Ulrich Schibler

Transcriptionally active nuclear extracts have been prepared from rat liver, brain, and spleen. The adenovirus-2 major late promoter directs efficient transcription by RNA polymerase II in all of these extracts, whereas the promoter of the mouse albumin gene is significantly used only in the liver extract. Albumin sequences located between -170 and -55 are required for this liver-specific in vitro transcription, since deletion of this region results in almost a 100-fold reduction in transcription. In addition, insertion of these sequences in either orientation upstream of the parotid-specific Amy-1 promoter, which is poorly transcribed in the liver extract, increases the activity of this promoter to a level comparable to that observed for the albumin promoter.

LAP, a transcriptional activator, and LIP, a transcriptional repressor, are translated from a single mRNA species by using two AUGs within the same reading frame. These two proteins share the 145 C-terminal amino acids that contain the basic DNA-binding domain and the leucine zipper dimerization helix. Probably owing to its higher affinity for its DNA cognate sequences, LIP can attenuate the transcriptional stimulation by LAP in substoichiometric amounts. As revealed by transient transfection experiments, a moderate increase in the ratio results in a significantly higher transcriptional activation of an appropriate target gene. The ratio increases about 5-fold during terminal rat liver differentiation and is thus likely to modulate the activity of LAP in the intact animal.

The mammalian circadian timing system consists of a master pacemaker in neurons of the suprachiasmatic nucleus (SCN) and clocks of a similar molecular makeup in most peripheral body cells. Peripheral oscillators are self-sustained and cell autonomous, but they have to be synchronized by the SCN to ensure phase coherence within the organism. In principle, the rhythmic expression of genes in peripheral organs could thus be driven not only by local oscillators, but also by circadian systemic signals. To discriminate between these mechanisms, we engineered a mouse strain with a conditionally active liver clock, in which REV-ERBα represses the transcription of the essential core clock gene Bmal1 in a doxycycline-dependent manner. We examined circadian liver gene expression genome-wide in mice in which hepatocyte oscillators were either running or arrested, and found that the rhythmic transcription of most genes depended on functional hepatocyte clocks. However, we discovered 31 genes, including the core clock gene mPer2, whose expression oscillated robustly irrespective of whether the liver clock was running or not. By contrast, in liver explants cultured in vitro, circadian cycles of mPer2::luciferase bioluminescence could only be observed when hepatocyte oscillators were operational. Hence, the circadian cycles observed in the liver of intact animals without functional hepatocyte oscillators were likely generated by systemic signals. The finding that rhythmic mPer2 expression can be driven by both systemic cues and local oscillators suggests a plausible mechanism for the phase entrainment of subsidiary clocks in peripheral organs.

The promoter of the mouse albumin gene contains at least six binding sites for specific DNA-binding proteins (A to F). Four of these sites (A, D, E, and F) can be occupied by transcription factors that are considerably enriched in liver nuclei, as compared to spleen or brain nuclei. These factors consist of a heat-stable protein that fills sites A, D, and F, and a member of a family of nuclear factor I (NF-I) related proteins that occupies site E. Site C binds a protein that is equally abundant in liver, brain, and spleen nuclei. Occupancy of this site and the binding of the heat-stable factor to the immediately adjacent site D appear to be mutually exclusive. However, both of these competing binding sites are required for maximal in vitro transcription.

The PAR-domain basic leucine zipper (PAR bZip) transcription factors DBP, TEF, and HLF accumulate in a highly circadian manner in several peripheral tissues, including liver and kidney. Mice devoid of all three of these proteins are born at expected Mendelian ratios, but are epilepsy prone, age at an accelerated rate, and die prematurely. In the hope of identifying PAR bZip target genes whose altered expression might contribute to the high morbidity and mortality of PAR bZip triple knockout mice, we compared the liver and kidney transcriptomes of these animals to those of wild-type or heterozygous mutant mice. These experiments revealed that PAR bZip proteins control the expression of many enzymes and regulators involved in detoxification and drug metabolism, such as cytochrome P450 enzymes, carboxylesterases, and constitutive androstane receptor (CAR). Indeed, PAR bZip triple knockout mice are hypersensitive to xenobiotic compounds, and the deficiency in detoxification may contribute to their early aging.

In mammals, all overt circadian rhythms are thought to be coordinated by a central pacemaker residing in the hypothalamic suprachiasmatic nucleus (SCN) [1]. The phase of this pacemaker is entrained by photic cues via the retino-hypothalamic tract. Circadian clocks probably rely on a feedback loop in the expression of certain clock genes (reviewed in [2,3]). Surprisingly, however, such molecular oscillators are not only operative in pacemaker cells, such as SCN neurons, but also in many peripheral tissues and even in cell lines kept in vitro [4-7]. For example, a serum shock can induce circadian gene expression in cultured Rat-1 fibroblasts [5]. This treatment also results in a rapid surge of expression of the clock genes Per1 and Per2, similar to that observed in the SCNs of animals receiving a light pulse [8-10]. Serum induction of Per1 and Per2 transcription does not require ongoing protein synthesis [5] and must therefore be accomplished by direct signaling pathways. Here, we show that cAMP, protein kinase C, glucocorticoid hormones and Ca2+ can all trigger a transient surge of Per1 transcription and elicit rhythmic gene expression in Rat-1 cells. We thus suspect that the SCN pacemaker may exploit multiple chemical cues to synchronize peripheral oscillators in vivo.

Article17 December 2001free access Glucocorticoid hormones inhibit food-induced phase-shifting of peripheral circadian oscillators Nguyet Le Minh Nguyet Le Minh Département de Biologie Moléculaire, Sciences II, Université de Genève, 30 Quai Ernest Ansermet, CH-1211 Genève, Switzerland Present address: CNRS FRE2401, Génétique Moléculaire, Neurophysiologie et Comportement, Institut de Biologie, Collège de France, 11 place Marcelin Berthelot, F-75231 Paris, Cedex 5, France Search for more papers by this author Francesca Damiola Francesca Damiola Département de Biologie Moléculaire, Sciences II, Université de Genève, 30 Quai Ernest Ansermet, CH-1211 Genève, Switzerland Present address: Signalisations et identités cellulaires, Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, Université Claude Bernard Lyon I, Bat. G. Mendel, 43, boulevard du 11 Novembre 1918, F-69622 Villeurbanne, Cedex, France Present address: CNRS FRE2401, Génétique Moléculaire, Neurophysiologie et Comportement, Institut de Biologie, Collège de France, 11 place Marcelin Berthelot, F-75231 Paris, Cedex 5, France Search for more papers by this author François Tronche François Tronche Molecular Biology of the Cell, Deutsches Krebsforschungzentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Günther Schütz Günther Schütz Molecular Biology of the Cell, Deutsches Krebsforschungzentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Ueli Schibler Corresponding Author Ueli Schibler Département de Biologie Moléculaire, Sciences II, Université de Genève, 30 Quai Ernest Ansermet, CH-1211 Genève, Switzerland Search for more papers by this author Nguyet Le Minh Nguyet Le Minh Département de Biologie Moléculaire, Sciences II, Université de Genève, 30 Quai Ernest Ansermet, CH-1211 Genève, Switzerland Present address: CNRS FRE2401, Génétique Moléculaire, Neurophysiologie et Comportement, Institut de Biologie, Collège de France, 11 place Marcelin Berthelot, F-75231 Paris, Cedex 5, France Search for more papers by this author Francesca Damiola Francesca Damiola Département de Biologie Moléculaire, Sciences II, Université de Genève, 30 Quai Ernest Ansermet, CH-1211 Genève, Switzerland Present address: Signalisations et identités cellulaires, Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, Université Claude Bernard Lyon I, Bat. G. Mendel, 43, boulevard du 11 Novembre 1918, F-69622 Villeurbanne, Cedex, France Present address: CNRS FRE2401, Génétique Moléculaire, Neurophysiologie et Comportement, Institut de Biologie, Collège de France, 11 place Marcelin Berthelot, F-75231 Paris, Cedex 5, France Search for more papers by this author François Tronche François Tronche Molecular Biology of the Cell, Deutsches Krebsforschungzentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Günther Schütz Günther Schütz Molecular Biology of the Cell, Deutsches Krebsforschungzentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Ueli Schibler Corresponding Author Ueli Schibler Département de Biologie Moléculaire, Sciences II, Université de Genève, 30 Quai Ernest Ansermet, CH-1211 Genève, Switzerland Search for more papers by this author Author Information Nguyet Le Minh1,4, Francesca Damiola1,2,4, François Tronche3, Günther Schütz3 and Ueli Schibler 1 1Département de Biologie Moléculaire, Sciences II, Université de Genève, 30 Quai Ernest Ansermet, CH-1211 Genève, Switzerland 2Present address: Signalisations et identités cellulaires, Centre de Génétique Moléculaire et Cellulaire, UMR CNRS 5534, Université Claude Bernard Lyon I, Bat. G. Mendel, 43, boulevard du 11 Novembre 1918, F-69622 Villeurbanne, Cedex, France 3Molecular Biology of the Cell, Deutsches Krebsforschungzentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany 4Present address: CNRS FRE2401, Génétique Moléculaire, Neurophysiologie et Comportement, Institut de Biologie, Collège de France, 11 place Marcelin Berthelot, F-75231 Paris, Cedex 5, France ‡N.Le Minh and F.Damiola contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:7128-7136https://doi.org/10.1093/emboj/20.24.7128 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The circadian timing system in mammals is composed of a master pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus and slave clocks in most peripheral cell types. The phase of peripheral clocks can be completely uncoupled from the SCN pacemaker by restricted feeding. Thus, feeding time, while not affecting the phase of the SCN pacemaker, is a dominant Zeitgeber for peripheral circadian oscillators. Here we show that the phase resetting in peripheral clocks of nocturnal mice is slow when feeding time is changed from night to day and rapid when switched back from day to night. Unexpectedly, the inertia in daytime feeding-induced phase resetting of circadian gene expression in liver and kidney is not an intrinsic property of peripheral oscillators, but is caused by glucocorticoid signaling. Thus, glucocorticoid hormones inhibit the uncoupling of peripheral and central circadian oscillators by altered feeding time. Introduction In mammals, most physiology and behavior is subject to daily oscillations that are driven by an endogenous circadian timing system. Current evidence suggests that this system has a hierarchical structure: a master pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus synchronizes slave oscillators in most tissues (for reviews see Brown and Schibler, 1999; Reppert and Weaver, 2001; Ripperger and Schibler, 2001). As indicated by its name, the circadian clock can measure time only approximately and therefore has to be reset every day by external time cues (Zeitgebers). Light, the major Zeitgeber for the central pacemaker, entrains the phase of oscillations in SCN neurons via the retinohypothalamic tract, and glutamate appears to be the major neurotransmitter in this process (Ding et al., 1994; Shirakawa and Moore, 1994; Ebling, 1996). Light pulses delivered to animals kept in constant darkness during the subjective night not only elicit phase shifts, but also induce a surge of immediate early gene expression in SCN neurons. The products of these immediate early genes include the circadian clock components PER1 and PER2, and several transcription factors, such as c-Fos, Jun-B and EGR3 (Albrecht et al., 1997; Shearman et al., 1997; Shigeyoshi et al., 1997; Morris et al., 1998). It is likely that some of these regulatory proteins participate in the light-induced phase shifting. Seven genes essential for mammalian circadian clock function have been identified. These include the two period isoforms mPer1 and mPer2 (Shearman et al., 1997; Sun et al., 1997; Tei et al., 1997; Zheng et al., 1999), the two cryptochrome isoforms mCry1 and mCry2 (Kume et al., 1999; van der Horst et al., 1999), two genes, Clock and Bmal1, encoding PAS helix–loop–helix transcription factors (King et al., 1997; Bunger et al., 2000) and Tau, encoding casein kinase 1ϵ (Lowrey et al., 2000). The products of these genes have been assembled into a model of a molecular oscillator, which is based on interlocked feedback loops in gene expression (Shearman et al., 2000). In this model, the two cryptochromes repress transcription of mCry and mPer genes via interference with the two PAS helix–loop–helix transcription factors Clock and Bmal1. The cyclic accumulation of mPER2 then acts to generate a rhythmic expression of Bmal1, whose oscillation may reinforce the negative feedback loop of mCry expression. Post-transcriptional regulation also appears to play an important role in rhythm generation (Keesler et al., 2000; Lowrey et al., 2000; Toh et al., 2001). Clock-controlled genes such as Arginine Vasopressin or Dbp, whose products display cyclic accumulation but are not required for clock function, can be regulated by the same interlocked feedback loops of gene expression that drive the rhythmic expression of bona fide pacemaker genes (Jin et al., 1999; Ripperger et al., 2000). Circadian gene expression of clock and clock-controlled genes is not only observed in the SCN, but also in most peripheral tissues. Moreover, circadian gene expression persists in organ explants for several consecutive days and can be induced in immortalized rat and mouse tissue culture cells (Balsalobre et al., 1998; Yamazaki et al., 2000; Yagita et al., 2001). Hence, it appears that peripheral cells contain bona fide circadian timekeepers that can generate circadian oscillations during several consecutive days. The phase angle relationship between different mRNA accumulation cycles is very similar in SCN neurons and peripheral cell types, and mutations in essential clock genes affect these oscillators in a similar way. Therefore, central and peripheral oscillators are likely to function according to similar molecular principles (Balsalobre et al., 1998; Yagita et al., 2001). In spite of their resemblance in molecular make-up, central and peripheral circadian clocks differ from each other in that only the former are self-sustained. Hence, it appears that the SCN must periodically re-entrain peripheral oscillators in order to prevent the dampening of circadian gene expression in organs such as liver, kidney, heart and pancreas (Yamazaki et al., 2000). The mechanisms by which the SCN accomplishes this task are not yet understood, but they are likely to be complex. In fact, ligands of the glucocorticoid receptor (Balsalobre et al., 2000a) and two other nuclear receptors, RARα and RXRα (McNamara et al., 2001), calcium ionophores and chemicals that activate either protein kinase A, protein kinase C and/or MAP kinases (Akashi and Nishida, 2000; Balsalobre et al., 2000b; Yagita and Okamura, 2000) can all trigger circadian gene expression in tissue culture cells. Moreover, some of these signals also provoke phase shifts in peripheral tissues of intact animals (e.g. Balsalobre et al., 2000a; McNamara et al., 2001). It is thus likely that multiple signaling pathways are used for phase resetting of circadian peripheral oscillators. Balsalobre et al. (2000a) have recently demonstrated that dexamethasone, a glucocorticoid receptor agonist, acts as a strong phase-shifting agent for peripheral oscillators in the mouse. However, since the phase of oscillators is the same in hepatocytes that do or do not express glucocorticoid receptors, glucocorticoid hormones cannot be the only Zeitgeber signal for peripheral clocks. Feeding time has recently been shown to be a dominant Zeitgeber for rhythmic gene expression in tissues such as liver, pancreas, kidney and heart (Damiola et al., 2000; Stokkan et al., 2001). Thus, if nocturnal animals like mice or rats are fed exclusively during the day for extended periods, the phase of circadian gene expression in several examined organs becomes completely inverted. Interestingly, however, restricted feeding has no influence on the phase of rhythmic gene expression in the SCN (Damiola et al., 2000; Stokkan et al., 2001). These observations raise the possibility that the SCN entrains peripheral clocks indirectly by imposing activity and resting phases, and thus feeding behavior. According to this model, food metabolites or signals elicited by feeding behavior or food processing would be the principal timing cues for peripheral oscillators. Upon switching nocturnal animals to daytime feeding, it takes several days until the reversed phase is established (Damiola et al., 2000). This relatively slow phase adaptation is reminiscent of the time-consuming photic entrainment of the SCN master clock, which manifests itself in jet lag after long-distance east- or westbound trips. Here we demonstrate that the sluggish phase resetting of peripheral clocks by altered feeding time is not caused by an intrinsic inertia of circadian oscillators to adapt to the new Zeitgeber time, but by active signaling pathways that counteract rapid phase resetting. Thus, in the absence of glucocorticoid hormones or the glucocorticoid hormone receptor, feeding time can induce large phase shifts in peripheral tissues such as the liver, thereby inverting the phase of their oscillators within a few days. Results Daytime feeding phase-shifts peripheral oscillators faster in adrenalectomized compared with sham-operated mice As shown previously, food is a dominant Zeitgeber for circadian oscillators in several mouse tissues, including liver, pancreas, kidney and heart. Mice, which are nocturnal animals, ingest ∼80% of their daily food intake during the night (N.Preitner, unpublished results). As a consequence, restricting feeding time to the dark period does not significantly alter the phase angle of cyclic gene expression (Damiola et al., 2000). However, when food is offered only during the light phase during several consecutive days, the phase of circadian gene expression of clock and clock-controlled genes becomes completely inverted after 7–10 days (Damiola et al., 2000). Among several tissues examined, this phase resetting occurred most rapidly in the liver. We wished to determine whether glucocorticoid hormones, which efficiently induce circadian gene expression in cultured cells and elicit potent phase shifts in peripheral tissues of intact animals, participate in the food-induced resetting of peripheral oscillators. To this end, the kinetics of phase changes in the rhythmic expression of the clock genes mPer1, mPer2 and mCry1, and the clock-controlled gene Dbp, were compared in livers and kidneys of normal (sham-operated) and adrenalectomized mice. As shown in Figure 1A and B, the accumulation profiles of the mRNA encoded by these genes are strikingly different during the second day after initiation of daytime feeding. In sham-operated animals (right panels), the peak and trough levels of some mRNAs are offset by only ∼4 h when compared with mice fed ad libitum or during the night (Figure 2A and C). For example, in control animals, Dbp mRNA, whose circadian transcription is regulated directly by components of the molecular oscillator (Ripperger et al., 2000), reaches peak and trough levels in liver and kidney at about Zeitgeber times ZT14 and ZT2, respectively, during the second day of daytime feeding (ZT0 = time when the lights are switched on). However, much larger phase shifts are observed in tissues of adrenalectomized mice. Thus, during the second day after restricted feeding, Dbp mRNA peak levels are observed in liver between ZT0 and ZT4, which is significantly closer to the time at which daytime feeding-induced phase resetting is completed (Figure 2B and Damiola et al., 2000). Figure 1.Accumulation of circadian transcripts in adrenalectomized and sham-operated mice at the onset of daytime feeding. Adrenalectomized mice and sham-operated mice, kept under a 12 h light–dark regimen (lights on ZT0), were switched from ad libitum to daytime feeding. On day D, food was removed at ZT12 and provided exclusively during the light phase of the following days. During the second day (D+1) of restricted feeding, animals were killed at 4 h intervals and the livers, kidneys and brains were collected for monitoring the accumulation of mRNAs specified by the three clock genes mPer1, mPer2 and mCry1, and the clock output gene Dbp. Relative transcript levels in liver (A) and kidney (B) were determined by RNase protection assays using radiolabeled antisense RNA probes. Tbp (Tatabox-binding protein) mRNAs served as a control for a transcript whose accumulation does not oscillate during the day (not shown). The approximate times at which peak levels of mRNA accumulation are observed in animals fed ad libitum or during the night are ZT12 (mPer1), ZT17 (mPer2), ZT11 (Dbp) and ZT22 (mCry1) (see Damiola et al., 2000 and Figure 2C). These times are indicated by arrows on top of the respective panels. The accumulation of mPer1 and mPer2 mRNA in the SCN was assessed by in situ hybridization to coronal brain sections, using 35S-labeled antisense RNA probes (C). Only the hypothalamus regions containing the SCN (small pairwise structures at the base of the hypothalamus) are depicted. At ZT20, the signal obtained for mPer2 mRNA is somewhat higher in the adrenalectomized animal compared with sham-operated animals, but this difference does not significantly change the phase of mPer2 mRNA accumulation. Download figure Download PowerPoint Figure 2.Hepatic accumulation of mCry1 and Dbp transcripts in night-time- and daytime-fed adrenalectomized and sham-operated mice. Adrenalectomized mice and sham-operated mice fed ad libitum were kept under a 12 h light–dark regimen (lights on ZT0) before they were switched to night-time feeding [(A) and (C)] or daytime feeding (B). One day (A) or 1 week [(B) and (C)] after restricted feeding had been initiated, animals were killed at 4 h intervals and the livers were collected for monitoring the accumulation of Dbp and mCry1 transcripts. Tbp mRNA was included as a transcript whose accumulation is neither circadian- nor feeding time-dependent. Download figure Download PowerPoint In kidney, phase reversal by daytime feeding takes somewhat longer than in liver (Damiola et al., 2000). Thus, as we have observed previously for the transition from ad libitum to daytime feeding, the expression of some genes (e.g. Dbp, mPer2 and mCry1) is not yet rhythmic in kidney during the second day of daytime feeding. Nevertheless, mPer1 mRNA, which in kidney is the sole examined transcript with a clearly cyclic accumulation during the second day of restricted feeding, already follows a profile close to that observed after complete phase reversal. In contrast to the observations made in liver and kidney, daytime feeding does not affect circadian gene expression in the SCN (Damiola et al., 2000; Stokkan et al., 2001). As shown by the in situ hybridization experiments presented in Figure 1C, this holds true for rhythmic mPer1 and mPer2 mRNA accumulation in both sham-operated and adrenalectomized mice. Hence, as reported in previous studies (Balsalobre et al., 2000a; Damiola et al., 2000; Stokkan et al., 2001), neither feeding time nor glucocorticoid signaling appear to influence the phase of the central molecular pacemaker. Similar experiments to those presented in Figure 1 were also performed with mice fed exclusively during the night, or for extended time periods during the day. The experiments presented in Figure 2 indicate that glucocorticoid signaling does not influence the phase of circadian gene expression in animals subjected to night-time feeding for either 1–2 days (Figure 2A) or 1 week (Figure 2C). This is in keeping with the previously published finding that the phases of circadian gene expression are indistinguishable in animals fed ad libitum or exclusively during the night (Damiola et al., 2000). We also noticed that after extended periods of daytime feeding, the steady-state phase of cyclic mRNA accumulation is the same in the presence or absence of glucocorticoid hormone signaling (Figure 2B). We draw two major conclusions from the results presented thus far. First, glucocorticoid signaling strongly slows down—but does not prevent—the adaptation of circadian gene expression to a feeding schedule that is in conflict with the normal activity phase of the animal. Secondly, glucocorticoids are not required for the phase setting of circadian oscillators in liver and kidney under conditions in which food is available throughout the 24 h day (see also Damiola et al., 2000) or during the normal activity phase. Daytime feeding changes the profile of daily glucocorticoid secretion In animals fed ad libitum, glucocorticoid hormones are secreted by the adrenal gland in a circadian and episodic fashion (Carrillo et al., 1980; Tronche et al., 1998). The episodes, consisting of short bouts of hormone secretion (Shiraishi et al., 1984), are not synchronized between animals. The SCN pacemaker is believed to control the daily fluctuations of corticosterone levels via the hypothalamus–pituitary gland–adrenal axis and additional routes (Lejeune-Lenain et al., 1987). In both diurnal and nocturnal mammals, serum glucocorticoids reach peak levels around or just before the onset of the activity phase. Given the strong inhibition of food-induced phase resetting by glucocorticoid signaling, we wished to examine whether daytime feeding affects the temporal pattern of serum glucorticoids. To this end we monitored the serum concentrations of corticosterones at 4 h intervals around the clock in mice fed ad libitum (Figure 3A), in mice at the onset of daytime feeding (Figure 3C) and in mice exposed for 1 week to daytime feeding (Figure 3B). After 1 week, the inverted phase of circadian gene expression has been fully established in the peripheral organs examined. Because of the episodic nature of corticosterone secretion (Carrillo et al., 1980), the values obtained for the 5–7 individuals killed at a given time point vary greatly at times when these hormones reach high circadian levels, but are low in all animals at nadir times of glucocorticoid secretion. As a consequence, the circadian phase of glucocorticoid secretion is characterized best by the trough levels, which are highly reproducible. In accordance with published data (Cheifetz, 1971), corticosterone plasma levels are lowest at the dark–light transition (ZT0) and highest at the light–dark transition (ZT12) in animals kept under a 12 h light–dark regimen (Figure 3A). Interestingly the temporal glucocorticoid profile is bimodal in mice stably entrained to daytime feeding, with a peak of short duration between ZT20 and ZT4 (zenith at ZT0) and a broader peak between ZT4 and ZT20 (zenith between ZT8 and ZT12) (Figure 3B). The average levels of both peaks are in the range of 200 ng/ml, which resembles the average peak level measured for animals fed ad libitum. Figure 3.Diurnal corticosterone levels in animals fed ad libitum or during the day. Daytime-dependent corticosterone serum levels were determined in mice fed ad libitum (A), or in mice fed exclusively during the day for 7 days (B) or 1–2 days (C). For each time point, the blood was collected from five [(B) and (C)] to seven (A) individuals. As indicated in the text, due to the episodic nature of glucocorticoid secretion, the individual variability is small during nadir times but large during zenith times. In the steady-state profiles shown in (A) and (B), the values obtained for ZT16 and ZT20 are repeated to visualize the bimodal distributions. Download figure Download PowerPoint Based on these results, we conclude that daytime feeding establishes a bimodal temporal pattern of cortico sterone secretion, of which only the first peak appears to depend upon feeding time. The second oscillation is akin to the glucocorticoid profile recorded in animals fed ad libitum in that it reaches maximal values just before the light–dark transition. This second wave of corticosterone secretion may be controlled primarily by the SCN pacemaker, which does not change its phase in daytime-fed animals. At the onset of daytime feeding, corticosterone levels can attain particularly high levels during the dark phase (Figure 3C). Conceivably, these elevated glucocorticoid concentrations could be responsible for slowing down daytime feeding-induced resetting of peripheral oscillators (see Discussion). The inhibition of food-entrained phase resetting by glucocorticoids requires the glucocorticoid receptor and is tissue autonomous The observed inhibitory effect of glucocorticoid signaling on feeding time-induced phase resetting could be accomplished via direct cell-autonomous mechanisms or indirect mechanisms involving systemic signals whose production and/or release into the blood is controlled by glucocorticoid hormones. To discriminate between these two scenarios, we examined phase resetting of circadian gene expression in livers and kidneys of wild-type mice and GRAlfpCre mice, carrying glucocorticoid receptor null alleles exclusively in the hepatocytes (Kellendonk et al., 2000). As shown in Figure 4A (left panel), on the second day of daytime feeding the phases of circadian expression of Dbp, mPer1, mPer2 and mPer3 in the livers of GRAlfpCre mice are already shifted to values close to those observed 1 week after the animals had been subjected to the new feeding regimen. In contrast, the cyclic gene expression of these genes is only moderately advanced in the kidneys of GRAlfpCre mice (Figure 4B, left panel) and in kidneys or livers of wild-type mice (Figure 4A and B, right panels). Figure 4.Accumulation of circadian transcripts in GRAlfpCre and wild-type mice at the onset of daytime feeding. GRAlfpCre and wild-type mice were switched to daytime feeding as explained in the legend to Figure 1. The hepatocytes of GRAlfpCre mice harbor a glucocorticoid receptor null allele and thus cannot respond to corticosterone signaling. The relative mRNA levels were determined by RNase protection assays for liver (A) and kidney (B), and by in situ hybridization for the SCN (C). For comparison, the approximate peak times of mRNA accumulation in animals fed ad libitum or during the night are indicated by arrows (see Figure 1). Download figure Download PowerPoint The expression of mPer1 behaves somewhat differently when compared with that of the other circadian genes examined. Under normal circumstances, it is high during the dark phase of the first day and low throughout the period of daytime feeding examined in the livers of GRAlfpCre mice. Relatively low levels of mPer1 mRNA have also been observed in the livers of adrenalectomized animals (Figure 1A). As outlined in the previous section, corticosterone levels are particularly high during the dark phase at the onset of daytime feeding, when mPer1 mRNA accumulates to high cellular concentrations. This correlation could indicate that mPer1 is directly regulated by glucocorticoid hormones (see Balsalobre et al., 2000a and Discussion). We noticed, however, that in kidneys of adrenalectomized animals, mPer1 expression is not significantly diminished when compared with sham-operated animals (Figure 1B). Thus, in contrast to the observations made with liver, in kidney glucocorticoid signaling does not appear to be required for high mPer1 mRNA expression. We also compared the circadian mPer1 and mPer2 mRNA accumulation profiles in the SCN of GRAlfpCre and wild-type mice on the second day of daytime feeding (Figure 4C). As expected from the data presented in Figure 1, rhythmic gene expression is not significantly altered in the SCNs of mice subjected to restricted feeding, in spite of the high corticosterone levels measured during the dark phase at the onset of daytime feeding. In fact, neither glucocorticoid signaling nor restricted feeding causes phase shifts in circadian gene expression in SCN neurons (Balsalobre et al., 2000a; Damiola et al., 2000; Stokkan et al., 2001). The switch from daytime to night-time feeding results in a rapid phase resetting of circadian gene expression in liver and kidney For nocturnal animals, daytime feeding is in conflict with their activity phase, and although feeding time is a dominant Zeitgeber for circadian clocks in several peripheral tissues, it takes several days to adapt the phase of these oscillators to the unusual feeding schedule. In the results above, we demonstrated that the inertia in this phase resetting is not an intrinsic property of peripheral oscillators, but is caused principally by glucocorticoid signaling. We wished to determine whether the phase resetting in peripheral organs proceeds with different kinetics when mice are switched from an abnormal to a normal feeding schedule than when they are switched in the reverse direction. To this end, we subjected mice to 8 days of daytime feeding and then switched them to night-time feeding. Circadian gene expression was recorded at 4 h intervals between 32 and 64 h after the switch from daytime to night-time feeding. The data depicted in Figure 5 show that, in liver, the night-time entrained phase of circadian gene expression is already approached 2–3 days after changing the feeding regimen. For example, on the second day after switching from daytime to night-time feeding, Dbp mRNA reaches zenith levels between ZT8 and ZT12, similar to what had been observed in animals fed ad libitum or during the night. The phase changes proceed with slightly slower kinetics in kidney, in which the phases of several mRNA accumulation profiles lag behind those monitored in liver by ∼2 h. We have previously noticed that circadian kidney gene expression also adapts somewhat more slowly to daytime feeding, at least for certain examined genes. Figure 5.Accumulation of circadian transcripts in mice switched from daytime to night-time feeding or vice versa. Mice kept under a 12 h LD regimen were entrained to daytime feeding or night-time feeding for 8 days, and then switched to night-time or daytime feeding, respectively. At the times after feeding time reversal (re-entrainment) indicated, liver and kidney RNAs were subjected to RNase protection assays with the probes indicated on the left-hand side of the panels. Tbp mRNA was included as a transcript whose accumulation is neither circadian- nor feeding time-dependent (not shown). For comparison, the approximate peak times of mRNA accumulation in animals fed ad libitum or during the night are indicated by arrows.

The full-length cDNA for a transcriptional activator, DBP, that binds to the D site of the albumin promoter has been cloned. DBP belongs to a family of related transcription factors including Fos, Jun, CREB, and C/EBP, which share a conserved basic domain. However, unlike most other members of this family, DBP does not contain a "leucine zipper" structure. Among several rat tissues tested, significant levels of its protein are only observed in liver; yet, with the exception of testis, DBP mRNA is present in all of the examined tissues. DBP as well as its mRNA accumulate to significant levels only in adult animals. During chemically induced liver regeneration, DBP expression is rapidly down-regulated, suggesting that DBP may be involved in the proliferation control of hepatocytes. This cell growth-dependent expression of DBP, in contrast to its tissue specificity, appears to be controlled at the level of mRNA accumulation.

In mammals, many aspects of behavior and physiology, and in particular cellular metabolism, are coordinated by the circadian timing system. Molecular clocks are thought to rely on negative feedback loops in clock gene expression that engender oscillations in the accumulation of transcriptional regulatory proteins, such as the orphan receptor REV-ERBalpha. Circadian transcription factors then drive daily rhythms in the expression of clock-controlled output genes, for example genes encoding enzymes and regulators of cellular metabolism. To gain insight into clock output functions of REV-ERBalpha, we carried out genome-wide transcriptome profiling experiments with liver RNA from wild-type mice, Rev-erbalpha knock-out mice, or REV-ERBalpha overexpressing mice. On the basis of these genetic loss- and gain-of-function experiments, we concluded that REV-ERBalpha participates in the circadian modulation of sterol regulatory element-binding protein (SREBP) activity, and thereby in the daily expression of SREBP target genes involved in cholesterol and lipid metabolism. This control is exerted via the cyclic transcription of Insig2, encoding a trans-membrane protein that sequesters SREBP proteins to the endoplasmic reticulum membranes and thereby interferes with the proteolytic activation of SREBPs in Golgi membranes. REV-ERBalpha also participates in the cyclic expression of cholesterol-7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in converting cholesterol to bile acids. Our findings suggest that this control acts via the stimulation of LXR nuclear receptors by cyclically produced oxysterols. In conclusion, our study suggests that rhythmic cholesterol and bile acid metabolism is not just driven by alternating feeding-fasting cycles, but also by REV-ERBalpha, a component of the circadian clockwork circuitry.

Most living beings, including humans, must adapt to rhythmically occurring daily changes in their environment that are generated by the Earth's rotation. In the course of evolution, these organisms have acquired an internal circadian timing system that can anticipate environmental oscillations and thereby govern their rhythmic physiology in a proactive manner. In mammals, the circadian timing system coordinates virtually all physiological processes encompassing vigilance states, metabolism, endocrine functions and cardiovascular activity. Research performed during the past two decades has established that almost every cell in the body possesses its own circadian timekeeper. The resulting clock network is organized in a hierarchical manner. A master pacemaker, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, is synchronized every day to the photoperiod. In turn, the SCN determines the phase of the cellular clocks in peripheral organs through a wide variety of signalling pathways dependent on feeding cycles, body temperature rhythms, oscillating bloodborne signals and, in some organs, inputs of the peripheral nervous system. A major purpose of circadian clocks in peripheral tissues is the temporal orchestration of key metabolic processes, including food processing (metabolism and xenobiotic detoxification). Here, we review some recent findings regarding the molecular and cellular composition of the circadian timing system and discuss its implications for the temporal coordination of metabolism in health and disease. We focus primarily on metabolic disorders such as obesity and type 2 diabetes, although circadian misalignments (shiftwork or 'social jet lag') have also been associated with the aetiology of human malignancies.

The liver-enriched transcription factor DBP is expressed with a stringent circadian rhythm. We present evidence that DBP is a regulator of the circadian expression of the rat gene encoding cholesterol 7 alpha hydroxylase (C7 alpha H), the rate-limiting enzyme in the conversion of cholesterol to bile acids. As with DBP, C7 alpha H mRNA reaches peak levels in the evening, and its cycling is independent of daily food and light cues. As predicted for a DBP target gene, the primary level of C7 alpha H circadian expression is at the transcriptional level. DBP can activate the C7 alpha H promoter in cotransfection assays through a cognate DNA site centered around -225. In nuclear extracts prepared by a novel method that, in contrast to conventional techniques, yields near-quantitative recovery of DBP and other non-histone proteins, the DNA site required for DBP activation is the predominant site of occupancy by nuclear factors on the C7 alpha H promoter. At this site, the predominant binding activity is an evening-specific complex of which DBP is a component. These data suggest that DBP may play an important role in cholesterol homeostasis through circadian transcriptional regulation of cholesterol 7 alpha hydroxylase.

HNF1 is a liver-specific transcription factor that plays the dominant role in determining the cell type-specific in vitro transcription of the albumin gene. Here we report the purification and preliminary characterization of HNF1. HNF1 appears to be heavily glycosylated since it is retained on a wheat germ agglutinin-agarose column and can be eluted from it with N-acetylglucosamine, a property not observed with other factors binding to the albumin promoter. Using in vitro transcription assays we demonstrate that purified HNF1 strongly stimulates albumin promoter activity in spleen nuclear extracts, which are devoid of this factor. Likewise, an artificial promoter consisting of two HNF1 recognition sites in front of a TATA motif is strongly activated by HNF1 in such extracts. In addition to stimulating transcription directly by binding to its cognate site, HNF1 may further enhance albumin promoter activity by interacting cooperatively with other trans-acting factors.

The liver-enriched transcriptional activator protein DBP accumulates in hepatocytes of adult rats according to a strictly controlled circadian rhythm. DBP is not detectable in liver nuclei during the morning hours. Its level raises sharply during the afternoon and reaches a maximum at about 8 p.m. During the night the cellular DBP concentration decreases below detectability. This oscillation is "free running," transcriptionally regulated, and may be under the negative control of glucocorticoid hormones. In keeping with the rhythmicity of DBP accumulation, the albumin gene, a putative target of DBP, is transcribed more efficiently in the evening than in the morning.

The protein kinase MO15/CDK7 has recently been shown to be associated with the general transcription factor TFIIH and to be capable of phosphorylating the RNA polymerase II carboxy-terminal domain. Here, we show that a monoclonal MO15/CDK7 antibody coimmunoprecipitates, from a rat liver nuclear extract, all components of the RNA polymerase II transcription apparatus required for initiation at the albumin and adenovirus major late promoters. The immunoprecipitate includes RNA polymerase II, TFIID, TFIIB, TFIIH, TFIIF, and TFIIE, but is devoid of transcriptional activator proteins, such as HNF1, HNF4, and C/EBP alpha. The finding of an autonomously initiating RNA polymerase II holoenzyme in mammalian cells suggests conceptual similarities between transcription initiation in prokaryotes and eukaryotes.

Hepatocyte nuclear factor 1 (HNF-1) is a transcriptional regulatory protein possibly involved in the activation of many liver-specifically expressed genes. HNF-1 mRNA is restricted to a small number of tissues, suggesting that the HNF-1 gene itself is regulated at the transcriptional level. We have isolated and characterized the promoter region of this gene and have determined its transcriptional potential in several cell types by cell-free transcription and transient transfection experiments. In in vitro transcription assays, an HNF-1 promoter is active in nuclear extracts from liver and kidney, two tissues that contain HNF-1, but silent in nuclear extracts from spleen and lung, which are devoid of this transcription factor. Likewise, in transfection experiments, HNF-1 promoter-chloramphenicol acetyltransferase (CAT) fusion genes are expressed in Hep G2 cells, which express HNF-1, but not in mouse L cells or Hela cells, which do not express HNF-1. In both cell-free transcription and transient transfection assays, a relatively short promoter segment located between positions -82 and -40 is necessary and sufficient to direct cell type-specific HNF-1 transcription. This region contains a single site for a DNA-binding protein that has been tentatively identified as hepatocyte nuclear factor 4, a member of the steroid hormone receptor family.

Cell sizes can differ vastly between cell types in individual metazoan organisms. In rat liver, spleen, and thymus, differences in average cell size roughly reflect differences in RNA:DNA ratios. For example, hepatocytes were found to have a cytoplasmic:nuclear volume ratio and an RNA:DNA ratio which were 34- and 21-fold higher, respectively, than those in thymocytes. RNA synthesis per DNA-equivalent in the hepatocytes was 25-fold greater than that in thymocytes, suggesting that differences in overall transcriptional activity, not differences in overall RNA stability, were primarily responsible for determining cellular RNA:DNA ratios. The mechanisms determining the capacity of large cells to synthesize and accumulate more ubiquitous cytoplasmic macromolecules, such as ribosomes, than smaller cells is entitled "cell size regulation." Cell size regulation may have important consequences on the tissue distribution of transcription factors. Thus, in liver, lung, kidney, spleen, and brain, cellular levels of the mRNA encoding the leucine zipper protein DBP correlate closely to cellular RNA:DNA ratios. Our results suggest that DBP mRNA levels, like rRNA levels, are transcriptionally determined. Thus the dbp gene, like the ribosomal genes, may be subject to cell size regulation. As a consequence, nuclei from liver, a tissue with a very large average cell size, accumulated higher levels of DBP protein than nuclei from small-celled tissues, such as spleen or lung. In contrast to DBP, the ubiquitous transcription factors Oct1 and NF-Y escaped cell size control. Nuclei from most tissues contained similar amounts of these factors irrespective of cell size. Likewise, tissues with large or small average cell sizes contained similar levels of the mRNAs encoding Oct1 or NF-Ya, one of the subunits of the heteromeric CCAAT-binding factor NF-Y, per DNA-equivalent. Interestingly, mRNA encoding NF-Yb, another subunit of NF-Y, was subject to cell size regulation. Our results suggest that NF-Yb protein escapes cell size regulation at a posttranslational level.

The two highly related PAR basic region leucine zipper proteins TEF and DBP accumulate according to a robust circadian rhythm in liver and kidney. In liver nuclei, the amplitude of daily oscillation has been estimated to be 50-fold and 160-fold for TEF and DBP, respectively. While DBP mRNA expression is the principal determinant of circadian DBP accumulation, the amplitude of TEF mRNA cycling is insufficient to explain circadian TEF fluctuation. Conceivably, daily variations in TEF degradation or nuclear translocation efficiency may explain the discrepancy between mRNA and protein accumulation. In vitro, TEF and DBP bind the same DNA sequences. Yet, in co-transfection experiments, these two proteins exhibit different activation potentials for two reporter genes examined. While TEF stimulates transcription from the albumin promoter more potently than DBP, only DBP is capable of activating transcription efficiently from the cholesterol 7 alpha hydroxylase (C7alphaH) promoter. However, a TEF-DBP fusion protein, carrying N-terminal TEF sequences and the DNA binding/dimerization domain of DBP, enhances expression of the C7alphaH-CAT reporter gene as strongly as wild-type DBP. Our results suggest that the promoter environment, rather than the affinity with which PAR proteins recognize their cognate DNA sequences in vitro, determines the promoter preferences of TEF and DBP.

Mammalian physiology has to adapt to daily alternating periods during which animals either forage and feed or sleep and fast. The adaptation of physiology to these oscillations is controlled by a circadian timekeeping system, in which a master pacemaker in the suprachiasmatic nucleus (SCN) synchronizes slave clocks in peripheral organs. Because the temporal coordination of metabolism is a major purpose of clocks in many tissues, it is important that metabolic and circadian cycles are tightly coordinated. Recent studies have revealed a multitude of signaling components that possibly link metabolism to circadian gene expression. Owing to this redundancy, the implication of any single signaling pathway in the synchronization of peripheral oscillators cannot be assessed by determining the steady-state phase, but instead requires the monitoring of phase-shifting kinetics at a high temporal resolution.

In most mammals, daily rhythms in physiology are driven by a circadian timing system composed of a master pacemaker in the suprachiasmatic nucleus (SCN) and peripheral oscillators in most body cells. The SCN clock, which is phase-entrained by light-dark cycles, is thought to synchronize subsidiary oscillators in peripheral tissues, mainly by driving cyclic feeding behavior. Here, we examined the expression of circadian clock genes in the SCN and the liver of the common vole Microtus arvalis, a rodent with ultradian activity and feeding rhythms. In these animals, clock-gene mRNAs accumulate with high circadian amplitudes in the SCN but are present at nearly constant levels in the liver. Interestingly, high-amplitude circadian liver gene expression can be elicited by subjecting voles to a circadian feeding regimen. Moreover, voles with access to a running wheel display a composite pattern of circadian and ultradian behavior, which correlates with low-amplitude circadian gene expression in the liver. Our data indicate that, in M. arvalis, the amplitude of circadian liver gene expression depends on the contribution of circadian and ultradian components in activity and feeding rhythms.

ABSTRACT DBP, a liver-enriched transcriptional activator protein of the leucine zipper protein family, accumulates according to a very strong circadian rhythm (amplitude approx. 1000-fold). In rat parenchymal hepatocytes, the protein is barely detectable during the morning hours. At about 2 p.m., DBP levels begin to rise, reach maximal levels at 8 p.m. and decline sharply during the night. This rhythm is free-running: it persists with regard to both its amplitude and phase in the absence of external time cues, such as daily dark/light switches. Also, fasting of rats for several days influences neither the amplitude nor the phase of circadian DBP expression. Since the levels of DBP mRNA and nascent transcripts also oscillate with a strong amplitude, circadian DBP expression is transcriptionally controlled. While DBP mRNA fluctuates with a similar phase and amplitude in most tissues examined, DBP protein accumulates to high concentrations only in liver nuclei. Hence, at least in nonhepatic tissues, cyclic DBP transcription is unlikely to be controlled by a positive and/or negative feedback mechanism involving DBP itself. More likely, the circadian DBP expression is governed by hormones whose peripheral concentrations also oscillate during the day. Several lines of evidence suggest a pivotal role of glucocorticoid hormones in establishing the DBP cycle. Two genes whose mRNAs and protein products accumulate according to a strong circadian rhythm with a phase compatible with regulation by DBP encode enzymes with key functions in cholesterol metabolism: HMG-coA reductase is the rate-limiting enzyme in cholesterol synthesis; cholesterol 7-a hydroxylase performs the rate-limiting step in the conversion of cholesterol to bile acid. DBP may thus be involved in regulating cholesterol homeostasis.

The efficiency and tissue-specificity of transgene expression in animals is usually subject to the position of integration into the host chromatin. We have discovered that a 2.8kbp fragment flanking the rat gene encoding the transcription factor LAP (C/EBP beta) directs position-independent, copy number-dependent expression in transgenic-mouse livers. Concomitantly, the DNAse I hypersensitivity pattern normally observed in the liver is established in the integrated transgene construct demonstrating that this region is capable of creating chromatin structures equivalent to the endogenous situation. These observations are reminiscent of the locus control regions (LCR) described for several genes. Additionally, this LAP element functions with both intron-less and intron-containing genes. The tissue specificity of this element, however, is not restricted to liver. The 2.8kbp region is capable of allowing position-independent, copy number-dependent expression in brain, kidney, heart, spleen, and lung, but in a construct-dependent manner. This is, to our knowledge, the first transcription factor gene with which a cis-linked LCR-like element has been associated.

Mammalian physiology is governed by a complex circadian timing system that involves interacting positive and negative transcriptional feedback loops. A key role in this feedback loop was attributed to the PAS domain helix–loop–helix protein CLOCK, on the basis of a dominant-negative mutation in this transcription factor. However, recent experiments by Reppert and coworkers with Clock knockout mice suggest that CLOCK is dispensable for rhythmic gene expression and behavior, presumably because other proteins can substitute for CLOCK in these animals. Mammalian physiology is governed by a complex circadian timing system that involves interacting positive and negative transcriptional feedback loops. A key role in this feedback loop was attributed to the PAS domain helix–loop–helix protein CLOCK, on the basis of a dominant-negative mutation in this transcription factor. However, recent experiments by Reppert and coworkers with Clock knockout mice suggest that CLOCK is dispensable for rhythmic gene expression and behavior, presumably because other proteins can substitute for CLOCK in these animals.

Already in 1729, Jean-Jacques d'Ortous de Mairan, a French astronomer, noticed that the mimosa plants in his backyard opened and closed their leaves at similar times during the day. Intriguingly, these daily cycles of leaf movements persisted in plants kept in nearly constant darkness (Figure 1), and de Mairan thus concluded that they must be driven by endogenous clocks rather than environmental light-dark cycles. In the meantime, self-sustained circadian oscillators have been found in nearly all light-sensitive organisms from cyanobacteria to mammals. In the latter. they influence most aspects of physiology and behaviour and thus play essential roles in health and disease. Molecular research on these biological timekeepers has been initiated in the 1980s in the fruit fly Drosophila melanogaster by Jeffrey Hall, Michael Rosbash, and Michael Young, and their discovery of the first clock genes has been honoured with the 2017 Nobel Prize in Physiology or Medicine. As elaborated below, the Drosophila and mammalian clocks are very similar, and the work on Drosophila clocks has been pivotal in the identification of mammalian clock genes and in their functional analysis (Figure 1). The molecular work by the three Nobel Prize awardees is based on genetic studies conducted in the early 1970s by Ronald Konopka and Seymour Benzer.1 The latter already played a pivotal role in the functional definition of a gene. By generating a large number of T4 recombinant strains with mutations in the rII gene, he concluded in the 1950s that the gene must have a linear structure and that the minimal unit of recombination is one nucleotide. Some years later, he became fascinated by the behaviour, in particular phototaxis, of Drosophila melanogaster. His new research direction to study the fly brain was neither approved nor understood by his most famous colleagues. His mom was worried as well: “From this you can make a living?” she wondered. This did not, however, discourage Benzer to continue his ambitious scientific endeavours. Ron Konopka, Benzer's graduate student at that time, initiated a genetic screen for genes on the X chromosome affecting circadian behaviour. This screen was both risky and elegant. It was risky, because the X chromosome might not encompass genes relevant for the phenotype of interest. But if such X chromosome-linked genes indeed existed, the screening strategy was elegant for two reasons. First, males only contain one X chromosome, and the examination of male flies should thus reveal even recessive mutations. Second, fruit flies were known to have a highly robust circadian eclosion rhythm. Hence, chemically mutagenized male flies emerging from their pupae “at the wrong time” (after a few rounds of genetic purification) were likely to carry a “circadian timing mutation” on the X chromosome. To the excitement of Konopka and Benzer, the study culminated in a dream result, in that a single locus, period (per), carried mutations causing short-period rhythms (pershort), long-period rhythms (perlong), and arrhythmicity (per0). Pleasingly, the same mutations also engendered corresponding changes in the rhythms of locomotor activity, indicating that one and the same clock drives different physiological outputs. This fundamental discovery by Benzer and Konopka reached far beyond chronobiology. It represented the first experimental demonstration that defined Mendelian loci participate in determining phenotypic traits as complex as behaviour (Figure 1). In 1984, Jeffrey Hall and Michael Rosbash at Brandeis University physically isolated the Drosophila per gene by positional cloning and thereby provided compelling evidence for per being directly responsible for circadian behaviour. Moreover, they demonstrated that the accumulation of per mRNA and PER protein exhibits a high-amplitude circadian oscillation even in flies kept in constant darkness. Even more importantly, the authors postulated that this cycle is driven by a negative feedback loop (Figure 1),2, 3 a concept that has stood the test of time in many organisms from cyanobacteria to humans. Almost simultaneously, Michael Young also cloned the per gene and connected its mutants to circadian behaviour.4 These pioneering studies reported that circadian locomotor and eclosion rhythms can be restored upon introduction of cloned per sequences into the genome of arrhythmic per0 null flies. During the following years Young and coworkers discovered timeless (tim), another essential core clock gene, and demonstrated that its protein product formed a heterodimer with PER.5 Moreover, Young's laboratory found that PER phosphorylation by the Doubletime protein kinase regulated PER stability and thus critically affected the period length of the Drosophila clock. Mammalian core clock genes have escaped detection and isolation until the late 1990s. Many of us remember 1997 as the year of mammalian clock molecular biology. In this year, Joseph Takahashi, then at Northwestern University in Chicago and now at UT South Western Medical Center in Dallas, published the results of a heroic forward genetic screen of mice whose gametes were mutagenized by the injection of N-ethyl-N-nitrosourea. These endeavours resulted in the identification of a semi-dominant mutation in a locus dubbed Clock (for circadian locomotor output cycles kaput), affecting the period length in wheel-running activity (Figure 1).6 Positional cloning and sequencing of the Clock gene revealed that it encodes a large PAS domain helix-loop-helix transcription factor. Subsequent work by his and other laboratories demonstrated that CLOCK forms an obligatory heterodimer with BMAL1 and, as such, activates transcription of Per and Cryptochrome (Cry) genes, which specify the negative limb members of the feedback loop. Owing to comparative genomics and PCR assays with degenerate primers in the laboratories of Cheng Chi Lee and Hajime Tei, respectively, Per1, the first mammalian ortholog of Drosophila per, was discerned in the same magic year: 1997. We now know that Per1 and Per2, two of the three mammalian Per isoforms, are indispensable for circadian rhythm generation. A year later, clk and cyc, the two Drosophila orthologs of mammalian Clock and Bmal1, respectively, were unveiled in the laboratories of Hall and Rosbash. This further emphasized the similarity of the canonical molecular oscillators operative in flies and mammals. As it turned out, the most noteworthy difference between the two is the substitution of tim by Cry's in the mammalian clockwork. Interestingly CRY also plays a role in the Drosophila system, where it serves primarily as a photoreceptor participating in the phase entrainment of the clock by light. The discovery of mammalian core clock genes allowed studying their activity in different tissues. Somewhat surprisingly, they were not only expressed rhythmically in the brain's suprachiasmatic nucleus (SCN), known to harbour the circadian pacemaker, but also in virtually all peripheral tissues. By itself, this did not provide compelling evidence for the existence of circadian oscillators in the latter, as the daily ups and downs of clock gene expression in the periphery could have been driven by rhythmic systemic signals orchestrated by the SCN. In 1998, however, the robust circadian expression of clock genes was demonstrated in serum-shocked Rat-1 fibroblasts cultured in vitro (Figure 1).7 A few years later, single-cell recordings revealed the self-sustained and cell-autonomous nature of these fibroblast clocks. As explants of many tissues kept in tissue culture also displayed circadian gene expression, it became evident that most body cells harbour circadian timekeepers. This led to the concept of a hierarchically organized clock network, in which the SCN synchronizes peripheral oscillators by a variety of signalling pathways (Figure 1). One of the fundamental roles of the mammalian circadian system is the temporal orchestration of metabolism and other physiological processes. In humans, chronic circadian misalignments provoked by rotation shiftwork schedules, “social jetlag” or frequent time-zone changes have been reported to result in metabolic and cardiovascular diseases, depression and cancer. Seymour Benzer has announced to his colleagues “that he wanted to work on humans because they show such interesting and bizarre behaviors.” Owing to the pioneering studies on Drosophila and mammalian tissues and cultured cells, we now can dissect molecular clocks in human individuals. Such studies will hopefully lead to new strategies for treating metabolic and other diseases, even though the journey from bench to bedside might be very long. Once again, the attribution of the Nobel Prize in Physiology or Medicine to scientists studying fruit flies underscores the importance of basic research in lower, genetically amenable organisms for the understanding of human physiology and pathophysiology. We entirely subscribe to the statement of our colleague Martha Merrow, Chair of Medical Psychology at the Ludwig Maximilian University in Munich: “It's just a fantastic choice. It will be great for our field.” We have no conflict to declare.

When used in an in vitro transcription assay, the promoter of a cloned alpha 2u globulin gene is much more active in liver than in spleen nuclear extracts. Promoter deletion experiments suggest that both positive and negative regulatory mechanisms may be involved in the differential in vitro transcription from the alpha 2u globulin promoter in these two nuclear extracts. Interestingly, removal of promoter elements upstream from position -74 results in a significant increase of in vitro transcription in spleen but not in liver nuclear extracts, and thus reduces the difference in transcription observed with longer alpha 2u promoters in these two extracts. Deletion of additional nucleotides to position -43 strongly reduces the in vitro transcription efficiency of the promoter in extracts from both tissues. None of the examined promoters containing between 3000 and 22 nucleotides of 5' flanking regions are differentially transcribed in liver nuclear extracts from either male or female rats. Thus, in contrast to cell-type specificity, sex-specificity could not be observed in our in vitro transcription experiments. DNase I protection experiments with crude nuclear extracts and partially or highly purified nuclear proteins suggests the presence of six recognition sites for DNA-binding factors between the TATA element and position -210. Some of these factors could be identified as proteins that also bind to elements within the albumin gene promoter.