In mammals, the circadian and stress systems (both centers of which are located in the hypothalamus) are involved in adaptation to predictable and unpredictable environmental stimuli, respectively. Although the interaction and relationship between these two systems are intriguing and have been studied in different ways since the "pre-clock gene" era, the molecular interaction between them remains largely unknown. Here, we show by systematic molecular biological analysis that acute physical stress elevated only Period1 (Per1) mRNA expression in mouse peripheral organs. Although behavioral rhythms in vivo and peripheral molecular clocks are rather stable against acute restraint stress, the results of a series of promoter analyses, including chromatin immunoprecipitation assays, indicate that a glucocorticoid-responsive element in the Per1 promoter is indispensable for induction of this mRNA both in vitro and in vivo. These results suggest that Per1 can be a potential stress marker and that a third pathway of Per1 transcriptional control may exist in addition to the clock-regulated CLOCK-BMAL1/ E-box and light-responsive cAMP-responsive element-binding protein/cAMP-responsive element pathways.

Although circadian transcription of Period2 (Per2) is fundamental for the generation of circadian rhythm, the molecular mechanism remains unclear. Here we report that cell-autonomous circadian transcription of Per2 is driven by two transcriptional elements, one for rhythm generation and the other for phase control. The former contains the E-box-like sequence (CACGTT) that is sufficient and indispensable to drive oscillation, and indeed circadian transcription factors site-specifically bind to it. Furthermore, the nature of this atypical E-box is different from that of the classical circadian E-box. The current feedback loop model is based mainly on Period1. Our results provide not only compelling evidence in support of this model but also an explanation for a general basic mechanism to produce various patterns in the phase and amplitude of cell-autonomous circadian gene expression. INTRODUCTIONIn nearly all organisms, behavioral and physiological processes display ϳ24 h rhythms that are controlled by circadian pacemakers (Pittendrigh, 1993). The circadian organization of physiology and behavior in mammals is governed by the suprachiasmatic nuclei (SCN), a defined pair of cell clusters in the anteroventral hypothalamus (Ralph et al., 1990). Circadian clocks can count time only approximately and must be adjusted every day by the photoperiod in order to be in harmony with the outside world (Menaker, 2003). Circadian oscillators also exist in most peripheral cells and even in cultured cells (Balsalobre et al., 1998;Yamazaki et al., 2000). It is thought that the phase of these peripheral timekeepers is reset by signals regulated by the SCN pacemaker (Akashi and Nishida, 2000;Schibler and Sassone-Corsi, 2002).The molecular makeup of circadian clocks has been the subject of intense genetic and biochemical investigation in various organisms, including cyanobacteria, Neurospora, higher plants, Drosophila, and mammals (Dunlap, 1999;Kondo and Ishiura, 2000;Allada et al., 2001;Williams and Sehgal, 2001;Young and Kay, 2001;Reppert and Weaver, 2002). Over the last several years, orthologues of most Drosophila circadian clock genes have been cloned from mammals (Albrecht and Eichele, 2003;Lowrey and Takahashi, 2004). Although mPer2 was literally cloned as a secondary mammalian period gene (Albrecht et al., 1997;Takumi et al., 1998), gene-knockout analysis revealed that an mPer2 mutant displays a loss of circadian rhythmicity, revealing a prominent role for mPER2 in the mammalian clock (Zheng et al., 1999). Additionally familial advanced sleep phase syndrome has been attributed to a missense mutation in hPer2 (Toh et al., 2001). These studies demonstrate that a robust circadian fluctuation in Per2 transcription is an essential event for the generation of circadian rhythm.Circadian oscillators appear to have been highly conserved throughout evolution and to involve transcriptiontranslation negative feedback loops for the regulation of clock genes (Dunlap, 1999;Young and Kay, 2001). In mammals, in vitro studies have shown that th...

Background: The circadian rhythm of about 24 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 (SCN) 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.

Circadian rhythms, which period is approximately one day, are generated by endogenous biological clocks. These clocks are found throughout the animal kingdom, as well as in plants and even in prokaryotes. Molecular mechanisms for circadian rhythms are based on transcriptional oscillation of clock component genes, consisting of interwoven autoregulatory feedback loops. Among the loops, the nuclear transport of clock proteins is a crucial step for transcriptional regulation. In the present study, we showed that the nuclear entry of mCRY2, a mammalian clock component, is mediated by the importin ␣/␤ system through a bipartite nuclear localization signal in its carboxyl end. In vitro transport assay using digitonin-permeabilized cells demonstrated that all three importin ␣s, ␣1 (Rch1), ␣3 (Qip-1), and ␣7 (NPI-2), can mediate mCRY2 import. mCRY2 with the mutant nuclear localization signal failed to transport mPER2 into the nucleus of mammalian cultured cells, indicating that the nuclear localization signal identified in mCRY2 is physiologically significant. These results suggest that the importin ␣/␤ system is involved in nuclear entry of mammalian clock components, which is indispensable to transcriptional oscillation of clock genes.