neuroblastoma mouse, and human 293 kidney cells were kind gifts from Drs. U. Ziegler, K. Pajusola, and O. Georgiev (Zürich, Switzerland), respectively. The rat-1 cell line was kindly provided by U. Schibler (Geneva, Switzerland). All cells were cultured in DMEM medium (high glucose; Gibco-BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (Boehringer Mannheim, Mannheim, Germany), 100 U/ml penicillin, 100 µg/ml streptomycin, 1x MEM nonessential amino acids, 2 mM L-glutamine, and 1 mM Na-pyruvate (all Gibco-BRL) in a humidified atmosphere containing 5% CO2 at 37°C. G418 (400 µg/ml; Calbiochem, San Diego, CA) was supplemented to the culture medium for HeLa Tet-Off cells (HT42).

RNA blot analysis
RNA was isolated using TRIzol Reagent (Gibco-BRL) and analyzed by RNA blotting using DIG Northern Starter Kit (Roche, Nutley, NJ) according to the manufacturer’s protocol. The PER1 and CLOCK probes were a kind gift from U. Albrecht and the L28 probe was obtained as described previously (57 , 58) .

Protein extractions
Cellular nuclear extracts and cytoplasmic fractions were prepared as follows: 1 x 108 cells were washed twice, collected in ice-cold PBS, and pelleted. After incubation in cell lysis buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 150 mM NaCl, 0.2% Nonidet P-40) on ice for 5 min, the cells were centrifuged for 5 min at 3000 g to obtain cytoplasmic fractions. The pelleted nuclei were extracted with nuclear extraction buffer (400 mM NaCl, 20 mM HEPES pH 8, 1 mM EDTA) at 4°C for 15 min with gentle agitation.

Nuclear fraction from mouse tissues were prepared as follows: frozen tissue was rapidly triturated with pestle and mortar and homogenized with 10 strokes in 1 ml of buffer C1 (10 mM HEPES pH 8, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA) using Dounce manual type grinder. After incubation on ice for 15 min, homogenate was centrifuged for 5 min at 1000 g, supernatant was discarded, and the pellet was washed three times with buffer C1. After final centrifugation at 1000 g, the pellet was resuspended in buffer C2 (20 mM HEPES pH 8, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol). KCl (4 M) equal to 1/10th of the buffer C2 volume used for resuspension was added to the pellet. After 30 min incubation on ice, nuclear fraction was derived by centrifugation for 5 min at 12,000 g.

Whole brain homogenates were obtained as described (59) . The homogenate was centrifuged for 5 min at 1500 g to obtain cytoplasmic fraction and nuclear pellet. Nuclear fraction was obtained by incubating the pellet with the buffer C2 for 30 min with gentle agitation, followed by centrifugation for 5 min at 12,000 g. Immediately before use, all buffers were supplemented with 1 mM dithiothreitol, 1 mM PMSF, 1 mM Na3VO4, and a protease inhibitor mixture consisting of 2 µg/ml each of leupeptin, pepstatin, and aprotinin (all obtained from Sigma, St. Louis, MO). Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA) or the BCA assay (Pierce, Rockford, IL) using bovine serum albumin as a standard.

Western blot analysis
Protein extracts were electrophoresed through SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH) using standard procedures. Equal protein loading and transfer was verified by Ponceau staining (Sigma). The membrane was blocked with 4% (w/v) instant non-fat milk powder and incubated for 2 h with the indicated antibodies diluted in PBS containing 4% milk powder. An anti-HIF-1{alpha} polyclonal immunoglobulin Y (IgY) antibody was raised against a bacterially expressed GST-HIF-1{alpha} fusion protein containing amino acids 530 to 825 of human HIF-1{alpha}. Generation and purification of these antibodies are described in more detail elsewhere (60) . Anti-PER1 and anti-CLOCK antibodies were kindly provided by J. Stiehr and B. Rollmam (Affinity BioReagents, Neshanic Station, NJ). The respective horseradish peroxidase-coupled secondary antibodies were rabbit anti-chicken (Promega, Madison, WI) or goat anti-rabbit (Sigma). Super Signal Chemiluminescent Substrate (Pierce) was used for detection.

Immunoprecipitation
Nuclear extracts (200 µg) from cultured cells or mouse brain were incubated with the affinity-purified anti-HIF-1{alpha} IgY antibody (300 ng) for 2 h on ice, followed by incubation with a rabbit anti-chicken antibody (Promega) or a rabbit anti-PER1-N antibody. Protein A-Sepharose (Pharmacia, Piscataway, NJ) was added and incubated with rotation for 30 min at 4°C. After centrifugation of the precipitate at 15,000 g, the pellet was washed three times with buffer C2 (see above) and twice with 10 mM Tris-HCl pH 7.5. Finally, the precipitates were analyzed by immunoblotting using either the anti-HIF-1{alpha} IgY or anti-PER1-N.

Cell transfections
Cells were transfected with 20 µg of plasmid DNA using the calcium phosphate-mediated transfection method. Two days after transfection, the cells were harvested and analyzed for protein expression using Western blotting.

Plasmids and generation of the HIF-1{alpha} stably expressing HeLa Tet-Off cell line
Two oligonucleotides (5'-CATGACGCGTCATGAGAGGAATCG(CATCAC)3GC-3' and 5'-CATGGC(GTGATG)3CGATCCTCTCATGACGCGT-3') were annealed and cloned into NcoI opened plasmid pBSKhHIF-1{alpha}T7 encoding the human HIF-1{alpha} cDNA. The hHIF-1{alpha}-HisTag cDNA contained in Mlu/DraI fragment was cloned into pBI vector (Clontech, Palo Alto, CA), resulting in pBI-HisHIF. An Eco47III/SmaI fragment from pEGFP-c1 (Clontech) encoding green fluorescent protein was cloned into NotI opened pBI-HisHIF. Resulted construct was termed pBI-HisHIF-EGFP and used for transfection into a HeLa-derived cell line, termed HeLa Tet-Off (Clontech), which expresses the chimeric tTa (61) . Cells were electroporated (gene pulser; Bio-Rad) with 25 µg pBI-HisHIF-EGFP mixed with 1 µg pTK-Hyg encoding the hygromycin B resistance gene. One day after treatment, selection was initiated with hygromycin B (Calbiochem). Positive clones were selected with 200 µg/ml hygromycin B and 1 µg/ml doxycycline (Fluka; Buchs, Switzerland) according to Clontech’s protocol. Hygromycin B-resistant clones were screened for HIF-1{alpha} overexpression using SDS-PAGE and Western blot analysis. Among several clones, one expressing high levels of HIF-1{alpha} was termed HT42 and used here. mPER1 plasmid was a kind gift from Dr. U. Albrecht and has been described (20) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PER1 protein isoforms show distinct cellular compartmentalization and tissue-specific size differences
PER1 protein levels were analyzed by Western blotting of nuclear (n) and cytoplasmic (c) fractions obtained from human 293 kidney cells, rat-1 and mouse Swiss3T3 fibroblasts, and mouse N2A neuroblastoma cells transfected or nontransfected with an expression plasmid encoding full-length mouse PER1. Rabbit anti-PER1-N antibody was raised against a synthetic peptide corresponding to amino acids 39–51 of mouse PER1. As shown in Fig. 1 (upper left), this anti-PER1-N antibody recognized a 55 kDa band in nuclear fractions obtained from both transfected and untransfected 293 cells. Two bands of 140 and 75 kDa were present only in transfected 293 kidney cells. Rat-1 fibroblasts shown to express detectable levels of endogenous PER1 mRNA (17) revealed strong expression of the 140 kDa PER1 protein, predominantly in nuclear fraction (Fig. 1A , upper right, anti-PER1-N antibody). Detection of high and low molecular weight species was significantly reduced by preadsorption of the antiserum with the synthetic peptide used for immunization. In nuclear and cytoplasmic fractions obtained from transfected mouse Swiss3T3 and N2A cell lines, anti-PER1-N antibody recognized a 140 kDa band, showing very weak endogenous protein in untransfected N2A cells but not in 3T3 fibroblasts. A prominent 45 kDa band was detected only in nuclear but not in cytoplasmic fractions of transfected and parental Swiss3T3 and N2A cells. Overexpression of PER1 protein and antibody preadsorption experiments showed that anti-PER1-N antibody is capable of specifically detecting PER1 protein. To our surprise, PER1 protein varied in size depending on cell type and intracellular location. In addition to bands of the predicted molecular mass of ~140 kDa, additional PER1 species of 55 and 45 kDa were detected that were predominantly nuclear.



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  		Figure 1. Recognition of endogenous and transiently expressed PER1 and of endogenous CLOCK proteins in mouse and human cell lines and in mouse adult tissues. A) Human 293 kidney cells, mouse Swiss3T3 fibroblasts, and N2A neuroblastoma cell line were transfected with an expression plasmid encoding full-length mouse PER1 and harvested 2 days after transfection. Nuclear (n) and cytoplasmic (c) fractions were prepared from transfected or untransfected 293, Swiss3T3, N2A, and rat-1 cells and analyzed by Western blotting. Detection was performed using either rabbit anti-PER1-N (amino-terminal peptide) and anti-PER1-C (carboxyl-terminal peptide) antibodies or antibody depleted of specific anti-PER1-N antibody by preincubation with the peptide antigen (amino-terminal peptide). B) 6- to 7-wk-old NMRI mice were killed by cervical dislocation and organs were rapidly isolated and frozen in liquid nitrogen. Nuclear fractions were prepared from brain, liver, kidney, and spleen, followed by Western blot analysis. Detection was performed using either rabbit anti-PER1-N and rabbit anti-CLOCK antibodies or the anti-PER1-N and anti-CLOCK antibodies that were depleted of specific antibodies by preincubation with PER1 or CLOCK peptide antigens, respectively. One predominant band was detected in all organs analyzed by anti-CLOCK antibody. C) Nuclear and cytoplasmic fractions from either parental Swiss3T3 and N2A cells or cells transfected with an expression plasmid encoding full-length mouse PER1 cDNA were analyzed by Western blotting. Detection was performed with anti-PER1-C antibody (carboxyl-terminal peptide). D) Brains from NMRI mice were isolated and processed to obtain whole brain homogenates (lane 1) or crude cytoplasmic (lane 2) and nuclear (lane 3) fractions. The fractions were analyzed by Western blotting and detection with either anti-PER1 antibodies directed either against amino-terminal (anti-PER1-N) or carboxyl-terminal (anti-PER1-C) peptides or the antibody that was depleted of specific anti-PER1-N and anti-PER1-C antibodies by preincubation with the peptide antigen (amino- or carboxyl-terminal peptides). An arrow indicates the PER1 species.

Based on these in vitro data, we tested whether multiple PER1 protein isoforms could also be observed in different adult mouse tissues. Nuclear fractions were prepared from brain, liver, kidney, and spleen of 6- to 7-wk-old NMRI mice and analyzed by Western blotting. In the brain, the anti-PER1-N antibody recognized a double band of 48 kDa (Fig. 1B , left). In liver and kidney, the detected PER1 species have the size of ~45–48 and 55 kDa, respectively. A very weak band of 140 kDa was observed in the spleen. The anti-CLOCK antibody detected in nuclear fractions of all mouse tissues analyzed showed one prominent band of ~100 kDa corresponding to the predicted molecular mass of the CLOCK protein (Fig. 1B , right). Detection of these species was abolished by preadsorption of anti-PER1-N (Fig. 1B , middle) and anti-CLOCK antibodies (data not shown) with synthetic PER1 and CLOCK peptides.

We used an anti-PER1-C antibody raised against a peptide spanning the carboxyl-terminal amino acids 1156–1174 of mouse PER1 to examine in greater detail the expression of the PER1 protein isoforms. As shown in Fig. 1A (upper right, anti-PER1-C antibody), in rat-1 cells this antibody detected a protein of approximately 200 kDa, located both in nuclear and cytoplasmic fractions. On the other hand, anti-PER1-C antibody detected major bands of ~150 kDa present predominantly but not exclusively in cytoplasmic fractions of the PER1-overexpressing Swiss3T3 and N2A cells. As expected, a faint band was detected in untransfected cells revealing the presence of endogenous PER1 protein (Fig. 1C ). We next tested whether the longer form of PER1 protein is also cytoplasmic in mouse adult tissues. As shown in Fig. 1D, in whole brain homogenates (fraction 1), anti-PER1-N antibody detected a very faint band of ~110 kDa. A crude enrichment of cytoplasm by centrifugation of the homogenate allowed detection of a prominent cytoplasmic (fraction 2) 110 kDa form of PER1 protein. Extraction of the pellet with a buffer of high NaCl concentration (fraction 3) revealed the presence of the 48 kDa PER1 isoform and small amounts of the 110 kDa isoform, the latter being present due to the impurity of this extract. The anti-PER1-C antibody showed immunoreactivity in whole brain homogenate (fraction 1) to a double band of 140 kDa. This band was dramatically increased in crude cytoplasmic extract (fraction 2) but absent in nuclear fraction 3. Specificity of detected PER1 protein forms was confirmed by preadsorption of antibodies with synthetic peptides used for immunization (Fig. 3D ).



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  		Figure 3. Hypoxia regulates PER1 mRNA and PER1 and CLOCK protein levels in the brain of NMRI mice. Animals (n=3–4) were entrained to 12 h LD for 2 wk and at indicated ZT exposed to decreasing amounts of oxygen (from 21% to 6%) for 1 h, followed by cervical dislocation. Brains were isolated and processed to obtain either nuclear protein fractions or total RNA. A) Northern blot analysis of mouse brain RNA probed with in vitro synthesized DIG-labeled mouse PER1 antisense mRNA. Hybridization of the ribosomal protein L28 mRNA was used to normalize for equal loading and blotting efficiency. Relative amounts of PER1 mRNA in brains of normoxic and hypoxic mice are represented. B) Western blot analysis of nuclear fraction derived from brains of hypoxically exposed mice. Detection was carried out by anti-PER1-N, anti-CLOCK, anti-Sp-1, and anti-HIF-1{alpha} IgY antibodies, followed by densitometry. Amounts of PER1 and CLOCK were normalized to amounts of Sp-1 protein and expressed as relative units. Each value represents an average of at least 3 experiments.

Taken together, these results suggest that PER1 protein exists as several isoforms of 140, 110, and 48 kDa in the mouse brain. The polypeptides show distinct cellular compartmentalization with only a shorter, amino-terminal 45–48 kDa form present in nuclear fraction. This indicates that the carboxyl terminus of PER1 protein is important for cytoplasmic retention. The isoforms appear to have tissue-specific size differences: nuclear forms of PER1 in brain and liver show a double band of 45–48 kDa; in kidney and spleen, 55 and 140 kDa bands, respectively. In N2A, Swiss3T3, and rat-1 cells, the longer form has a size of ~150 kDa in former cells and 200 kDa in the later (see above). Only minute amounts of a shorter nuclear form were detected in rat-1 fibroblasts (data not shown), whereas in mouse Swiss3T3 fibroblasts and the N2A neuroblastoma cell line, a 45 kDa polypeptide was a major nuclear isoform of PER1 (Fig. 1A ).

Nuclear isoforms of PER1 protein show daily and circadian oscillation
The PER1 mRNA has been shown to undergo a robust circadian rhythmicity in the SCN with a peak at 3–4 CT (circadian time) and trough at 16–18 CT (reviewed in ref 4 ). Based on the postulated role of PER1 as a negative feedback regulator of its own expression (62 , 63) , the levels of PER1 protein should be in anti-phase to the PER1 mRNA amounts in vivo. To prove that the detected PER1 nuclear isoform is capable of daily oscillation, we quantified the amounts of the 48 kDa isoform of PER1 in the brain of mice entrained to 12 h LD (light-dark cycle). As shown in Fig. 2 A, the PER1 48 kDa polypeptide shows a daily rhythmicity in mouse brain with a peak at ~12 ZT (zeitgeber time: 12 h after light is on). The PER1 protein levels begin to decline early at night, rising slightly toward the morning. No daily rhythmicity of CLOCK protein was observed.



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  		Figure 2. PER1 protein shows daily and circadian oscillation in nuclear fractions obtained from total brain lysates of NMRI mice and serum-shocked rat-1 fibroblasts, respectively. A) 6- to 7-wk-old NMRI mice were entrained to 12 h LD for 2 wk and killed at the indicated zeitgeber times (ZT). Nuclear fractions from isolated brains were prepared and analyzed by Western blotting and detection by either anti-PER1-N or anti-CLOCK antibodies, followed by densitometry. Amounts of PER1 protein were normalized to the unspecific band (constitutive protein of 40 kDa) and plotted on a graph as a function of ZT. Each time point represents a mean value of 5 experiments. (*P<0.05, t test). The light/dark bar represents diurnal cycle. B) Rat-1 cells were grown to confluence in a medium containing 5% fetal calf serum. After being maintained for 6 days in the same medium, cells were shifted to a medium containing 50% adult horse serum (17) for 2 h, followed by replacement with serum-free medium. The cells were harvested at the indicated times after serum shock, and nuclear fractions were isolated and analyzed by Western blotting. Detection was performed with either anti-PER1-N or anti-CLOCK antibodies.

Recently, Balsalobre et al. showed that treatment of cultured rat-1 fibroblasts with high serum concentrations induced the circadian expression of several genes, including the PER1 mRNA, which was capable of oscillating for up to 3 days, reaching maximum levels at ~20, 44, and 68 h after serum stimulation (17) . Assuming that the rise in protein production would lag at least 1–2 h behind mRNA synthesis, one would predict the PER1 protein to peak 21–22 h after serum shock. Indeed, levels of nuclear PER1 protein began to decline rapidly after rat-1 cells were treated with 50% horse serum, becoming nearly undetectable after 10 h. At 22 h, nuclear PER1 protein amounts reached maximal levels, which declined again 28 h after serum shock. CLOCK protein remained constant throughout the experiment (Fig. 2B ). These results show that the 48 kDa nuclear isoform of PER1 in adult mouse brain and the 140 kDa nuclear form of PER1 in rat-1 fibroblasts exhibit daily and circadian rhythmicity, respectively.

Effect of hypoxia on the circadian clock in mouse adult brain
To explore the possibility that PER1 might be regulated by hypoxia, we examined the PER1 and CLOCK mRNA and protein levels in mice exposed to decreasing concentrations of oxygen (21%–6%) within 1 h at ZT0–1 and ZT12–13 and compared them to normoxic controls. Figure 3 A (upper part) shows a representative Northern blot of the PER1 mRNA isolated from brain of normoxic or hypoxic mice. Pixel density of the PER1 mRNA signal was normalized to that of a ribosomal protein L28 mRNA and represented as relative units in a graph at a bottom part of the Fig. 3A . The graph shows that reduced oxygenation during ZT0–1 results in a fivefold up-regulation of the PER1 mRNA. On the contrary, when a similar hypoxic insult was conducted between ZT12 and ZT13, no statistically significant changes in the PER1 mRNA levels were observed. The CLOCK mRNA was not significantly affected by hypoxia [relative abundance of the CLOCK mRNA in mouse normoxic vs. hypoxic brain was 2.1(1.2)/3.15(0.8) at ZT1 and 6.8(4)/10.5(3.12) at ZT13 (with SD in parentheses].

A representative Western blot analysis of nuclear fractions obtained from the same mouse brains as above is shown in Fig. 3B (upper part). The graphs indicate that the PER1 protein is increased by twofold after a hypoxic insult at ZT0–1. No significant differences were found between normoxic and hypoxic mice after treatment at ZT12–13 (Fig. 3B , bottom part). The CLOCK protein appeared to be induced three- to sixfold by hypoxia in mice treated at both ZT0–1 and ZT12–13. As expected, HIF-1{alpha} protein accumulated in hypoxic nuclear fractions.

Formation of HIF-1{alpha} and PER1 heterodimers
The PER1 mRNAs and protein increase in hypoxia could be due to hypoxia-dependent activation of the PER1 gene transcription and to post-translational stabilization of the protein product, respectively. In the former case, HIF-1 might mediate up-regulation of the PER1 mRNA through several putative HIF binding sites present in the PER1 promoter (unpublished observations). On the other hand, An et al. recently reported that wild-type p53 is stabilized through protein–protein interaction with HIF-1{alpha} (64) . To test whether PER1 protein is interacting with HIF-1{alpha}, we performed a series of the HIF-1{alpha}:PER1 coimmunoprecipitation experiments using either N42 cells, transiently transfected with an expression plasmid encoding the PER1 cDNA, rat-1 cells, or protein extracts from mouse brain. The HT42 cell line is a doxycycline-repressible, HeLa Tet-Off-derived subline, which is stably expressing high levels of the HIF-1{alpha} protein in normoxia in the absence of doxycycline (64a) . Western blot analysis of HT42 cell line (Fig. 4 A) shows highly increased levels of predominantly nuclear 43 kDa and cytoplasmic 200 kDa bands in fractions obtained from the PER1 transfected cells. The detection of these bands was abolished by preadsorption of anti-PER1-N antibody with the corresponding antigen peptide. The anti HIF-1{alpha} antibody immunoprecipitated minute amounts of HIF-1{alpha} protein from the nuclear fraction obtained from repressed N42 cells grown in the presence of doxycycline. However, in induced cells, the HIF-1{alpha} protein is drastically up-regulated. The anti-PER1-N antibody coimmunoprecipitated the HIF-1{alpha} protein only in the PER1 transfected cells (Fig. 4B , left). A weak coimmunoprecipitation of HIF-1{alpha} protein by anti-PER1-N antibody and strong immunoprecipitation of HIF-1{alpha} protein by anti-HIF-1{alpha} IgY antibody were also observed in nuclear fractions isolated from hypoxic rat-1 cells (Fig. 4B , middle).



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  		Figure 4. Coimmunoprecipitation of HIF-1{alpha}:PER1 complexes. A) A doxycycline-repressible, HeLa Tet-Off-derived cell line HT42 stably expressing HIF-1{alpha} in normoxia in the absence of doxycycline was transfected with an expression plasmid encoding full-length mouse PER1 cDNA and cells were harvested 2 days after transfection. Nuclear (n) and cytoplasmic (c) fractions were prepared from either transfected or untransfected cells and analyzed by Western blotting. Detection was performed with anti-PER1-N antibody or anti-PER1-N antibody preincubated with PER1 peptide antigen. B) NMRI mice were exposed to decreasing amounts of oxygen (from 21% to 6%) for 1 h, followed by cervical dislocation. Nuclear fractions were prepared from normoxic and hypoxic animals. For immunoprecipitation, nuclear fractions obtained from either HT42 and rat-1 cells or mouse brain were incubated with either the anti-HIF-1{alpha} IgY antibody, followed by a rabbit anti-IgY antibody or anti-PER1-N antibody, and the immune complexes were precipitated by addition of protein A-Sepharose. The precipitates were analyzed by Western blotting using anti-HIF-1{alpha} antibody (WB, Western blotting).

We tested next whether HIF-1{alpha}:PER1-complexes could be detected in vivo in mice. The nuclear fractions obtained from brains of normoxic or hypoxic mice were analyzed by immunoprecipitation using either anti-PER1-N or anti-HIF-1{alpha} IgY antibodies, followed by Western blotting with anti-HIF-1{alpha} IgY antibody. HIF-1{alpha} was detected by Western blot analysis in normoxic mouse brain, as expected (65 , 66) , with up-regulation of the protein in hypoxia (Fig. 4B , right, WB). Correspondingly, HIF-1{alpha} is immunoprecipitated by the anti-HIF-1{alpha} antibody from normoxic nuclear fraction (Fig. 4B , right). Minute amounts of normoxic HIF-1{alpha} are precipitated by anti-PER1 antibody. In hypoxia, a strong increase of HIF-1{alpha} coimmunoprecipitating with PER1 is observed. No CLOCK could be detected in either HIF-1{alpha} or PER1 immunoprecipitates (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we visualized the PER1 protein in nuclear and cytoplasmic fractions obtained from mouse tissues and cell lines. Application of two new anti-PER1 antibodies, raised against synthetic peptides spanning either amino-terminal or carboxyl-terminal amino acids of mouse PER1 enabled us to detect several PER1 protein isoforms, which showed distinct cellular compartmentalization. The full size PER1 protein of ~150 kDa resides predominantly in the cytoplasm. In addition, we observed intermediate cytoplasmic polypeptides and a nuclear double band of 45–48 kDa. The shorter isoform exhibits daily oscillation in mouse brain, indicating that it might function as a component of the circadian clock. Indeed, daily and circadian oscillation of nuclear specific PER1 immunoreactivity in the SCN of mouse brain have been reported (59) , consistent with nuclear localization of PER1 recombinant protein when transfected into cell lines (32 , 35) . PER1 has a cytoplasmic localization domain (CLD) spanning amino acids 330–389 of mouse PER1, but no consensus nuclear localization signal (67) . Thus, cleavage of CLD would release the protein from cytoplasmic retention and facilitate its nuclear translocation, probably in cooperation with a nuclear transporter.

The origin of the PER1 protein isoforms is not clear but could be due to alternative mRNA splicing or post-translational processing. The latter plays an important role in regulation of Drosophila circadian clock (reviewed in ref 68 ) and other processes such as nuclear entry of Notch protein in Drosophila embryos, regulated by ligand-induced cleavage (69) , activation of mammalian transcription factor ATF6 by proteolysis (70) , sterol-regulated proteolysis and nuclear translocation of sterol regulatory element binding protein 1 (71) , and regulation of receptor specificity and activity of vascular endothelial growth factor C by proteolytic processing (72) . The function of PER1 in the circadian system is still elusive but presumably the protein has a role in a transcription cycle, and thus must be nuclearly localized. Our data indicate that PER1 might not only be regulated at the level of protein abundance, but also via modulating its nuclear entry. The molecular weight variations between the PER1 protein isoforms might reflect a differential role of PER1 in a circadian clock of peripheral tissues. Whether the longer cytoplasmic form of PER1 is also rhythmic is a matter of further investigation.

The circadian clock can be modulated by a variety of environmental stimuli including light, temperature (73) , or behavior (74 , 75) in vivo and serum (17) , forskolin (76 , 77) , or TPA (78) in vitro. We demonstrated that hypoxia modulates the PER1 mRNA and the PER1 and CLOCK protein levels in mouse brain. As the sleep-wake cycle is under control of the circadian clock, the circadian master genes are expected to influence sleeping behavior (79) . Physiological studies indicated that hypoxia affects the sleep-waking pattern of rats (80 , 81) and humans (82) and that the structure of sleep of high-altitude dwellers experiencing hypoxic conditions throughout life is different from that of lowland inhabitants (83) . Our observations indicate that oxygen supply modulates the circadian clock at the molecular level, probably via HIF-1. It will be important to monitor locomotor activity of mice exposed to hypoxia and kept in DD (constant darkness) conditions. We will delineate the effect of oxygen on the circadian system using as an in vitro model the rat-1 cells that harbor a functional circadian mechanism (17) .

Our results provide evidence for the existence of a cross-talk between members of the PAS family of proteins participating in hypoxic and circadian pathways. The interaction could occur by HIF-1 dependent transcriptional regulation of circadian gene expression or HIF-1{alpha} protein–protein interactions altering the stability of circadian proteins by protecting them from proteolytical degradation. On the other hand, there could be a PAS protein involved in both hypoxia and circadian signaling, for example, the newly discovered brain-specific basic helix-loop-helix/PAS protein MOP9 (84) . MOP9 displays sequence similarity with MOP3/BMAL1 and CYCLE, has elevated levels of expression in mouse SCN, and was shown to form transcriptionally active heterodimers with CLOCK and HIF-1{alpha} proteins (30 , 84) . The targets of putative HIF-1{alpha}:MOP9 heterodimer are so far unknown. Based on this study, transcriptional regulation of PER1 might be such a candidate target. This hypothesis is now under investigation.


   ACKNOWLEDGMENTS
 
We are grateful to U. Schibler, U. Ziegler, K. Pajusola, O. Georgiev, and U. Albrecht for the generous gift of cell lines and plasmids, J. Stiehr and B. Rollman from the Affinity BioReagents for kindly providing the antibodies, J. C. Dunlap for valuable discussions, C. Gasser for the artwork, P. Spielmann and F. Parpan for excellent technical assistance, and I. Desbaillets for critically reading the manuscript. This work has been supported by grants from the Swiss National Science Foundation (31–56743.99), the Roche Research Foundation, the Käthe Zingg-Schwichtenberg Fonds, the Hartmann Müller-Stiftung (810), and the EMDO-Stiftung, all to M.G.


   FOOTNOTES
 
1 Present address: Institute of Physiology, Medical University Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany.

Received for publication February 27, 2001. Revision received July 20, 2001.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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