BACKGROUND
the circadian rhythm of about  <dig> hours is a fundamental physiological function observed in almost all organisms from prokaryotes to humans.
circadian rhythms have been known to be generated in pacemaker cells, the suprachiasmatic nuclei  of hypothalamus in mammals, and entrained by environmental cues, such as light, temperature, noise, feeding or social cues, whereas a recent analysis using mper2luciferase knockin mice has demonstrated that peripheral tissues express self-sustained circadian oscillations  <cit> .
the output of circadian oscillation appears as locomotive activity, hormonal secretion, the sleep-wake cycle, and many other physiological functions.
disruption of the circadian rhythms has been associated with various kinds of diseases, such as cardiovascular diseases, psychiatric diseases and cancer in humans  <cit> .
identification of clock genes has allowed study of the molecular bases for circadian behaviors and temporal physiological processes and has prompted the idea that molecular clocks reside not only in a central pacemaker, but also in peripheral tissues, even in immortalized cells  <cit> .
furthermore, previous molecular dissection revealed that the mechanism of circadian oscillation at a molecular level is based on transcriptional regulation of clock and clock-controlled genes, which consists of interwoven positive and negative feedback loops  <cit> .
there is a distinct connection between genes and behaviors in circadian rhythms, which is conserved from fly or other lower organisms to humans  <cit> .
the drosophila period mutants, originally identified as a circadian mutant brought us the first clock gene, period  <cit> , while a point mutation of hper <dig> was recently shown to cause a familial advanced sleep phase syndrome  <cit> .
as described above, circadian rhythms rely on a negative feedback loop in gene expression that involves a limited number of clock genes.
recent molecular dissection has increased our understanding of the molecular nature of the transcriptional regulation of some clock genes.
the circadian phenotypes at the cellular level may be represented as temporal mrna expression.
global gene expression profiling using microarrays has led to the discovery of many circadian-regulated genes, but there is only a minor overlap of cycling transcripts between tissues  <cit> .
thus, circadian rhythms are an appropriate study target for systems biology.
in this study, we systematically examined the mrna expression of common circadian-regulated genes in several mouse peripheral tissues and made oscillatory profiles of canonical clock genes.
moreover, by bioinformatics, we identified  <dig> clock elements for circadian transcription .
these  <dig> elements and their combination would suffice to explain the biological timing of expression of these clock and clock-controlled genes.
RESULTS
to examine the circadian expression of mouse clock and clock-related genes in peripheral tissues, we performed the quantitative real-time reverse transcription-polymerase chain reaction  method on mrnas from  <dig> different mouse peripheral tissues .
after entrainment of housed mice for  <dig> weeks under a light-dark  cycle, samples were collected every  <dig> hr starting at circadian time   <dig>  in the third dark-dark  cycle.
out of  <dig> mouse genes examined,  <dig> genes  showed robust circadian of mrna expression in all tissues except the testis.
these genes should thus be considered to be core molecules of the circadian clock.
circadian mrna expression patterns were similar in each tissue with the exception of testis, where weak or no rhythm was observed.
this common pattern of the rhythm throughout many peripheral tissues implies that there may exist a universal mechanism for resetting the peripheral clock.
the peak transcript level of each circadian rhythm was as follows: mbmal <dig> and mnpas <dig>  in subjective night at ct20-ct0; mrev-erbα, in subjective day at ct4-8; mdbp and mrev-erbβ, at ct8; mper <dig>  at ct8-12; mper <dig>  at ct12; and mper <dig>  at ct12- <dig> .
in the peripheral tissues mrna peaks occurred approximately  <dig> hr later than those in the central pacemaker, scn  <cit> .
in the testis,  <dig> genes  showed weak rhythms of their mrna expression, while no other genes, including the mper family, showed any clear oscillation.
surprisingly, the expression of mper <dig> transcripts in the testis, which did not show a circadian rhythm, was substantially higher than other tissues, and exceeded rna expression of other genes studied in the testis.
this is consistent with data recently reported  <cit> ; although a previous report indicated circadian rhythm of mper <dig> in the testis  <cit> .
these findings suggest that mper <dig> may play an alternative role in the testis, including developmental regulation during spermatogenesis.
the rhythm of mcry <dig> mrna expression was obviously circadian  except in the testis, but the peak-trough amplitude was relatively smaller than that of the above genes.
mcry <dig> and mclock rna levels seemed to be rhythmic except in the testis, but the rhythm was rather weak and not clearly circadian.
no circadian rhythms were observed in the remaining  <dig> genes examined, i.e.
mcki-δ, mcki-ε, and mtim.
despite a central role in drosophila for timeless , the mammalian homologue  was originally found to show weak or no rhythm in the scn  <cit> ; and its functional role in mammalian clocks remains controversial  <cit> .
as described above, the expression of  <dig> genes fluctuated in an overt circadian fashion; and so we aligned them in the order of the peak of their oscillatory phase .
among them, the transcriptional oscillation of  <dig> representative clock genes, bmal <dig> and per <dig>  was examined by using the real-time luciferase reporter assay.
nih3t <dig> cells were transfected with the hbmal1-luc or mper2-luc construct and then stimulated with a high concentration of serum.
after the serum shock, in the presence of luciferin, light emission was measured and integrated for  <dig> min at intervals of  <dig> min.
both promoters fused to luciferase showed circadian rhythms .
the phase of bmal <dig> oscillation in cultured cells was almost the opposite of that of per <dig>  which is consistent with data of mrna expression in mouse peripheral tissues obtained by real-time rt-pcr .
this result indicates that the promoter regions used in the real-time luciferase reporter assay are sufficient for producing circadian transcriptional oscillation.
the promoter analyses of several clock genes have been reported, as described below.
a molecular mechanism of canonical clock genes is based on transcriptional regulation via interlocked feedback and/or feed-forward loops  <cit> .
one example of the regulation of a known characterized gene in mammals is that of per <dig>  the transcription of per <dig> is activated by binding of the clock/bmal <dig> hetero-complex, both members of which are bhlh-pas  proteins, to the e-boxes in the promoter region of per <dig>  <cit> .
the translated per <dig> is posttranslationally modified by cki-ε  <cit>  and, together with other clock proteins such as crys  <cit> , is returned to the nucleus to suppress its own transactivation, resulting in closure of the per <dig> loop.
e-box elements are also known to be essential for transcriptional regulation of many clock-controlled output genes including the vasopression genes  <cit> .
on the other hand, one of the positive elements, bmal <dig>  whose mrna expression is cycled antiphase to pers, as described above, forms another loop  <cit> .
the orphan nuclear receptors rorα and rev-erbα regulate circadian transcription positively and negatively, respectively, through ror/rev-erb elements  in the promoter region of bmal <dig>  <cit> .
moreover, an in silico search identified these  <dig> rores in the promoter region of npas <dig> , the expression pattern of which was very similar to that of bmal <dig>  these findings gave us the idea that the circadian pattern of rna expression might be dependent on transcriptional regulation by specific transcription factors.
using the ncbi database and celera database system, we systematically searched for the above  <dig> elements and another clock element, a dbp-binding element , described below.
these elements are conserved among human, mouse, and rat genome sequences in the regions 9-kb upstream and 5-kb downstream of the transcription start site .
intriguingly, the transcriptional elements corresponded to the cyclic pattern of the clock genes shown in figure  <dig>  bmal <dig> and npas <dig>  the circadian peaks of which were both in subjective night at ct20- <dig>  included rores in their promoters, as described above.
in the promoter of per <dig> and per <dig>  the peaks of which were in subjective day at ct12- <dig>  e-boxes were found.
in fact, the conserved e-box in per <dig> is not a typical e-box of the molecular clock, cacgtg, but an atypical element, cacgtt.
compared with per <dig>  which contains  <dig> conserved e-boxes, per <dig> may have other unknown factors and elements responsible for its robust transcriptional oscillation.
per <dig>  whose peak was a bit earlier than that of per <dig>  has another element, a dbpe, in its promoter region, in addition to the  <dig> e-boxes well studied in vitro  <cit> .
per <dig>  the peak of which was even earlier at ct8- <dig>  did not have conserved e-boxes but instead contained dbpes in its gene.
three genes, rev-erbα, dbp, and rev-erbβ, the peaks of which were between those of bmal <dig> and per, had a mixed combination of the elements.
the rev-erbα genome sequence included  <dig> rore,  <dig> dbpe, and  <dig> e-boxes, whereas rev-erbβ included all  <dig> elements only in the mouse genome sequence.
dbp contained  <dig> rores and  <dig> e-boxes in each genome.
among the elements described above, some of them in  were experimentally studied and confirmed  <cit> .
transcripts of  <dig> genes  showed a robust circadian rhythm in different peripheral tissues .
the amount of mrna in the trough was nearly zero and the peak-trough amplitude of these genes was clearly higher than that of the others examined.
thus, in terms of mrna expression among the canonical clock genes examined, these  <dig> genes likely constitute the core molecules of a molecular circadian clock.
the expression timing in a 24-h period appears to be conveyed through  <dig> kinds of sequence elements bound by specific transcription factors .
recent genome-wide analyses using microarrays revealed that many genes  oscillated but only several tens of common genes overlapped between two tissues examined  <cit> .
among the core candidate genes with similar circadian regulation in those  <dig> tissues, we examined the circadian transcription of  <dig> candidate genes besides the known clock genes, but could not find genes with oscillatory behavior in different peripheral tissues comparable to that in the  <dig> genes described above.
the  <dig> genes studied here may approximate the entirety of the core oscillatory genes in the genome.
if so, the  <dig> elements described here may be sufficient for explaining the biological timing of mrna expression of clock genes.
however, our preliminary results showed that an atypical e-box in per <dig> promoter may be insufficient for full transcriptional oscillation .
further detailed studies of each promoter, combined with systematic analyses using microarrays and real-time rt-pcr, will give us a more detailed comprehension of the intertwined positive and negative regulatory loops of molecular biological clocks.
CONCLUSIONS
the current study has clarified the detailed circadian expression of mrnas for clock and clock-related genes in different peripheral tissues of the mouse.
the observation of oscillatory profiles of canonical clock genes is not only useful for physiological and pathological examination of the circadian clock in various organs but also important for systematic understanding of transcriptional regulation on a genome-wide basis.
our finding of the oscillatory expression of canonical clock genes in a temporal order provides us an interesting hypothesis, that cyclic timing of all clock and clock-controlled genes may be dependent on several transcriptional elements including  <dig> known elements, e-box, rore, and dbpe.

 the circadian rhythm of about hours is a fundamental physiological function observed in almost all organisms from prokaryotes to humans. circadian rhythms have been known to be generated in pacemaker cells, the suprachiasmatic nuclei of hypothalamus in mammals, and entrained by environmental cues, such as light, temperature, noise, feeding or social cues, whereas a recent analysis using mper2luciferase knockin mice has demonstrated that peripheral tissues express self-sustained circadian oscillations. the output of circadian oscillation appears as locomotive activity, hormonal secretion, the sleep-wake cycle, and many other physiological functions. disruption of the circadian rhythms has been associated with various kinds of diseases, such as cardiovascular diseases, psychiatric diseases and cancer in humans. identification of clock genes has allowed study of the molecular bases for circadian behaviors and temporal physiological processes and has prompted the idea that molecular clocks reside not only in a central pacemaker, but also in peripheral tissues, even in immortalized cells. furthermore, previous molecular dissection revealed that the mechanism of circadian oscillation at a molecular level is based on transcriptional regulation of clock and clock-controlled genes, which consists of interwoven positive and negative feedback loops. there is a distinct connection between genes and behaviors in circadian rhythms, which is conserved from fly or other lower organisms to humans. the drosophila period mutants, originally identified as a circadian mutant brought us the first clock gene, period, while a point mutation of hper was recently shown to cause a familial advanced sleep phase syndrome. as described above, circadian rhythms rely on a negative feedback loop in gene expression that involves a limited number of clock genes. recent molecular dissection has increased our understanding of the molecular nature of the transcriptional regulation of some clock genes. the circadian phenotypes at the cellular level may be represented as temporal mrna expression. global gene expression profiling using microarrays has led to the discovery of many circadian-regulated genes, but there is only a minor overlap of cycling transcripts between tissues. thus, circadian rhythms are an appropriate study target for systems biology. in this study, we systematically examined the mrna expression of common circadian-regulated genes in several mouse peripheral tissues and made oscillatory profiles of canonical clock genes. moreover, by bioinformatics, we identified clock elements for circadian transcription. these elements and their combination would suffice to explain the biological timing of expression of these clock and clock-controlled genes.  to examine the circadian expression of mouse clock and clock-related genes in peripheral tissues, we performed the quantitative real-time reverse transcription-polymerase chain reaction method on mrnas from different mouse peripheral tissues. after entrainment of housed mice for weeks under a light-dark cycle, samples were collected every hr starting at circadian time in the third dark-dark cycle. out of mouse genes examined, genes showed robust circadian of mrna expression in all tissues except the testis. these genes should thus be considered to be core molecules of the circadian clock. circadian mrna expression patterns were similar in each tissue with the exception of testis, where weak or no rhythm was observed. this common pattern of the rhythm throughout many peripheral tissues implies that there may exist a universal mechanism for resetting the peripheral clock. the peak transcript level of each circadian rhythm was as follows: mbmal and mnpas in subjective night at ct20-ct0; mrev-erbα, in subjective day at ct4-8; mdbp and mrev-erbβ, at ct8; mper at ct8-12; mper at ct12; and mper at ct12-. in the peripheral tissues mrna peaks occurred approximately hr later than those in the central pacemaker, scn. in the testis, genes showed weak rhythms of their mrna expression, while no other genes, including the mper family, showed any clear oscillation. surprisingly, the expression of mper transcripts in the testis, which did not show a circadian rhythm, was substantially higher than other tissues, and exceeded rna expression of other genes studied in the testis. this is consistent with data recently reported; although a previous report indicated circadian rhythm of mper in the testis. these findings suggest that mper may play an alternative role in the testis, including developmental regulation during spermatogenesis. the rhythm of mcry mrna expression was obviously circadian except in the testis, but the peak-trough amplitude was relatively smaller than that of the above genes. mcry and mclock rna levels seemed to be rhythmic except in the testis, but the rhythm was rather weak and not clearly circadian. no circadian rhythms were observed in the remaining genes examined, i.e. mcki-δ, mcki-ε, and mtim. despite a central role in drosophila for timeless, the mammalian homologue was originally found to show weak or no rhythm in the scn; and its functional role in mammalian clocks remains controversial. as described above, the expression of genes fluctuated in an overt circadian fashion; and so we aligned them in the order of the peak of their oscillatory phase. among them, the transcriptional oscillation of representative clock genes, bmal and per was examined by using the real-time luciferase reporter assay. nih3t cells were transfected with the hbmal1-luc or mper2-luc construct and then stimulated with a high concentration of serum. after the serum shock, in the presence of luciferin, light emission was measured and integrated for min at intervals of min. both promoters fused to luciferase showed circadian rhythms. the phase of bmal oscillation in cultured cells was almost the opposite of that of per which is consistent with data of mrna expression in mouse peripheral tissues obtained by real-time rt-pcr. this result indicates that the promoter regions used in the real-time luciferase reporter assay are sufficient for producing circadian transcriptional oscillation. the promoter analyses of several clock genes have been reported, as described below. a molecular mechanism of canonical clock genes is based on transcriptional regulation via interlocked feedback and/or feed-forward loops. one example of the regulation of a known characterized gene in mammals is that of per the transcription of per is activated by binding of the clock/bmal hetero-complex, both members of which are bhlh-pas proteins, to the e-boxes in the promoter region of per. the translated per is posttranslationally modified by cki-ε and, together with other clock proteins such as crys, is returned to the nucleus to suppress its own transactivation, resulting in closure of the per loop. e-box elements are also known to be essential for transcriptional regulation of many clock-controlled output genes including the vasopression genes. on the other hand, one of the positive elements, bmal whose mrna expression is cycled antiphase to pers, as described above, forms another loop. the orphan nuclear receptors rorα and rev-erbα regulate circadian transcription positively and negatively, respectively, through ror/rev-erb elements in the promoter region of bmal. moreover, an in silico search identified these rores in the promoter region of npas, the expression pattern of which was very similar to that of bmal these findings gave us the idea that the circadian pattern of rna expression might be dependent on transcriptional regulation by specific transcription factors. using the ncbi database and celera database system, we systematically searched for the above elements and another clock element, a dbp-binding element, described below. these elements are conserved among human, mouse, and rat genome sequences in the regions 9-kb upstream and 5-kb downstream of the transcription start site. intriguingly, the transcriptional elements corresponded to the cyclic pattern of the clock genes shown in figure bmal and npas the circadian peaks of which were both in subjective night at ct20- included rores in their promoters, as described above. in the promoter of per and per the peaks of which were in subjective day at ct12- e-boxes were found. in fact, the conserved e-box in per is not a typical e-box of the molecular clock, cacgtg, but an atypical element, cacgtt. compared with per which contains conserved e-boxes, per may have other unknown factors and elements responsible for its robust transcriptional oscillation. per whose peak was a bit earlier than that of per has another element, a dbpe, in its promoter region, in addition to the e-boxes well studied in vitro. per the peak of which was even earlier at ct8- did not have conserved e-boxes but instead contained dbpes in its gene. three genes, rev-erbα, dbp, and rev-erbβ, the peaks of which were between those of bmal and per, had a mixed combination of the elements. the rev-erbα genome sequence included rore, dbpe, and e-boxes, whereas rev-erbβ included all elements only in the mouse genome sequence. dbp contained rores and e-boxes in each genome. among the elements described above, some of them in were experimentally studied and confirmed. transcripts of genes showed a robust circadian rhythm in different peripheral tissues. the amount of mrna in the trough was nearly zero and the peak-trough amplitude of these genes was clearly higher than that of the others examined. thus, in terms of mrna expression among the canonical clock genes examined, these genes likely constitute the core molecules of a molecular circadian clock. the expression timing in a 24-h period appears to be conveyed through kinds of sequence elements bound by specific transcription factors. recent genome-wide analyses using microarrays revealed that many genes oscillated but only several tens of common genes overlapped between two tissues examined. among the core candidate genes with similar circadian regulation in those tissues, we examined the circadian transcription of candidate genes besides the known clock genes, but could not find genes with oscillatory behavior in different peripheral tissues comparable to that in the genes described above. the genes studied here may approximate the entirety of the core oscillatory genes in the genome. if so, the elements described here may be sufficient for explaining the biological timing of mrna expression of clock genes. however, our preliminary results showed that an atypical e-box in per promoter may be insufficient for full transcriptional oscillation. further detailed studies of each promoter, combined with systematic analyses using microarrays and real-time rt-pcr, will give us a more detailed comprehension of the intertwined positive and negative regulatory loops of molecular biological clocks.  the current study has clarified the detailed circadian expression of mrnas for clock and clock-related genes in different peripheral tissues of the mouse. the observation of oscillatory profiles of canonical clock genes is not only useful for physiological and pathological examination of the circadian clock in various organs but also important for systematic understanding of transcriptional regulation on a genome-wide basis. our finding of the oscillatory expression of canonical clock genes in a temporal order provides us an interesting hypothesis, that cyclic timing of all clock and clock-controlled genes may be dependent on several transcriptional elements including known elements, e-box, rore, and dbpe. 
the observation of oscillatory profiles of canonical clock genes is not only useful for physiological and pathological examination of the circadian clock in various organs but also important for systematic understanding of transcriptional regulation on a genome-wide basis. identification of clock genes has allowed us to study the molecular bases for circadian behaviors and temporal physiological processes such as hormonal secretion, and has prompted the idea that molecular clocks reside not only in a central pacemaker, the suprachiasmatic nuclei of hypothalamus in mammals, but also in peripheral tissues, even in immortalized cells. our finding of the oscillatory expression of canonical clock genes with a temporal order provides us an interesting hypothesis, that cyclic timing of all clock and clock-controlled genes may be dependent on several transcriptional elements including known elements, e-box, rore, and dbpe. the bioinformatics analysis revealed that these genes have one or a combination of transcriptional elements, which are conserved among human, mouse, and rat genome sequences, and indicated that these elements may be responsible for the biological timing of expression of canonical clock genes. we systematically analyzed the mrna expression of clock and clock-controlled genes in mouse peripheral tissues. furthermore, previous molecular dissection revealed that the mechanism of circadian oscillation at a molecular level is based on transcriptional regulation of clock and clock-controlled genes. the circadian rhythm of about hours is a fundamental physiological function observed in almost all organisms from prokaryotes to humans. eight genes showed robust circadian expressions of mrnas in all tissues except testis, suggesting that these genes are core molecules of the molecular biological clock.
 the circadian rhythm of about hours is a fundamental physiological function observed in almost all organisms from prokaryotes to humans. identification of clock genes has allowed us to study the molecular bases for circadian behaviors and temporal physiological processes such as hormonal secretion, and has prompted the idea that molecular clocks reside not only in a central pacemaker, the suprachiasmatic nuclei of hypothalamus in mammals, but also in peripheral tissues, even in immortalized cells. furthermore, previous molecular dissection revealed that the mechanism of circadian oscillation at a molecular level is based on transcriptional regulation of clock and clock-controlled genes.  we systematically analyzed the mrna expression of clock and clock-controlled genes in mouse peripheral tissues. eight genes showed robust circadian expressions of mrnas in all tissues except testis, suggesting that these genes are core molecules of the molecular biological clock. the bioinformatics analysis revealed that these genes have one or a combination of transcriptional elements, which are conserved among human, mouse, and rat genome sequences, and indicated that these elements may be responsible for the biological timing of expression of canonical clock genes.  the observation of oscillatory profiles of canonical clock genes is not only useful for physiological and pathological examination of the circadian clock in various organs but also important for systematic understanding of transcriptional regulation on a genome-wide basis. our finding of the oscillatory expression of canonical clock genes with a temporal order provides us an interesting hypothesis, that cyclic timing of all clock and clock-controlled genes may be dependent on several transcriptional elements including known elements, e-box, rore, and dbpe. 