Genome-wide Expression Analysis of Mouse Liver Reveals CLOCK-regulated Circadian Output Genes
2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês
10.1074/jbc.m304564200
ISSN1083-351X
AutoresKatsutaka Oishi, Koyomi Miyazaki, Koji Kadota, Reiko Kikuno, Takahiro Nagase, Gen-ichi Atsumi, Naoki Ohkura, Takashi Azama, Miho Mesaki, Shima Yukimasa, Hisato Kobayashi, Chisato Iitaka, Takashi Umehara, Masami Horikoshi, Takashi Kudo, Yoshihisa Shimizu, Masahiko Yano, Morito Monden, Kazuhiko Machida, Juzo Matsuda, Shuichi Horie, Takeshi Todo, Norio Ishida,
Tópico(s)Light effects on plants
ResumoCLOCK is a positive component of a transcription/translation-based negative feedback loop of the central circadian oscillator in the suprachiasmatic nucleus in mammals. To examine CLOCK-regulated circadian transcription in peripheral tissues, we performed microarray analyses using liver RNA isolated from Clock mutant mice. We also compared expression profiles with those of Cryptochromes (Cry1 and Cry2) double knockout mice. We identified more than 100 genes that fluctuated from day to night and of which expression levels were decreased in Clock mutant mice. In Cry-deficient mice, the expression levels of most CLOCK-regulated genes were elevated to the upper range of normal oscillation. Most of the screened genes had a CLOCK/BMAL1 binding site (E box) in the 5′-flanking region. We found that CLOCK was absolutely concerned with the circadian transcription of one type of liver genes (such as DBP, TEF, and Usp2) and partially with another (such as mPer1, mPer2, mDec1, Nocturnin, P450 oxidoreductase, and FKBP51) because the latter were damped but remained rhythmic in the mutant mice. Our results showed that CLOCK and CRY proteins are involved in the transcriptional regulation of many circadian output genes in the mouse liver. In addition to being a core component of the negative feedback loop that drives the circadian oscillator, CLOCK also appears to be involved in various physiological functions such as cell cycle, lipid metabolism, immune functions, and proteolysis in peripheral tissues. CLOCK is a positive component of a transcription/translation-based negative feedback loop of the central circadian oscillator in the suprachiasmatic nucleus in mammals. To examine CLOCK-regulated circadian transcription in peripheral tissues, we performed microarray analyses using liver RNA isolated from Clock mutant mice. We also compared expression profiles with those of Cryptochromes (Cry1 and Cry2) double knockout mice. We identified more than 100 genes that fluctuated from day to night and of which expression levels were decreased in Clock mutant mice. In Cry-deficient mice, the expression levels of most CLOCK-regulated genes were elevated to the upper range of normal oscillation. Most of the screened genes had a CLOCK/BMAL1 binding site (E box) in the 5′-flanking region. We found that CLOCK was absolutely concerned with the circadian transcription of one type of liver genes (such as DBP, TEF, and Usp2) and partially with another (such as mPer1, mPer2, mDec1, Nocturnin, P450 oxidoreductase, and FKBP51) because the latter were damped but remained rhythmic in the mutant mice. Our results showed that CLOCK and CRY proteins are involved in the transcriptional regulation of many circadian output genes in the mouse liver. In addition to being a core component of the negative feedback loop that drives the circadian oscillator, CLOCK also appears to be involved in various physiological functions such as cell cycle, lipid metabolism, immune functions, and proteolysis in peripheral tissues. Many organisms display rhythms of physiology and behavior which are entrained to the 24-h cycle of light and darkness which prevails on Earth. In mammals, the suprachiasmatic nucleus (SCN) 1The abbreviations used are: SCN, suprachiasmatic nucleus; bHLH, basic helix-loop-helix; CT, circadian time(s); DBP, D site-binding protein; GADD, growth arrest and DNA damage-inducible; GC, glucocorticoid; HNF, hepatocyte nuclear factor; Id, inhibitors of differentiation/DNA-binding proteins; TEF, thyrotroph embryonic factor; WT, wild-type.1The abbreviations used are: SCN, suprachiasmatic nucleus; bHLH, basic helix-loop-helix; CT, circadian time(s); DBP, D site-binding protein; GADD, growth arrest and DNA damage-inducible; GC, glucocorticoid; HNF, hepatocyte nuclear factor; Id, inhibitors of differentiation/DNA-binding proteins; TEF, thyrotroph embryonic factor; WT, wild-type. is considered the master circadian pacemaker that controls most of the physical circadian rhythms including behavior (1Pando, M. P., and Sassone-Corsi, P. (2001) Sci. STKE http://www.stke.org/egi/content/full/OC_sigtrans;2001/107/re16.Google Scholar, 2Reppert S.M. Weaver D.R. Annu. Rev. Physiol. 2001; 63: 647-676Crossref PubMed Scopus (1203) Google Scholar). Studies of clock genes have recently implied that oscillatory mechanisms function in peripheral organs and isolated cells and that they appear to be entrained to the SCN (1Pando, M. P., and Sassone-Corsi, P. (2001) Sci. STKE http://www.stke.org/egi/content/full/OC_sigtrans;2001/107/re16.Google Scholar, 2Reppert S.M. Weaver D.R. Annu. Rev. Physiol. 2001; 63: 647-676Crossref PubMed Scopus (1203) Google Scholar). Although the peripheral oscillators seem to play an important role in regulating various physiological functions, little is known about the circadian oscillatory mechanism in peripheral tissues. Recent studies at the molecular level in bacteria, plants, and animals have provided a general model of the circadian pacemaker that is based on a self-sustained transcriptional/translational negative feedback loop (1Pando, M. P., and Sassone-Corsi, P. (2001) Sci. STKE http://www.stke.org/egi/content/full/OC_sigtrans;2001/107/re16.Google Scholar, 2Reppert S.M. Weaver D.R. Annu. Rev. Physiol. 2001; 63: 647-676Crossref PubMed Scopus (1203) Google Scholar). Clock was the first clock gene identified in vertebrates by forward mutagenesis using N-ethyl-N-nitrosourea in a behavioral screening (3Vitaterna M.H. King D.P. Chang A.M. Kornhauser J.M. Lowrey P.L. McDonald J.D. Dove W.F. Pinto L.H. Turek F.W. Takahashi J.S. Science. 1994; 264: 719-725Crossref PubMed Scopus (1318) Google Scholar). When transferred from a light-dark cycle to constant darkness, the periodicity of behavior exhibited by homozygous Clock mutants is abnormally long for the initial 5–15 cycles, and circadian rhythmicity is consequently lost (3Vitaterna M.H. King D.P. Chang A.M. Kornhauser J.M. Lowrey P.L. McDonald J.D. Dove W.F. Pinto L.H. Turek F.W. Takahashi J.S. Science. 1994; 264: 719-725Crossref PubMed Scopus (1318) Google Scholar). The Clock gene encodes a basic helix-loop-helix (bHLH)-PAS transcription factor (1Pando, M. P., and Sassone-Corsi, P. (2001) Sci. STKE http://www.stke.org/egi/content/full/OC_sigtrans;2001/107/re16.Google Scholar, 2Reppert S.M. Weaver D.R. Annu. Rev. Physiol. 2001; 63: 647-676Crossref PubMed Scopus (1203) Google Scholar). Like other bHLH transcription factors, CLOCK binds DNA and modulates transcription after dimerization with BMAL1 (a bHLH-PAS transcription factor) (1Pando, M. P., and Sassone-Corsi, P. (2001) Sci. STKE http://www.stke.org/egi/content/full/OC_sigtrans;2001/107/re16.Google Scholar, 2Reppert S.M. Weaver D.R. Annu. Rev. Physiol. 2001; 63: 647-676Crossref PubMed Scopus (1203) Google Scholar). As the Clock allele is truncated and causes a deletion of 51 amino acids, the mutation presumably would not have a significant effect on the N-terminal bHLH and PAS domains, leaving CLOCK dimerization and DNA binding intact (1Pando, M. P., and Sassone-Corsi, P. (2001) Sci. STKE http://www.stke.org/egi/content/full/OC_sigtrans;2001/107/re16.Google Scholar, 2Reppert S.M. Weaver D.R. Annu. Rev. Physiol. 2001; 63: 647-676Crossref PubMed Scopus (1203) Google Scholar). Actually, the mutant CLOCK protein can still form heterodimers with BMAL1 which bind to DNA, but the heterodimers are deficient in transactivation (1Pando, M. P., and Sassone-Corsi, P. (2001) Sci. STKE http://www.stke.org/egi/content/full/OC_sigtrans;2001/107/re16.Google Scholar, 2Reppert S.M. Weaver D.R. Annu. Rev. Physiol. 2001; 63: 647-676Crossref PubMed Scopus (1203) Google Scholar). The CLOCK/BMAL1 heterodimer drives the rhythmic transcription of period (mPer1, mPer2, and mPer3) and cryptochrome (mCry1 and mCry2) genes through the E box (CACGTG) in Per and Cry promoters (1Pando, M. P., and Sassone-Corsi, P. (2001) Sci. STKE http://www.stke.org/egi/content/full/OC_sigtrans;2001/107/re16.Google Scholar, 2Reppert S.M. Weaver D.R. Annu. Rev. Physiol. 2001; 63: 647-676Crossref PubMed Scopus (1203) Google Scholar). As the PER and CRY proteins are translated, they form multimeric complexes that are translocated to the nucleus. The CRY proteins are essential for the negative feedback loop that regulates the central clock (4Vitaterna M.H. Selby C.P. Todo T. Niwa H. Thompson C. Fruechte E.M. Hitomi K. Thresher R.J. Ishikawa T. Miyazaki J. Takahashi J.S. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12114-12119Crossref PubMed Scopus (560) Google Scholar, 5Kume K. Zylka M.J. Sriram S. Shearman L.P. Weaver D.R. Jin X. Maywood E.S. Hastings M.H. Reppert S.M. Cell. 1999; 98: 193-205Abstract Full Text Full Text PDF PubMed Scopus (1296) Google Scholar). The primary function of the CRY proteins in mammals is to inhibit CLOCK/BMAL1-mediated transactivation (1Pando, M. P., and Sassone-Corsi, P. (2001) Sci. STKE http://www.stke.org/egi/content/full/OC_sigtrans;2001/107/re16.Google Scholar, 2Reppert S.M. Weaver D.R. Annu. Rev. Physiol. 2001; 63: 647-676Crossref PubMed Scopus (1203) Google Scholar). However, how the CRY proteins negatively regulate CLOCK/BMAL1-mediated transactivation remains obscure. A recent study has shown that H3 histone acetylation is a potential target of CRY inhibitory action, as CRY proteins inhibit the increase in CLOCK/BMAL1-mediated transactivation induced by histone acetyltransferase p300 (6Etchegaray J.P. Lee C. Wade P.A. Reppert S.M. Nature. 2003; 421: 177-182Crossref PubMed Scopus (538) Google Scholar). Microarray analysis is a powerful tool with which to evaluate the expression of many genes in various experimental systems, and it can identify target genes for transcription factors. Hundreds of tissue-specific circadian clock-controlled genes, which regulate an impressive diversity of biological processes, have been identified by DNA microarray technology (7Panda S. Antoch M.P. Miller B.H. Su A.I. Schook A.B. Straume M. Schultz P.G. Kay S.A. Takahashi J.S. Hogenesch J.B. Cell. 2002; 109: 307-320Abstract Full Text Full Text PDF PubMed Scopus (1857) Google Scholar, 8Ueda H.R. Chen W. Adachi A. Wakamatsu H. Hayashi S. Takasugi T. Nagano M. Nakahama K. Suzuki Y. Sugano S. Iino M. Shigeyoshi Y. Hashimoto S. Nature. 2002; 418: 534-539Crossref PubMed Scopus (702) Google Scholar, 9Akhtar R.A. Reddy A.B. Maywood E.S. Clayton J.D. King V.M. Smith A.G. Gant T.W. Hastings M.H. Kyriacou C.P. Curr. Biol. 2002; 12: 540-550Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 10Chen L. Madura K. Mol. Cell. Biol. 2002; 22: 4902-4913Crossref PubMed Scopus (251) Google Scholar, 11Wang, Y., Osterbur, D. L., Megaw, P. L., Tosini, G., Fukuhara, C., Green, C. B., and Besharse, J. C. (2001) BMC Dev. Biol. 1, 9 www.biomedcentral.com/1471-213X/1/9.Google Scholar, 12Storch K.F. Lipan O. Leykin I. Viswanathan N. Davis F.C. Wong W.H. Weitz C.J. Nature. 2002; 417: 78-83Crossref PubMed Scopus (1233) Google Scholar, 13Delaunay F. Laudet V. Trends Genet. 2002; 18: 595-597Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). CLOCK protein is involved in the transcriptional regulation of several circadian output genes as well as in the core circadian clock (7Panda S. Antoch M.P. Miller B.H. Su A.I. Schook A.B. Straume M. Schultz P.G. Kay S.A. Takahashi J.S. Hogenesch J.B. Cell. 2002; 109: 307-320Abstract Full Text Full Text PDF PubMed Scopus (1857) Google Scholar). We speculated that transcripts that are rhythmic in a circadian manner, down-regulated in homozygous Clock mutant mice, and elevated at high or intermediate levels in Cry-deficient mice would potentially be direct targets of CLOCK protein. Thus, to examine the CLOCK-regulated genes that express in a circadian manner in mice, we performed oligonucleotide microarray analysis at circadian times (CT) 14 and CT2, when CLOCK/BMAL1 transcriptional activity is maximal and minimal, respectively (14Oishi K. Miyazaki K. Ishida N. Biochem. Biophys. Res. Commun. 2002; 298: 198-202Crossref PubMed Scopus (84) Google Scholar), using RNA isolated from the livers of wild-type (WT), Clock mutant, and Cry-deficient mice. We also searched for an E box element in the 10-kb region 5′-upstream from these genes and their respective human homologs. Our results revealed that several circadian expressing output genes are probably regulated by CLOCK and CRY proteins via the E box element(s) in the mouse liver. Mice—Clock mutant mice were derived from animals supplied by J. S. Takahashi (Northwestern University, Evanston IL). The animals had the Clock allele originally on a BALB/c and C57BL/6J background. A breeding colony was established by further backcrossing with Jcl:ICR mice (14Oishi K. Miyazaki K. Ishida N. Biochem. Biophys. Res. Commun. 2002; 298: 198-202Crossref PubMed Scopus (84) Google Scholar). Cry1 and Cry2 double knockout mice were generated as described previously (4Vitaterna M.H. Selby C.P. Todo T. Niwa H. Thompson C. Fruechte E.M. Hitomi K. Thresher R.J. Ishikawa T. Miyazaki J. Takahashi J.S. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12114-12119Crossref PubMed Scopus (560) Google Scholar, 5Kume K. Zylka M.J. Sriram S. Shearman L.P. Weaver D.R. Jin X. Maywood E.S. Hastings M.H. Reppert S.M. Cell. 1999; 98: 193-205Abstract Full Text Full Text PDF PubMed Scopus (1296) Google Scholar). C57BL/6 background mice were the WT control for the GeneChip analysis. Mice with WT Jcl:ICR and C57BL/6 backgrounds were Northern blot controls for Clock mutant and Cry-deficient mice, respectively. All male mice of 8–10 weeks of age were maintained under a 12:12-h light-dark cycle for at least 2 weeks before the day of the experiment. After being placed in constant darkness (DD), the animals were sacrificed at CT2 and CT14 on the 2nd day of DD. The livers were dissected, quickly frozen, and stored in liquid nitrogen. Samples and GeneChip Hybridization—Total RNA was purified from a pool of two to three animal tissues collected at each time point using ISOGEN (Nippon Gene Co., Ltd., Japan). Poly(A)+ RNA was purified from the total RNA using an Oligotex™-dT30 mRNA Purification kit (Takara). Double stranded cDNA containing the T7 RNA polymerase promoter at the 5′-end was synthesized from 1 μg of poly(A)+ RNA using a SuperScript double stranded cDNA synthesis kit (Invitrogen). Biotinylated amplified RNA was then synthesized using a BioArray™ HighYield™ RNA transcript labeling kit (Enzo Life Sciences) and purified using an RNeasy Mini kit (Qiagen). The labeled RNA was partially hydrolyzed and hybridized in duplicate (or more) to Affymetrix GeneChip (MG-U74Av2) arrays. The arrays were hybridized, washed, and analyzed using standard Affymetrix reagents and protocols. Northern Blotting—Northern blotting proceeded as described previously (14Oishi K. Miyazaki K. Ishida N. Biochem. Biophys. Res. Commun. 2002; 298: 198-202Crossref PubMed Scopus (84) Google Scholar). Random primed 32P-labeled probes were generated from cDNA fragments of mPer1 (bases 2358–3114; GenBank AB002108), mPer2 (bases 1123–1830; GenBank AF036893), FKBP51 (bases 191–1069; GenBank U16959), NADPH-P450 oxidoreductase (bases 60–1013; GenBank D17571), mDec1 (bases 722–1333; GenBank Y07836), Nocturnin (bases 901–1730; GenBank AF199491), TEF (bases 141–1020; GenBank BC036982), Usp2 (bases 89–384; GenBank AB041799), and DBP (bases 1138–1602; GenBank J03179). Samples were normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA (data not shown). Quantitative Reverse Transcription-PCR and in Situ Hybridization— Complementary DNA generated using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) was amplified by PCR using a LightCycler™ (Roche Applied Science). The primers were as follows: Usp2 forward, 5′-TGTATGCTGTGTCCAATCA-3′ and reverse 5′-TAGAAGAGCAAATAGGCGT-3′. To quantify and confirm the integrity of each RNA sample, glyceraldehyde-3-phosphate dehydrogenase was the internal standard. A standard curve was constructed with serial dilutions of cDNA obtained from hypothalamic RNA. Data were analyzed using LightCycler™ analysis software and are expressed as ratios to the highest value of the eight time points of the day. In situ hybridization proceeded as described previously (14Oishi K. Miyazaki K. Ishida N. Biochem. Biophys. Res. Commun. 2002; 298: 198-202Crossref PubMed Scopus (84) Google Scholar). In brief, mice were anesthetized with pentobarbital and perfused from the left ventricle with 4% paraformaldehyde in phosphate-buffered saline (pH 7.4). The animals were dissected under a dim red light in the dark. The tissues were fixed for 2.5 h at 4 °C, embedded in Tissue-Tek OCT compound (Miles), and cut into 8-μm cryosections. Digoxigenin-labeled RNA probes were generated from Usp2 (bases, 89–384; GenBank AB041799) using a DIG RNA labeling Kit (Roche Applied Science). Transcripts were hybridized and detected as described (14Oishi K. Miyazaki K. Ishida N. Biochem. Biophys. Res. Commun. 2002; 298: 198-202Crossref PubMed Scopus (84) Google Scholar). Screening of CLOCK-regulated Rhythmic Genes—Average difference values for each gene were calculated using Affymetrix microarray analysis software, and average difference values of 1 or lower were set to 1, to avoid division by 0 or a negative number. Other studies have demonstrated that CLOCK-regulated gene expression levels in Clock (Clk/Clk) mutant mice are generally lower than those in the WT (14Oishi K. Miyazaki K. Ishida N. Biochem. Biophys. Res. Commun. 2002; 298: 198-202Crossref PubMed Scopus (84) Google Scholar) and that an expression ratio of 2-fold is the approximate limit of sensitivity (15Su A.I. Cooke M.P. Ching K.A. Hakak Y. Walker J.R. Wiltshire T. Orth A.P. Vega R.G. Sapinoso L.M. Moqrich A. Patapoutian A. Hampton G.M. Schultz P.G. Hogenesch J.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4465-4470Crossref PubMed Scopus (1254) Google Scholar). Therefore, we applied three criteria to the selection of circadianrelated genes: WT14 (the value of WT at CT14)/WT02 > 2, (WT02 + WT14)/2 > Clk02, and (WT02 + WT14)/2 > Clk14. Under these criteria, not only the genes that show the damped expression in Clock mutants, but also the genes that show the changing in phase of circadian expression will be identified. Actually, known components of the CLOCK-regulated genes such as mPer1, mPer2, mDec1, and albumin D site-binding protein (DBP) were identified, suggesting that this screening strategy was appropriate for an examination of directly or indirectly CLOCK-regulated circadian gene expression in the mouse liver. Computational Analysis of E Box Motif—The protein-coding sequences of each cycling gene were used for BLAST as queries for corresponding genome contigs of mouse draft genome sequences (ftp.ensembl.org/pub/mouse-7.3/data/golden_path/). The consensus E box element (CACGTG) was searched against the 10-kb genome sequences upstream from the first methionine for the respective cycling genes (when the positions of coding regions were unavailable, we used the 10-kb genome sequences upstream from the mRNA start site instead). We identified 1216 genes that fluctuated day/night in the liver of WT mice. In these genes, 108 known genes (Fig. 1) and 27 unknown genes (Fig. 2) showed reduced expression levels in Clock mutant mice. Among them, known components of the circadian clock such as mPer1, mPer2, mDec1, and DBP were identified, suggesting that our current strategy was appropriate to examine CLOCK-regulated circadian gene expression in the mouse liver. Furthermore, BLAST search results of the screened expressed sequence tag clones revealed that our experiments were accurate because identical genes such as Nocturnin, long chain fatty acyl elongase (Lce), P450 oxidoreductase, a DnaJ homolog (mDj7), RAN GTPase-activating protein 1, and adenylate kinase 4 (Ak4) were screened using different probe sets (Fig. 1). The present study included known components of CLOCK-regulated circadian expressing genes such as mPer1, mPer2, and DBP (14Oishi K. Miyazaki K. Ishida N. Biochem. Biophys. Res. Commun. 2002; 298: 198-202Crossref PubMed Scopus (84) Google Scholar) in the screened genes (Fig. 1). Thus, our screening strategy could screen CLOCK-regulated circadian expressing genes in the liver. However, the timing of many systems such as locomotion (16Sei H. Oishi K. Morita Y. Ishida N. Neuroreport. 2001; 12: 1461-1464Crossref PubMed Scopus (43) Google Scholar), neuroendocrine (17Sei H. Sano A. Oishi K. Fujihara H. Kobayashi H. Ishida N. Morita Y. Neuroscience. 2003; 117: 785-789Crossref PubMed Scopus (13) Google Scholar), and feeding/drinking (14Oishi K. Miyazaki K. Ishida N. Biochem. Biophys. Res. Commun. 2002; 298: 198-202Crossref PubMed Scopus (84) Google Scholar) would be expected to change in the Clock mutant mice. Therefore, some of the observed changes in gene expression may be caused by alteration in these systems.Fig. 2Pseudo-color image of expressed sequence tag gene expression by WT, Clock mutant (Clk/Clk), and double knockout (Cry1 and Cry2) mice (WKO) under constant darkness (see Fig. 1 ).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We found that the expression of mDec1 mRNA is circadian in the liver (Fig. 3) as well as in the SCN (18Honma S. Kawamoto T. Takagi Y. Fujimoto K. Sato F. Noshiro M. Kato Y. Honma K. Nature. 2002; 419: 841-844Crossref PubMed Scopus (513) Google Scholar). DEC1 has shown to repress CLOCK/BMAL1-induced transactivation by direct protein-protein interaction and/or competition for E box elements (18Honma S. Kawamoto T. Takagi Y. Fujimoto K. Sato F. Noshiro M. Kato Y. Honma K. Nature. 2002; 419: 841-844Crossref PubMed Scopus (513) Google Scholar). In turn, the circadian expression of mDec1 mRNA was considered to be positively regulated by CLOCK/BMAL1 heterodimers (18Honma S. Kawamoto T. Takagi Y. Fujimoto K. Sato F. Noshiro M. Kato Y. Honma K. Nature. 2002; 419: 841-844Crossref PubMed Scopus (513) Google Scholar). However, mDec1 mRNA in Clock mutant mice was expressed in a circadian manner, although the expression phase was changed (Fig. 3). Interestingly, the peak expression levels of mDec1 mRNA were not changed by the Clock mutation. It seems that CLOCK-independent oscillating mechanisms exist in peripheral tissues (see below) (14Oishi K. Miyazaki K. Ishida N. Biochem. Biophys. Res. Commun. 2002; 298: 198-202Crossref PubMed Scopus (84) Google Scholar). Our present results indicated that the circadian expression of bHLH transcription factors such as mDec1, Arnt2, Hes6, and Hes3 is regulated by CLOCK, which is also a bHLH transcription factor (Figs. 1 and 3). We showed previously that the circadian expression of BMAL1 mRNA is blunted at the upper range of normal oscillation in the peripheral tissues of Clock mutant mice (14Oishi K. Miyazaki K. Ishida N. Biochem. Biophys. Res. Commun. 2002; 298: 198-202Crossref PubMed Scopus (84) Google Scholar). Thus, these bHLH transcription factors appear to be expressed in a circadian manner, and the regulation of their expression may be interdependent in the mouse liver. The inhibitors of differentiation/DNA-binding proteins (Id) act as dominant negative regulators of bHLH proteins and are important for the control of differentiation and cell cycle progression in organisms ranging from flies to humans (19Nakamura W. Honma S. Shirakawa T. Honma K. Nat. Neurosci. 2002; 5: 399-400Crossref PubMed Scopus (115) Google Scholar). We found that Id-1 mRNA levels were damped in the liver of Clock mutant mice (Fig. 1). Because Id lacks a basic DNA binding domain, heterodimers between Id and bHLH proteins cannot bind DNA. Primary targets for Id proteins are the bHLH transcription factors that regulate cell type-specific gene expression and the expression of cell cycle regulatory genes. Id-1 exhibits circadian rhythms of mRNA and protein expression in the pineal gland, whereas the related genes, Id-2 and Id-3, do not (20Humphries A. Klein D. Baler R. Carter D.A. J. Neuroendocrinol. 2002; 14: 101-108Crossref PubMed Scopus (49) Google Scholar). Dominant negative regulators of bHLH proteins might be involved in clock or clock-related circadian transcriptional regulation via the E box in various tissues including the pineal gland (20Humphries A. Klein D. Baler R. Carter D.A. J. Neuroendocrinol. 2002; 14: 101-108Crossref PubMed Scopus (49) Google Scholar) and liver. The present study shows the circadian expression of growth arrest and DNA damage-inducible (GADD) 45α, GADD45β, and GADD153 mRNAs in the mouse liver. Moreover, the circadian expression of these GADDs seems to be regulated by CLOCK (Fig. 1). In response to DNA-damaging stress, including UV radiation, γ-radiation, and exposure to the alkylating agent methyl methanesulfonate, mammalian cells can prevent cell cycle progression by controlling critical cell cycle regulators. The main role of GADDs is to block proliferation at G1 and G2 checkpoints in response to DNA damage. The GADD genes are up-regulated in response to a variety of stressors (21Fornace Jr., A.J. Jackman J. Hollander M.C. Hoffman-Liebermann B. Liebermann D.A. Ann. N. Y. Acad. Sci. 1992; 663: 139-153Crossref PubMed Scopus (174) Google Scholar). The G2/M transition is a crucial point for progression through the cell cycle and is controlled by the Cdc2 kinase-cyclin B complex. We also showed that the circadian mRNA expression of Wee1 (this kinase inhibits mitotic cell division by inactivating the M phase-promoting factor) was extremely reduced in the liver of Clock mutant mice, and the mRNA levels were continuously high in Cry-deficient mice (Fig. 1). A recent study has shown that mPER2 protein is involved in tumor suppression by regulating DNA damage-responsive pathways (22Fu L. Pelicano H. Liu J. Huang P. Lee C. Cell. 2002; 111: 41-50Abstract Full Text Full Text PDF PubMed Scopus (1016) Google Scholar). Growth control and the DNA damage response may be affected by Clock mutation because the circadian expression of mPer2 mRNA is regulated by the CLOCK protein (14Oishi K. Miyazaki K. Ishida N. Biochem. Biophys. Res. Commun. 2002; 298: 198-202Crossref PubMed Scopus (84) Google Scholar). In fact, some aged-Clock mutant mice developed salivary gland hyperplasia like mPer2 mutant mice (data not shown). Considering the current data and reported findings (22Fu L. Pelicano H. Liu J. Huang P. Lee C. Cell. 2002; 111: 41-50Abstract Full Text Full Text PDF PubMed Scopus (1016) Google Scholar), mammalian cell cycle-regulating mechanisms appear to be regulated by the circadian clock at the molecular level. The circadian expression of molecular chaperones such as Hsp105 and mDj7 was depressed in Clock mutant mice (Fig. 1). Many heat-shock genes are expressed in a circadian manner both in the SCN and in the liver (7Panda S. Antoch M.P. Miller B.H. Su A.I. Schook A.B. Straume M. Schultz P.G. Kay S.A. Takahashi J.S. Hogenesch J.B. Cell. 2002; 109: 307-320Abstract Full Text Full Text PDF PubMed Scopus (1857) Google Scholar, 8Ueda H.R. Chen W. Adachi A. Wakamatsu H. Hayashi S. Takasugi T. Nagano M. Nakahama K. Suzuki Y. Sugano S. Iino M. Shigeyoshi Y. Hashimoto S. Nature. 2002; 418: 534-539Crossref PubMed Scopus (702) Google Scholar, 9Akhtar R.A. Reddy A.B. Maywood E.S. Clayton J.D. King V.M. Smith A.G. Gant T.W. Hastings M.H. Kyriacou C.P. Curr. Biol. 2002; 12: 540-550Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 12Storch K.F. Lipan O. Leykin I. Viswanathan N. Davis F.C. Wong W.H. Weitz C.J. Nature. 2002; 417: 78-83Crossref PubMed Scopus (1233) Google Scholar). The decreased expression of these chaperones in Clock mutant mice suggests an altered response to external stress. Ubiquitylation and proteasome-mediated protein degradation are involved in the core mechanism of the circadian oscillator (23Yagita K. Tamanini F. Yasuda M. Hoeijmakers J.H. van der Horst G.T. Okamura H. EMBO J. 2002; 21: 1301-1314Crossref PubMed Scopus (225) Google Scholar). 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