Cloning and expression of Clock

Positional cloning was used to identify the circadian Clock gene in mice. Clock is a large transcription unit with 24 exons spanning 100,000 bp of DNA from which transcript classes of 7.5 and 10 kb arise. Clock encodes a member of the bHLH-PAS family of transcription factors. Clock PAS A and PAS B motifs as well as Clock's bHLH domain most closely resemble mouse Neuronal PAS2. The Clock PAS motifs are only distantly related to those of Drosophila Period, which has no bHLH domain. The Clock PAS motifs bear a closer sequence resemblance to the PAS motifs of Drosophila Single minded. In the Clock mutant allele, an AT nucleotide transversion in a splice donor site causes exon skipping and deletion of 51 amino acids in the Clock protein. Clock is a unique mammalian gene with known circadian function and with features predicting DNA binding, protein dimerization, and activation domains. In addition to being expressed in the hypothalamus and the eye, Clock is expressed in brain, testis, ovary, liver, heart, lung and kidney. Clock represents the second example of a PAS domain-containing Clock protein (in addition to Drosophila Period), which suggests that this motif may define an evolutionarily conserved feature of the circadian clock mechanism (King, 1997b).

As a complementary approach to positional cloning, in vivo complementation with bacterial artificial chromosome (BAC) clones expressed in transgenic mice was used to identify the circadian Clock gene. A 140 kb BAC transgene completely rescues both the long period and the loss-of-rhythm phenotypes in Clock mutant mice. Analysis with overlapping BAC transgenes demonstrates that a large transcription unit is, in fact, the Clock gene; it encodes a novel basic-helix-loop-helix-PAS domain protein. Overexpression of the Clock transgene can shorten period length beyond the wild-type range, which provides additional evidence that Clock is an integral component of the circadian pacemaking system. Taken together, these results provide a proof of principle that "cloning by rescue" is an efficient and definitive method in mice (Antoch, 1997).

The only vertebrate clock gene identified by mutagenesis is mouse Clock, which encodes a bHLH-PAS transcription factor. Clock has been cloned in zebrafish and in contrast to its mouse homologue, it is expressed with a pronounced circadian rhythm in the brain and in two defined pacemaker structures, the eye and the pineal gland. Clock oscillation is also found in other tissues, including kidney and heart. In these tissues, expression of Clock continues to oscillate in vitro. This demonstrates that self-sustaining circadian oscillators exist in several vertebrate organs, as has been reported for invertebrates (Whitmore, 1998).

The mammalian retina contains an endogenous circadian pacemaker that broadly regulates retinal physiology and function, yet the cellular origin and organization of the mammalian retinal circadian clock remains unclear. Circadian clock neurons generate daily rhythms via cell-autonomous autoregulatory clock gene networks. Thus, to localize circadian clock neurons within the mammalian retina, the cell type-specific expression of six core circadian clock genes was examined in individually identified mouse retinal neurons, and the clock gene expression rhythms in retinal degeneration (rd) mouse retinas were characterized. Individual photoreceptors, horizontal, bipolar, dopaminergic (DA) amacrines, catecholaminergic (CA) amacrines, and ganglion neurons were identified either by morphology or by a tyrosine hydroxylase (TH) promoter-driven red fluorescent protein (RFP) fluorescent reporter. Cells were collected, and their transcriptomes were subjected to multiplex single-cell RT-PCR for the core clock genes Period (Per) 1 and 2, Cryptochrome (Cry) 1 and 2, Clock, and Bmal1. Individual horizontal, bipolar, DA (dopaminergic), CA, and ganglion neurons, but not photoreceptors, were found to coordinately express all six core clock genes, with the lowest proportion of putative clock cells in photoreceptors (0%) and the highest proportion in DA neurons (30%). In addition, clock gene rhythms were found to persist for >25 days in isolated, cultured rd mouse retinas in which photoreceptors had degenerated. These results indicate that multiple types of retinal neurons are potential circadian clock neurons that express key elements of the circadian autoregulatory gene network and that the inner nuclear and ganglion cell layers of the mammalian retina contain functionally autonomous circadian clocks (Ruan, 2006).

Mutation of Clock

Clock is a semidominant mutation found in mice. Mice carrying the Clock mutation exhibit abnormalities of circadian behavior, including lengthening of the endogenous period and loss of rhythmicity. Clock segregates as a single autosomal locus and lengthens the period of the circadian rhythm by about 1 hour in heterozygotes. In homozygote mutants, period lengthens by about 4 hours upon initial transfer to constant darkness, after which these mice lose persistent circadian rhythms. To identify the gene affected by this mutation, a high-resolution genetic map (> 1800 meioses) was generated of the Clock locus. Clock is 0.7 cM distal to Kit on mouse chromosome 5. Mapping shows that Clock lies within the W19H deletion. Complementation analysis of different Clock and W19H compound genotypes indicates that the Clock mutation behaves as an antimorph. In an antimorph, the heterozygote (wild type/Clock) is more severe than the mutant allele plus a deletion (Clock/deletion) but less severe than the phenotype of Clock/Clock. The wild type/deletion heterozygote hemizygous phenotype is indistinguishable from the homozygous wild-type phenotype. This order of phenotypic severity is the defining characteristic of an antimorphic allele. That the Clock/deletion phenotype is significantly more severe than the Clock/wild type phenotype indicates that the wild-type allele is interacting with the Clock mutant allele to ameliorate the severity of the Clock mutant phenotype. This antimorphic behavior of Clock strongly argues that Clock defines a gene centrally involved in the mammalian circadian system (King, 1997a).

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker in mammals, and one molecular regulator of circadian rhythms is the Clock gene. The discharge patterns of SCN neurons isolated from Clock mutant mice has been studied. Long-term, multielectrode recordings show that heterozygous Clock mutant neurons have lengthened periods, while homozygous Clock neurons are arrhythmic, paralleling the effects on locomotor activity in the animal. In addition, cells in dispersals express a wider range of periods and phase relationships than cells in explants. These results suggest that the Clock gene is required for circadian rhythmicity in individual SCN cells and that a mechanism within the SCN synchronizes neurons and restricts the range of expressed circadian periods (Herzog, 1998).

Classic experiments have shown that ovulation and estrous cyclicity are under circadian control and that surgical ablation of the suprachiasmatic nuclei (SCN) results in estrous acyclicity in rats. Reproductive function has been characterized in the circadian Clock mutant mouse; the circadian Clock mutation both disrupts estrous cyclicity and interferes with the maintenance of pregnancy. Clock mutant females have extended, irregular estrous cycles, lack a coordinated luteinizing hormone (LH) surge on the day of proestrus, exhibit increased fetal reabsorption during pregnancy, and have a high rate of full-term pregnancy failure. Clock mutants also show an unexpected decline in progesterone levels at midpregnancy and a shortened duration of pseudopregnancy, suggesting that maternal prolactin release may be abnormal. In a second set of experiments, the function of each level of the hypothalamic-pituitary-gonadal (HPG) axis was interrogated in order to determine how the Clock mutation disrupts estrous cyclicity. Clock mutants fail to show an LH surge following estradiol priming in spite of the fact that hypothalamic levels of gonadotropin-releasing hormone (GnRH), pituitary release of LH, and serum levels of estradiol and progesterone are all normal in Clock/Clock females. These data suggest that Clock mutants lack an appropriate circadian daily-timing signal required to coordinate hypothalamic hormone secretion. Defining the mechanisms by which the Clock mutation disrupts reproductive function offers a model for understanding how circadian genes affect complex physiological systems (Miller, 2004).

Circadian regulator CLOCK is a histone acetyltransferase

The molecular machinery that governs circadian rhythmicity comprises proteins whose interplay generates time-specific transcription of clock genes. The role of chromatin remodeling in a physiological setting such as the circadian clock is yet unclear. The protein CLOCK, a central component of the circadian pacemaker, has been shown to have histone acetyltransferase (HAT) activity. CLOCK shares homology with acetyl-coenzyme A binding motifs within the MYST family of HATs. CLOCK displays high sequence similarity to ACTR, a member of SRC family of HATs, with which it shares also enzymatic specificity for histones H3 and H4. BMAL1, the heterodimerization partner of CLOCK, enhances HAT function. The HAT activity of CLOCK is essential to rescue circadian rhythmicity and activation of clock genes in Clock mutant cells. Identification of CLOCK as a novel type of DNA binding HAT reveals that chromatin remodeling is crucial for the core clock mechanism and identifies unforeseen links between histone acetylation and cellular physiology (Doi, 2006).

The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control

Circadian rhythms govern a large array of metabolic and physiological functions. The central clock protein CLOCK has HAT properties. It directs acetylation of histone H3 and of its dimerization partner BMAL1 at Lys537, an event essential for circadian function. The HDAC activity of the NAD+-dependent SIRT1 enzyme is regulated in a circadian manner, correlating with rhythmic acetylation of BMAL1 and H3 Lys9/Lys14 at circadian promoters. SIRT1 associates with CLOCK and is recruited to the CLOCK:BMAL1 chromatin complex at circadian promoters. Genetic ablation of the Sirt1 gene or pharmacological inhibition of SIRT1 activity lead to disturbances in the circadian cycle and in the acetylation of H3 and BMAL1. Finally, using liver-specific SIRT1 mutant mice, SIRT1 is shown to contribute to circadian control in vivo. It is proposed that SIRT1 functions as an enzymatic rheostat of circadian function, transducing signals originated by cellular metabolites to the circadian clock (Nakahata, 2008).

SIRT1 regulates circadian clock gene expression through PER2 deacetylation

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus of the brain that synchronizes countless subsidiary oscillators in peripheral tissues. The rhythm-generating mechanism is thought to rely on a feedback loop involving positively and negatively acting transcription factors. BMAL1 and CLOCK activate the expression of Period (Per) and Cryptochrome (Cry) genes, and once PER and CRY proteins accumulate to a critical level they form complexes with BMAL1-CLOCK heterodimers and thereby repress the transcription of their own genes. This study shows that SIRT1, an NAD(+)-dependent protein deacetylase, is required for high-magnitude circadian transcription of several core clock genes, including Bmal1, Rorgamma, Per2, and Cry1. SIRT1 binds CLOCK-BMAL1 in a circadian manner and promotes the deacetylation and degradation of PER2. Given the NAD(+) dependence of SIRT1 deacetylase activity, it is likely that SIRT1 connects cellular metabolism to the circadian core clockwork circuitry (Asher, 2008).

Clock protein interactions

The mouse Clock gene encodes a bHLH-PAS protein that regulates circadian rhythms and is related to transcription factors that act as heterodimers. Potential partners of CLOCK were isolated in a two-hybrid screen, and one, BMAL1, is coexpressed with CLOCK and PER1 at known circadian clock sites in brain and retina. BMAL1 is a bHLH-PAS protein that is expressed in brain and muscle. Strong coexpression of Clock and bmal1 are found in the inner nuclear layer of the retina, the outer nuclear (photoreceptor) layer, and a discrete subset of cells in the ganglion cell layer. CLOCK-BMAL1 heterodimers activate transcription from E-box elements (a type of transcription factor-binding site, found adjacent to the mouse per1 gene) and from an identical E-box known to be important for per gene expression in Drosophila. Mutant CLOCK from the dominant-negative Clock allele and BMAL1 form heterodimers that bind DNA but fail to activate transcription. Thus, CLOCK-BMAL1 heterodimers appear to drive the positive component of per transcriptional oscillations, which are thought to underlie circadian rhythmicity (Gekakis, 1998).

Mouse (mTim) and human (hTIM) orthologs of the Drosophila timeless (dtim) gene have been cloned and characterized. The mammalian Tim genes are widely expressed in a variety of tissues; however, unlike Drosophila, mTim mRNA levels do not oscillate in the suprachiasmatic nucleus (SCN) or retina. Importantly, hTIM interacts with the Drosophila PERIOD (dPER) protein as well as the mouse PER1 and PER2 proteins in vitro. In Drosophila (S2) cells, hTIM and dPER interact and translocate into the nucleus. Finally, hTIM and mPER1 specifically inhibit CLOCK-BMAL1-induced transactivation of the mPer1 promoter. Taken together, these results demonstrate that mTim and hTIM are mammalian orthologs of timeless and provide a framework for a basic circadian autoregulatory loop in mammals (Sangoram, 1998).

Most clock genes encode transcription factors that interact to elicit cooperative control of clock function. Using a two-hybrid system approach, two different partners of zebrafish (zf) CLOCK were isolated: both are similar to the mammalian BMAL1 (brain and muscle arylhydrocarbon receptor nuclear translocator-like protein 1). The two homologs, zfBMAL1 and zfBMAL2, contain conserved basic helix-loop-helix-PAS domains but diverge in the carboxyl termini, thus bearing different transcriptional activation potential. As for zfClock, the expression of both zfBmals oscillates in most tissues in the animal. However, in many tissues, the peak, levels, and kinetics of expression are different between the two genes and for the same gene from tissue to tissue. These results support the existence of independent peripheral oscillators and suggest that zfBMAL1 and zfBMAL2 may exert distinct circadian functions, interacting differentially with zfCLOCK at various times in different tissues. These findings also indicate that multiple controls may be exerted by the central clock and/or that peripheral oscillators can differentially interpret central clock signals (Cermakian, 2000).

In the brain both Bmals exhibit a robust rhythmic expression under LD conditions. The peak, however, is different for the two genes, Bmal1 reaching maximal expression at Zeitgeber time (ZT) 14, whereas Bmal2 peaks at ZT12. In addition, the kinetics of expression of the two genes differ, with the induction of Bmal2 being delayed but more rapid. The situation in the eye is similar, although the peak of Bmal1 expression is at ZT10-12 instead of ZT14 as in the brain. In situ hybridization analysis with zebrafish brains has revealed that the distribution of the two Bmal transcripts is very similar and that this distribution overlaps that of Clock. Bmals are expressed at high levels in the periventricular gray zone of the optic tectum, valvula cerebelli, corpus mammilare, and hypothalamus. Importantly, robust rhythmic expression of both genes is maintained when the animals are kept in a dark-dark regime, both in the brain and eye. The other clock structure, the pineal gland, also shows significant rhythmic expression of both genes, but Bmal1 expression appears to peak earlier than for Bmal2. When compared with the rhythmic pattern of Clock expression, it is evident that expression of the three genes is not synchronized in the brain and clock structures (Cermakian, 2000).

PAS (PER, ARNT, SIM) proteins play important roles in adaptation to low atmospheric and cellular oxygen levels, exposure to certain environmental pollutants, and diurnal oscillations in light and temperature. In an attempt to better understand how organisms sense environmental changes, a novel member of the PAS superfamily, MOP9 (member of PAS superfamily), that maps to human chromosome 12p11.22-11.23, has been characterized. This protein displays significant homology to the Drosophila circadian factor CYCLE and its putative mammalian ortholog MOP3/bMAL1. Like its homologs, MOP9 forms a transcriptionally active heterodimer with the circadian CLOCK protein, the structurally related MOP4, and hypoxia-inducible factors, such as HIF1alpha. In a manner consistent with its role as a biologically relevant partner of these proteins, MOP9 is coexpressed in regions of the brain such as the thalamus, hypothalamus, and amygdala. Importantly, MOP9 is coexpressed with CLOCK in the suprachiasmatic nucleus, the site of the master circadian oscillator in mammals (Hogenesch, 2000).

The mammalian genome encodes the originally identified CLOCK protein and a close homolog known as NPAS2 (also known as MOP4), as well as two close homologs of Drosophila CYCLE, MOP3, and MOP9. Circadian oscillations in mammalian physiology and behavior are regulated by an endogenous biological clock. Loss of the PAS protein MOP3 (also known as BMAL1) in mice results in immediate and complete loss of circadian rhythmicity in constant darkness. Additionally, locomotor activity in light-dark (LD) cycles is impaired and activity levels are reduced in Mop3-/- mice. Analysis of Period gene expression in the suprachiasmatic nucleus (SCN) indicates that these behavioral phenotypes arise from loss of circadian function at the molecular level. These results provide genetic evidence that MOP3 is the bona fide heterodimeric partner of mCLOCK. Furthermore, these data demonstrate that MOP3 is a nonredundant and essential component of the circadian pacemaker in mammals (Bunger, 2000).

MOP9 homolog is not functionally redundant with MOP3 but is dependent on MOP3 for circadian regulation. At the level of the circadian pacemaker, genetic evidence is provided that the MOP3 protein exerts its effects on the molecular feedback loop by acting as a positive regulator of gene expression. Given the effects of the dominant-negative Clock mutation, the effects of the Mop3 null allele on Per gene expression are consistent with an activator role of the CLOCK-MOP3 transcription factor complex. The observation that Mop3-/- mice do not entrain to a light cycle provides genetic evidence that MOP3 plays a functional role in the input pathway. In support of this idea, MOP3 (BMAL1) protein level displays a circadian rhythm in the SCN: it peaks at night and is reduced upon light stimulus. Finally, the critical role of MOP3 in the circadian output pathway is clearly supported by the arrhythmicity of transcriptional output gene mDBP in the liver of Mop3 mutant mice. Furthermore, Mop3-/- mice show decreased activity levels both in a light cycle and in constant darkness indicating that MOP3 may play a role in behavioral outputs beyond its role in generating behavioral rhythms (Bunger, 2000).

The effect of a null mutation of mMop3 on locomotor activity in constant dark conditions is similar to the phenotype seen in Cryptochrome 1 and 2 mutant mice. However, on light-dark cycles, Cry1-/-Cry2-/- double mutant mice do not show anticipatory activity as is seen in MOP3-/- mice. Furthermore, the Cry1-/-Cry2-/- double mutant mice also respond behaviorally to light pulses by immediately stopping activity at light onset and immediately starting activity at light offset. Such strong 'masking' effects of light are not observed in MOP3-/- mice (Bunger, 2000).

The effect of a Mop3 mutation on the level of activity is a novel phenotype that has not been reported in Clock, mPer2Brdm, and Cry1-/-Cry2-/- double mutant mice. Reduced activity has also been reported in mice with a targeted mutation at the dbp locus, a circadian regulated transcription factor that is controlled by MOP3. It is not clear from these results, however, whether the reduced activity phenotype in the Mop3-/- mice is caused by the loss of circadian rhythmicity or whether it may represent a novel role of MOP3 in regulation of metabolic or behavioral outputs. In conclusion, these data represent an example of a single-gene mutation that causes complete abolition of both behavioral and molecular circadian rhythms as well as disruptions in both input (entrainment) and output (activity level) pathways in mammals. Such widespread phenotypic consequences on the circadian system of mice argue that MOP3 rests near the top of the circadian gene hierarchy in mammals (Bunger, 2000).

A human cDNA encoding a novel member of the bHLH-PAS transcription factor superfamily, BMAL2, has been isolated that is highly similar to, but distinct from, BMAL1. The composite cDNA covers a 1720-bp sequence consisting of a putative 1653-bp open reading frame encoding a polypeptide of 551 amino acids. The deduced BMAL2 product contains a bHLH-PAS domain in its N-terminal region and a variable C-terminus. The overall identity of BMAL2 polypeptide to that of human BMAL1 is 49%. RNA analysis reveals that expression of BMAL2 transcripts is restricted to the fetal brain and to the adult liver in humans, while human BMAL1 mRNA is expressed in the brain and skeletal muscle. BMAL2 gene is localized on chromosome 12 at region p12.2-p11.2. These results suggest that BMAL2 may play different roles from BMAL1 in the embryonic brain and in adult mammals (Ikeda, 2000).

Recently discovered mammalian clock genes are believed to compose the core oscillator, which generates the circadian rhythm. BMAL1/CLOCK heterodimer is the essential positive element that drives clock-related transcription and self-sustaining oscillation by a negative feedback mechanism. BMAL1 protein expression was examined in the rat suprachiasmatic nuclei (SCN) by immunoblot analysis. Anti-BMAL1 antiserum raised against rBMAL1 recognizes 70 kDa mBMAL1b and detects a similar immunoreactivity (IR) as a major band in rat brains. Robust circadian BMAL1-IR oscillations with nocturnal peaks are detected in the SCN during a light/dark cycle and under constant darkness. A short duration light exposure at night acutely reduces BMAL1-IR in the SCN during photoentrainment. This might be attributable to the degradation of BMAL1 protein. Application of glutamate and NMDA to the SCN slices at projected night, a procedure mimicking photic phase delay shift, also acutely reduces BMAL1-IR in a similar manner. A rapid decrease of BMAL1 protein suggests that BMAL1 protein might be implicated in the light-transducing pathway within the SCN (Tamaru, 2000).

Circadian clock genes are expressed in the suprachiasmatic nucleus and in peripheral tissues to regulate cyclically physiological processes. Synchronization of peripheral oscillators is thought to involve humoral signals, but the mechanisms by which these are mediated and integrated are poorly understood. A hormone-dependent physical interaction of the nuclear receptors, RARalpha and RXRalpha, with CLOCK and the Cycle homolog MOP4 is reported. These interactions negatively regulate CLOCK/MOP4:BMAL1-mediated transcriptional activation of clock gene expression in vascular cells. MOP4 exhibits a robust rhythm in the vasculature, and retinoic acid can phase shift Per2 mRNA rhythmicity in vivo and in serum-induced smooth muscle cells in vitro, providing a molecular mechanism for hormonal control of clock gene expression. It is proposed that circadian or periodic availability of nuclear hormones may play a critical role in resetting a peripheral vascular clock (McNamara, 2001).

Clock:BMAL1 and NPAS2:BMAL1 are heterodimeric transcription factors that control gene expression as a function of the light-dark cycle. Although built to fluctuate at or near a 24-hour cycle, the clock can be entrained by light, activity, or food. The DNA-binding activity of the Clock:BMAL1 and NPAS2:BMAL1 heterodimers is regulated by the redox state of nicotinamide adenine dinucleotide (NAD) cofactors in a purified system. NAD(P)H can bind homologs of Clock and BMAL1, promoting their dimerization and DNA binding The reduced forms of the redox cofactors, NAD(H) and NADP(H), strongly enhance DNA binding of the Clock:BMAL1 and NPAS2:BMAL1 heterodimers, whereas the oxidized forms inhibit. These observations raise the possibility that food, neuronal activity, or both may entrain the circadian clock by direct modulation of cellular redox state (Rutter, 2001).

Mammalian CLOCK and BMAL1 are two members of bHLH-PAS-containing family of transcription factors that represent the positive elements of circadian autoregulatory feedback loop. In the form of a heterodimer, they drive transcription from E-box enhancer elements in the promoters of responsive genes. Abundance, posttranslational modifications, cellular localization of endogenous and ectopically expressed CLOCK and BMAL1 proteins have been examined. Nuclear/cytoplasm distribution of CLOCK was found to be under circadian regulation. Analysis of subcellular localization of CLOCK in embryo fibroblasts of mice carrying different germ-line circadian mutations show that circadian regulation of nuclear accumulation of CLOCK is BMAL1-dependent. Formation of CLOCK/BMAL1 complex following ectopic coexpression of both proteins is followed by their codependent phosphorylation, which is tightly coupled to CLOCK nuclear translocation and degradation. This binding-dependent coregulation is specific for CLOCK/BMAL1 interaction; no other PAS domain protein that can form a complex with either CLOCK or BMAL1 is able to induce similar effects. Importantly, all posttranslational events described in this study are coupled with active transactivation complex formation, which argues for their significant functional role. Altogether, these results provide evidence for an additional level of circadian system control, which is based on regulation of transcriptional activity or/and availability of CLOCK/BMAL1 complex (Kondratov, 2003).

These results suggest the novel function for one of the core clock components -- BMAL1 -- as a mediator of CLOCK nuclear accumulation. A hypothetical model of such regulation is outlined. Newly synthesized CLOCK protein is located predominantly in the cytoplasm, where it interacts with BMAL1 through the PAS-domain. This interaction by itself induces the cascade of posttranslational events potentially essential for regulating CLOCK/BMAL1 functional activities according to several possible scenarios. According to one of the options, the complex formation may generate a signal for phosphorylation of both partners, which occurs in the cytoplasm. This modification, in turn, triggers heterodimer nuclear translocation similar to previously described Shaggy-regulated PER/TIM nuclear translocation. After transactivation of responsive genes, one of the partners (CLOCK) is degraded, thus reducing nuclear concentration of active complex and preventing multiple rounds of activation (Kondratov, 2003).

The circadian clock is driven by cell-autonomous transcription/translation feedback loops. The BMAL1 transcription factor is an indispensable component of the positive arm of this molecular oscillator in mammals. A molecular genetic screening assay for mutant circadian clock proteins is presented that is based on real-time circadian rhythm monitoring in cultured fibroblasts. By using this assay, a domain was identified in the extreme C terminus of BMAL1 that plays an essential role in the rhythmic control of E-box-mediated circadian transcription. Remarkably, the last 43 aa of BMAL1 are required for transcriptional activation, as well as for association with the circadian transcriptional repressor Cryptochrome 1 (CRY1), depending on the coexistence of Clock protein. C-terminally truncated BMAL1 mutant proteins still associate with mPER2 (another protein of the negative feedback loop), suggesting that an additional repression mechanism may converge on the N terminus. Taken together, these results suggest that the C-terminal region of BMAL1 is involved in determining the balance between circadian transcriptional activation and suppression (Kiyohara, 2006).

The importance of histone methylation by the polycomb group proteins was examined in the mouse circadian clock mechanism. Endogenous EZH2, a polycomb group enzyme that methylates lysine 27 on histone H3, co-immunoprecipitates with CLOCK and BMAL1 throughout the circadian cycle in liver nuclear extracts. Chromatin immunoprecipitation revealed EZH2 binding and di- and tri-methylation of H3-K27 on both the Period 1 and Period 2 promoters. A role of EZH2 in cryptochrome-mediated transcriptional repression of the clockwork was supported by overexpression and RNA interference studies. Serum-induced circadian rhythms in NIH 3T3 cells in culture were disrupted by transfection of an RNA interfering sequence targeting EZH2. These results indicate that EZH2 is important for the maintenance of circadian rhythms and extend the activity of the polycomb group proteins to the core clockwork mechanism of mammals (Etchegaray, 2006).

CLOCK-mediated acetylation of BMAL1 controls circadian function

Regulation of circadian physiology relies on the interplay of interconnected transcriptional-translational feedback loops. The CLOCK-BMAL1 complex activates clock-controlled genes, including cryptochromes (Crys), the products of which act as repressors by interacting directly with CLOCK-BMAL1. CLOCK possesses intrinsic histone acetyltransferase activity and this enzymatic function contributes to chromatin-remodelling events implicated in circadian control of gene expression (Doi, 2006). This study shows that CLOCK also acetylates a non-histone substrate: its own partner, BMAL1, is specifically acetylated on a unique, highly conserved Lys 537 residue. BMAL1 undergoes rhythmic acetylation in mouse liver, with a timing that parallels the downregulation of circadian transcription of clock-controlled genes. BMAL1 acetylation facilitates recruitment of CRY1 to CLOCK-BMAL1, thereby promoting transcriptional repression. Importantly, ectopic expression of a K537R-mutated BMAL1 is not able to rescue circadian rhythmicity in a cellular model of peripheral clock. These findings reveal that the enzymatic interplay between two clock core components is crucial for the circadian machinery (Hirayama, 2007).

CLOCK and NPAS2 have overlapping roles in the suprachiasmatic circadian clock

Heterodimers of CLOCK and BMAL1, bHLH-PAS transcription factors, are believed to be the major transcriptional regulators of the circadian clock mechanism in mammals. However, a recent study shows that CLOCK-deficient mice continue to exhibit robust behavioral and molecular rhythms. This study reports that the transcription factor NPAS2 (MOP4) is able to functionally substitute for CLOCK in the master brain clock in mice to regulate circadian rhythmicity (DeBruyne, 2007).

Circadian rhythms regulate biological events like the timing of the sleep-wake cycle and are generated by a hierarchy of circadian clocks. Atop this hierarchy is a master clock that resides in the hypothalamic suprachiasmatic nuclei (SCN). The SCN clock is entrained by the daily light-dark cycle to the 24-h period by retina to SCN pathways, and it synchronizes the phase of circadian clocks in peripheral tissues that drive rhythmic changes in local physiology (DeBruyne, 2007).

The intracellular circadian clock mechanism in the mouse is regulated by transcriptional feedback loops that drive the self-sustaining clock mechanism in both the SCN and peripheral tissues. The critical molecular mechanism is thought to involve CLOCK:BMAL1 heterodimers that drive the rhythmic expression of three Period genes (mPer1-3) and two Cryptochrome genes (mCry1 and mCry2). The resulting proteins form PER:CRY complexes that translocate back into the nucleus to inhibit their own transcription, creating a negative feedback loop. A modulatory, interlocking positive transcriptional feedback loop involves the rhythmic regulation of Bmal1 transcription, through the coordinated actions of Rev-erbα (repressor) and Rora (activator), whose mRNA oscillations are antiphase to the mPer and mCry mRNA rhythms (DeBruyne, 2007).

Recent genetic evidence has shown that CLOCK is not essential for the circadian rhythm in locomotor activity in mice. However, compared with wild-type controls, CLOCK-deficient mice do have a slightly shortened circadian period in constant darkness and show altered circadian responses to light. Without CLOCK, molecular and biochemical rhythms are also altered, but most persist. Because BMAL1 is essential for the expression of circadian behavioral rhythms and homodimers are not transcriptionally active, an alternative dimerization partner for BMAL1 was sought (DeBruyne, 2007).

NPAS2 (also called MOP4) is a paralog of CLOCK that can dimerize with BMAL1 and appears to function in a clockwork mechanism in mouse forebrain. Its function in the SCN has been questioned, however, as previous studies were unable to detect Npas2 expression in the SCN. Homozygous Npas2-mutant mice (Npas2-/-), which do not express functional NPAS2, display robust circadian rhythms in locomotor behavior. Like CLOCK-deficient mice, Npas2-/- mice also have a slightly shortened circadian period and an altered response to perturbations in the light-dark cycle. These circadian phenotypes have been proposed to be a result of disrupted crosstalk between forebrain and SCN clocks, and not a result of NPAS2 deficiency in the SCN (DeBruyne, 2007).

To examine whether NPAS2 is the missing BMAL1 partner, CLOCK-deficient mice were generated carrying either one or no functional Npas2 alleles by interbreeding CLOCK-deficient mice with a previously generated null allele of Npas2. CLOCK-deficient animals carrying only one normal allele of Npas2 (Clock-/-; Npas2+/-) had substantially shorter circadian periods in constant darkness (22.7 h) compared with wild-type mice (23.8 h), with progressive rhythm instability. CLOCK-deficient mice with no functional NPAS2 (Clock-/-; Npas2-/-) exhibited arrhythmic locomotor behavior immediately on placement in constant darkness. These findings suggest that NPAS2 is the missing BMAL1 partner and that NPAS2 has a direct function in the SCN, the generator of rhythmic locomotor behavior. Notably, NPAS2 mutant mice carrying only one allele of Clock (Clock+/-; Npas2-/-) displayed behavioral rhythmicity in constant darkness that was similar to wild-type animals, suggesting that CLOCK may have a more prominent role than NPAS2 in the SCN clock (DeBruyne, 2007).

in situ hybridization was used to evaluate the status of the molecular clock in the SCN of the double mutant (Clock-/-; Npas2-/-) mice. It was found that the circadian rhythms of mPer1, mPer2, Rev-erbα and Bmal1 mRNA expressed in the SCN of wild-type mice were abolished in the double mutants. Consistent with the idea that the transcriptional drive of both the negative and positive feedback loops are disrupted in the double mutants, mPer1, mPer2 and Rev-erbα mRNA levels were at constant low levels over the circadian cycle, whereas Bmal1 mRNA levels were at constant high values, consistent with the idea that Bmal1 is repressed by REV-ERBα (DeBruyne, 2007).

Single knockout Npas2-/- mice displayed subtle alterations in the rhythmic expression of some genes in the SCN. For example, the mPer2 mRNA rhythm of Npas2-/- mice appeared to be slightly damped compared with wild-type mice. In addition, Bmal1 levels, although rhythmic, were increased throughout the circadian day in Npas2-/- mice, compared with wild types. The molecular defects in Npas2-/- SCN are more subtle than those observed in CLOCK-deficient mice, suggesting that CLOCK normally has a more prominent role than NPAS2 in controlling circadian gene expression (DeBruyne, 2007).

To further assess NPAS2 function in the SCN, CLOCK-deficient mice were generated carrying the mPer2Luciferase (mPer2Luc) allele. Mice carrying this allele at the mPer2 locus express a mPER2::LUC fusion protein, which allows real-time monitoring of circadian dynamics from tissue explants in culture. Using real-time reporting of bioluminescence from SCN explants, it was found that isolated SCN from CLOCK-deficient mice expressing the fusion protein (Clock-/-; mPer2Luc) still maintained self-sustained molecular oscillations in culture that were similar to those from wild-type SCN expressing the fusion protein (Clock+/+; mPer2Luc). These data suggest that NPAS2 maintains the SCN clockwork without CLOCK, independent of a major influence from other brain regions (DeBruyne, 2007).

To verify that Npas2 is actually expressed in the SCN of wild-type and CLOCK-deficient mice, a quantitative real-time PCR approach was used. The experiment clearly shows Npas2 expression in the SCN of both wild-type and Clock-/- mice (DeBruyne, 2007).

It is concluded that NPAS2 has a newly found, unexpected role in the SCN clock mechanism that controls circadian behavior. CLOCK and NPAS2 can independently heterodimerize with BMAL1 in the SCN to maintain molecular and behavioral rhythmicity. Whether NPAS2 normally functions to regulate circadian gene expression in the SCN of wild-type mice, or whether NPAS2 only has functionally relevant effects on gene expression in the absence of CLOCK, cannot be distinguished. Nevertheless, the results show that NPAS2 maintains circadian function in the absence of CLOCK. The differences in gene expression profiles between the Clock-/- and Npas2-/- single-knockouts suggests that different circadian promoters may have different affinities or requirements for CLOCK:BMAL1 versus NPAS2:BMAL1 heterodimers. Thus, NPAS2 may function coordinately with CLOCK in the SCN. These findings show a new level of transcriptional control in the SCN clockwork (DeBruyne, 2007).

HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability

The human homologue of the C. elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. It is concluded that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome (Collins, 2007).

Transcriptional targets of Clock

How might PER-dependent negative feedback inhibit or override per gene transcriptional activation by the CLOCK-BMAL1 heterodimer? One possibility is the PER-dependent sequestration of either CLOCK, BMAL1, or both, leading to a loss of CLOCK-BMAL1 DNA-binding activity, similar to the mechanism by which the Id protein inhibits myogenic differentiation. Other possibilities include a PER-dependent repressor that binds either to the E-box or to a nearby site with the per gene upstream region. It is not known if PER (including any other associated proteins) acts directly to achieve negative feedback, but the observation that coexpression of Drosophila PER and its partner TIM is sufficient to inhibit Drosophila Clock-dependent per gene activation in cultured cells suggests a direct mechanism (Gekakis, 1998).

The transcriptional regulation of the clock-controlled arginine vasopressin gene in the suprachiasmatic nuclei (SCN) has been studied. Vasopressin is synthesized and released in a circadian manner from neurons in the SCN. The SCN-derived peptide rhythm is driven by rhythmic transcription of the vasopressin gene. Peptide released from axons within the SCN acts locally through vasopressin-V1 receptors to modulate the amplitude of the SCN neuronal firing rate rhythm. Vasopressinergic efferent projections from the SCN also transmit circadian output signals to distant hypothalamic and extrahypothalamic regions. Vasopressin of SCN origin also appears to have a specific output function in driving rhythmic hypothalamo-pituitary-adrenal axis (HPA) activity. Vasopressin released from SCN terminals in the dorsomedial hypothalamus and subparaventricular zone has a pronounced inhibitory effect on the release of the adrenal hormone corticosterone. Vasopressin release might affect descending projections from brain stem and spinal cord, thereby decreasing splanchnic neural activity and corticosterone release. The vasopressin-mediated inhibition of corticosterone release is one aspect of a two-part regulatory mechanism controlling the corticosterone rhythm. The second mechanism involves a vasopressin-independent stimulatory input from the SCN to the HPA (Jin, 1999 and references).

A core clock mechanism in mouse SCN appears to involve a transcriptional feedback loop in which CLOCK and BMAL1 are positive regulators and three mPeriod (mPer) genes are involved in negative feedback. The RNA rhythm of each mPer gene is severely blunted in Clock/Clock mice. The vasopressin RNA rhythm is abolished in the SCN of Clock/Clock animals, leading to markedly decreased peptide levels. Luciferase reporter gene assays show that CLOCK-BMAL1 heterodimers act through an E box enhancer in the vasopressin gene to activate transcription; this activation can be inhibited by the mPER and mTIM proteins. Deletion of the protein dimerization PAS domain and cytoplasmic localization domain from mPER1 (amino acids 179-495) eliminates the inhibitory effect of mPER1 on CLOCK-BMAL1-mediated transcription. This finding suggests that mPER1-mediated inhibition is dependent on protein-protein interactions. These data indicate that the transcriptional machinery of the core clockwork directly regulates a clock-controlled output rhythm (Jin, 1999).

Transcript levels of DBP, a member of the PAR leucine zipper transcription factor family (see Drosophila Par-domain protein 1), exhibit a robust rhythm in suprachiasmatic nuclei, the mammalian circadian center. DBP is able to activate the promoter of a putative clock oscillating gene, mPer1, by directly binding to the mPer1 promoter. The mPer1 promoter is cooperatively activated by DBP and CLOCK-BMAL1. However, dbp transcription is activated by CLOCK-BMAL1 through E-boxes and inhibited by the mPER and mCRY proteins, as is the case for mPer1. Thus, a clock-controlled dbp gene may play an important role in central clock oscillation (Yamaguchi, 2000).

D-element binding protein (DBP), the founding member of the PAR family of basic leucine zipper (bZip) transcription factors, is expressed according to a robust daily rhythm in the suprachiasmatic nucleus and several peripheral tissues. Other members of this family include TEF (thyroid embryonic factor), its avian ortholog VBP (vitellogenin promoter-binding protein), and HLF (hepatocyte leukemia factor). All of these proteins share high amino acid sequence similarities within a amino-terminal activation domain, a PAR domain rich in proline and acidic amino acid residues, and a carboxy-terminal moiety encompassing the bZip region necessary for DNA binding and dimerization. In vitro all PAR bZip proteins avidly bind the consensus DNA recognition sequence 5'-RTTAYGTAAY-3' as homo- or hetero-dimers. In rat and mouse liver the expression of all three PAR bZip proteins is subject to strong circadian regulation, peak and trough levels being reached in the early evening and morning, respectively. In the case of Dbp the amplitude of circadian mRNA oscillation can largely account for the daily amplitude in protein oscillation. The mRNA accumulation oscillates not only in peripheral tissues such as liver, but also in neurons of the SCN, believed to harbor the central circadian pacemaker. Moreover, run-on experiments in isolated nuclei and physical mapping of nascent RNA chains suggest that circadian transcription plays a pivotal role in rhythmic DBP expression (Ripperger, 2000 and references therein).

Previous studies with mice that have been deleted for the Dbp gene have established that DBP participates in the regulation of several clock outputs, including locomotor activity, sleep distribution, and liver gene expression. Evidence that circadian Dbp transcription requires the basic helix-loop-helix-PAS protein CLOCK, an essential component of the negative-feedback circuitry generating circadian oscillations in mammals and fruit flies. Genetic and biochemical experiments suggest that CLOCK regulates Dbp expression by binding to E-box motifs within putative enhancer regions located in the first and second introns. Similar E-box motifs have been found previously in the promoter sequence of the murine clock gene mPeriod1. Hence, the same molecular mechanisms generating circadian oscillations in the expression of clock genes may directly control the rhythmic transcription of clock output regulators such as Dbp (Ripperger, 2000).

In mammals, circadian control of physiology and behavior is driven by a master pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Gene expression profiling has been used to identify cycling transcripts in the SCN and in the liver. To test whether the core circadian transcriptional activator Clock is involved in the regulation of most of the circadianly regulated output genes, gene expression of Clock mutant mice was examined at a time when Clock/MOP3 transcriptional activity is normally at its peak. The Clock mutant allele is an antimorph (a type of dominant-negative mutation) that antagonizes transcription induced by the CLOCK/MOP3 activator complex. This analysis revealed ~650 cycling transcripts and showed that the majority of these are specific to either the SCN or the liver. Genetic and genomic analysis suggests that a relatively small number of output genes are directly regulated by core oscillator components. Major processes regulated by the SCN and liver were found to be under circadian regulation. Importantly, rate-limiting steps in these various pathways are key sites of circadian control, highlighting the fundamental role that circadian clocks play in cellular and organismal physiology. In each tissue type examined, the major clusters of genes regulated by circadian rhythms participate in the principal functions of the organ, and many of these genes code form proteins that function in the rate-limiting steps in their respective pathways. The peak phases of expression of these key transcripts are also appropriately timed. A comparison of circadian transcription in mammals and flies suggests that circadian control of several key processes and pathways including heme biosynthesis (Alas1), cholesterol metabolism (HMGCoA lyase), neuropeptide signaling (Dbi), neuronal excitability (Kcnma1/slo), energy metabolism (hexokinase), and xenobiotic metabolism (glutathione-s-transferase and cytochrome p450s) have been conserved over more than 600 million years of evolution. The circadian control of transcription in higher organisms is integrated with the spatial control of gene expression to target rate-limiting steps in major pathways in their relevant organs, resulting in a systems-level temporal orchestration of behavior and physiology for optimal adaptation of the organism to its environment (Panda, 2002).

Mammalian circadian rhythms are generated by a feedback loop in which BMAL1 and CLOCK, players of the positive limb, activate transcription of the cryptochrome and period genes, components of the negative limb. Bmal1 and Per transcription cycles display nearly opposite phases and are thus governed by different mechanisms. The orphan nuclear receptor REV-ERBalpha is identified as the major regulator of cyclic Bmal1 transcription in the suprachiasmatic nuclei and in the liver. Circadian Rev-erbalpha expression is controlled by components of the general feedback loop. Thus, REV-ERBalpha constitutes a molecular link through which components of the negative limb drive antiphasic expression of components of the positive limb. While REV-ERBalpha influences the period length and affects the phase-shifting properties of the clock, it is not required for circadian rhythm generation (Preitner, 2002).

In previous studies, Bmal1 transcription has been postulated to be controlled by mechanisms that are the opposite of those involved in Cry1 and Per1/Per2 expression. Thus, circadian Bmal1 expression appears to be positively controlled by PER and CRY proteins, and transfection studies suggest that Bmal1 transcription may be negatively autoregulated by BMAL1 and CLOCK. The model proposed in this study offers a simple explanation for this regulatory circuit, by proposing that Rev-erbalpha expression is negatively regulated by PER and CRY proteins and positively regulated by BMAL1 and CLOCK. The cyclic accumulation of REV-ERBalpha then imposes circadian regulation on Bmal1 transcription. Several observations support this model. (1) Rev-erbalpha transcription appears to be positively regulated by CLOCK and BMAL1, the molecular targets of CRY/PER-mediated repression. Indeed, the Rev-erbalpha promoter contains three evolutionarily conserved E boxes between the major transcriptional start site and position -500, and in cotransfection assays, transcription from the Bmal1 promoter is activated by CLOCK and BMAL1. Furthermore Rev-erbalpha mRNA accumulates to constitutively low levels in homozygous Clock mutant mice. (2) The accumulation of Rev-erbalpha mRNA transcripts is lowest at times when PER2 protein reaches high nuclear levels. (3) Rev-erbalpha is constitutively expressed at intermediate levels in Per1Brdm1/Per2Brdm1 or Cry1/Cry2 mutant mice. Zenith levels of Rev-erbalpha transcripts are not expected in these mutant mice. Rev-erbalpha transcription itself is activated by CLOCK and BMAL1. At a high concentration, REV-ERBalpha is expected to extinguish the expression of its own activators, which in turn would result in diminished Rev-erbalpha transcription. Hence, Rev-erbalpha mRNA levels should be frozen at intermediate rather than maximal levels in Per1/Per2 and Cry1/Cry2 double mutant mice (Preitner, 2002).

Mechanisms similar to the one proposed here for the mouse oscillator might also apply for the circadian timing systems of the zebrafish and the fruit fly. Thus, in zebrafish, Rev-erbalpha (zfRev-erbalpha) also displays circadian expression with a phase opposite to that of zClock/zBmal1. Two interconnected feedback loops driving the nearly antiphasic circadian expression of the positive and negative limb components were first proposed in Drosophila. Apparently, the two feedback loops in Drosophila are also coupled by an indirect mechanism, in which PER and TIM downregulate the expression of a repressor that inhibits clk transcription in a circadian fashion (Preitner, 2002).

REV-ERBalpha belongs to the large family of transcription factors called 'orphan nuclear receptors' -- nuclear receptors for which no ligand has yet been found. REV-ERBalpha does not contain the ligand-dependent C-terminal activation domain AF2. Thus, in cotransfection studies with reporter genes carrying RORE sequences, REV-ERBalpha acts as a repressor rather than an activator. This repression might occur by multiple mechanisms. When two REV-ERBalpha molecules bind either to two closely spaced RORE sequences or to a direct repeat element with the sequence RGGTCANNRGGTCA (DR-2), they can bind the corepressor NCoR1. In turn, NCoR1 might recruit a histone deacetylase, which promotes the conversion of accessible into inaccessible chromatin. REV-ERBalpha might also inhibit transcription more directly by competing with transcriptional activators (e.g., the orphan receptors RORalpha, RORß, and/or RORalpha) for the occupancy of RORE sequences (Preitner, 2002).

The vasopressin gene is expressed in the suprachiasmatic nucleus where the basic helix-loop-helix (bHLH)-PAS factors CLOCK and MOP3 regulate circadian expression through interactions with E-box sequences. Vasopressin gene regulation by HIF-1alpha, a bHLH-PAS factor involved in responses to hypoxia, has been studied. By transfecting Neuro-2A cells with 5' flanking regions of the vasopressin gene driving a luciferase reporter, it has been shown that CLOCK and HIF-1alpha cooperate in the induction of expression from 1000 bp and 350 bp of the vasopressin promoter but do not activate a 120-bp promoter fragment. The region between -191 and -128 contains an E-box A that appears to be essential for HIF-1alpha/CLOCK-mediated transcriptional activity. However, gel-shift analysis shows that the cooperative effect of HIF-1alpha and CLOCK results in MOP3 binding, but does not involve heterodimerization of HIF-1alpha/CLOCK, at E-box A. These data indicate that cross-talk between mediators of hypoxic and circadian pathways can regulate target genes (Ghorbel, 2003).

In non-mammalian vertebrates, the pineal gland is photoreceptive and contains an intrinsic circadian oscillator that drives rhythmic production and secretion of melatonin. These features require an accurate spatiotemporal expression of an array of specific genes in the pineal gland. Among these is the arylalkylamine N-acetyltransferase, a key enzyme in the melatonin production pathway. In zebrafish, pineal specificity of zfaanat2 is determined by a region designated the pineal-restrictive downstream module (PRDM), which contains three photoreceptor conserved elements (PCEs) and an E-box, elements that are generally associated with photoreceptor-specific and rhythmic expression, respectively. By using in vivo and in vitro approaches, it has been found that the PCEs and E-box of the PRDM mediate a synergistic effect of the photoreceptor-specific homeobox OTX5 and rhythmically expressed clock protein heterodimer, BMAL/CLOCK, on zfaanat2 expression. Furthermore, the distance between the PCEs and the E-box was found to be critical for PRDM function, suggesting a possible physical feature of this synergistic interaction. OTX5-BMAL/CLOCK may act through this mechanism to simultaneously control pineal-specific and rhythmic expression of zfaanat2 and possibly also other pineal and retinal genes (Appelbaum, 2005).

Mammalian circadian rhythms are based on transcriptional and post-translational feedback loops. Essentially, the activity of the transcription factors BMAL1 (also known as MOP3) and CLOCK is rhythmically counterbalanced by Period (PER) and Cryptochrome (CRY) proteins to govern time of day-dependent gene expression. Circadian regulation of the mouse albumin D element-binding protein (Dbp) gene involves rhythmic binding of BMAL1 and CLOCK and marked daily chromatin transitions. Thus, the Dbp transcription cycle is paralleled by binding of BMAL1 and CLOCK to multiple extra- and intra-genic E boxes, acetylation of Lys9 of histone H3, trimethylation of Lys4 of histone H3 and a reduction of histone density. In contrast, the antiphasic daily repression cycle is accompanied by dimethylation of Lys9 of histone H3, the binding of heterochromatin protein 1alpha and an increase in histone density. The rhythmic conversion of transcriptionally permissive chromatin to facultative heterochromatin relies on the presence of functional BMAL1-CLOCK binding sites (Ripperger, 2006).

microRNA modulation of circadian-clock period and entrainment

microRNAs (miRNAs) are a class of small, noncoding RNAs that regulate the stability or translation of mRNA transcripts. Although recent work has implicated miRNAs in development and in disease, the expression and function of miRNAs in the adult mammalian nervous system have not been extensively characterized. This study examined the role of two brain-specific miRNAs, miR-219 and miR-132, in modulating the circadian clock located in the suprachiasmatic nucleus. miR-219 is a target of the CLOCK and BMAL1 complex, exhibits robust circadian rhythms of expression, and the in vivo knockdown of miR-219 lengthens the circadian period. miR-132 is induced by photic entrainment cues via a MAPK/CREB-dependent mechanism, modulates clock-gene expression, and attenuates the entraining effects of light. Collectively, these data reveal miRNAs as clock- and light-regulated genes and provide a mechanistic examination of their roles as effectors of pacemaker activity and entrainment (Cheng, 2007).

As a starting point to examine miRNA expression in the SCN, data from a genome-wide screening technique was used to identify CREB-regulated miRNAs. CREB was the initial focus because photic stimulation has been shown to elicit robust CRE-dependent gene expression in the SCN. For these studies, miR-132, a CREB-regulated miRNA that is induced by neurotrophins, was studied. First, the CREB Serial Analysis of Chromatin Occupancy (SACO) results were verified by using a combination of chromatin immunoprecipitation (ChIP) and real-time PCR. The miR-132 enhancer region was selectively immunoprecipitated by a CREB antibody but not IgG. The abundance of a bona fide CREB target, c-Fos, was analyzed as a positive control. The CREB ChIP did not enrich for a locus near the 18S ribosomal-RNA repeat or other negative control regions. Given the role of CREB as a light-inducible transcriptional factor within the SCN, these data raised the possibility that miR-132 expression might be activated by light (Cheng, 2007).

To discover miRNAs that are regulated by the E box-dependent core timing mechanism, miRNAs were screened by using a CLOCK ChIP. Interestingly, the enhancer region of miR-219-1 was significantly enriched in the CLOCK IP fraction, whereas that of miR-132 was not. The mperiod1 gene, a bona fide target of CLOCK- and BMAL1-mediated transcription, was used as the positive control, and 18S rRNA was used as the negative control. Notably, the miR-219-1 enhancer region was also enriched in the CREB antiserum IP fraction (Cheng, 2007).

Sequence alignment of pre-miR-132 across multiple species, including mouse, human, dog, zebrafish, and puffer fish indicates that the mature miR-132 sequence is highly conserved throughout evolution. With respect to the miR-219-1 gene, vertebrates possess multiple copies, and sequence alignment of pre-miR-219 from varied species shows a high degree of conservation. Zebrafish in situ hybridization has shown that miR-219 is enriched in the brain. As predicted by the ChIP assays, an E box motif (noncanonical) is found in the promoter region of the miR-219-1 gene and two consensus CRE motifs were also identified. These response elements are found in mice, rats, humans, and dogs (Cheng, 2007).

To determine whether the CLOCK and BMAL1 heterodimer regulates miR-219-1 expression, CLOCK and BMAL1 were expressed in PC12 cells and miR-219-1 expression was examined by semiquantitative reverse transcription (RT)-PCR and real-time (RT) PCR approaches. Overexpression of CLOCK and BMAL1 together resulted in a significant increase in pre-miR-219-1 transcript levels as determined by semiquantitative RT-PCR and real-time PCR, whereas expression of CLOCK or BMAL1 singly had limited effect. Both pre-miR-132 and gapdh levels were largely unaffected by CLOCK and BMAL1 coexpression (Cheng, 2007).

The role of CREB as a regulator of miR-132 transcript was examined. In primary cortical neurons, both forskolin (an adenylate cyclase agonist) and KCl depolarization induced expression of pre-miR-132 as assessed by RT-PCR. Expression of A-CREB, which blocks CREB-mediated transcription, suppressed forskolin- and KCl-induced pre-miR-132 expression (Cheng, 2007).

Cryptochrome and Clock

Two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop. In other words, mCRY1/mPER and mCRY2/mPER dimers inhibit CLOCK:BMAL1-mediated transcriptional activation, thus resulting in the negative regulation of PER and TIM in the nucleus. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1-mediated transcription. When mPER3 is coexpressed with either mPER1 or mPER2, mPER3 is dramatically redistributed from cytoplasm to become expressed in both cytoplasm and nucleus. mPER1 is more effective than mPER2 in promoting nuclear entry of mPER3; that is, nucleus-only location was found in 3× more cells with mPER1 cotransfections, as compared with mPER2. Despite trying all possible combinations of mPER proteins with mTIM, including adding all four proteins at once, a nucleus-only location of mPER1 or mPER2 could not be induced in greater than 30% of NIH3T3 cells. This differs dramatically from the in vivo situation in which both mPER1 and mPER2 are entirely nuclear in SCN cells when detectable. Thus, it would appear that mPER function cannot be fully reconstituted in NIH3T3 cells. Since mTim fails to drive mPER nuclear localization, it is concluded that the mTim function is not conserved between flies and mammals. This suggests that there are other clock-relevant factors important for the nuclear translocation of the mPER proteins (Kume, 1999).

This suggests cryptochrome might be involved in the negative limb of the feedback loop that results in the lowering of transcriptional levels of clock related genes. Indeed, mCry1 and mCry2 RNA levels are reduced in the central and peripheral clocks of Clock/Clock mutant mice. mCRY1 and mCRY2 are nuclear proteins that interact with each of the mPER proteins, translocate each mPER protein from cytoplasm to nucleus, and are rhythmically expressed in the suprachiasmatic circadian clock. Luciferase reporter gene assays show that mCRY1 or mCRY2 alone abrogates CLOCK:BMAL1-E box-mediated transcription. The mPER and mCRY proteins appear to inhibit the transcriptional complex differentially (Kume, 1999).

In addition to a potential direct inhibitory effect of the mCRY proteins on the CLOCK:BMAL1-E box complex, the cryptochromes could also inhibit transcription by directly interacting with the mPER proteins and translocating them to the nucleus for subsequent transcriptional effects. To evaluate the potential for protein-protein interactions between the mCRY and mPER families, coimmunoprecipitation using epitope-tagged proteins was carried out. COS7 cells cotransfected with expression plasmids encoding mCRY1-HA and either mPER1-V5, mPER2-V5, mPER3-V5, or mTIM-V5 expressed each V5-tagged protein prior to immunoprecipitation. Immunoprecipitation with the HA antibody and analysis of the immunoprecipitated material with anti-V5 antibodies indicates the presence of heterodimeric interactions between mCRY1 and each of the mPER and mTIM proteins. Coimmunoprecipitation experiments using mCRY2-HA instead of mCRY1-HA similarily show the presence of heterodimeric interactions between mCRY2 and each of the mPER and mTIM proteins. Having shown that mCRY:mPER heterodimers could exist, it was next determined whether such interactions translocate the mPER proteins to the nucleus. In marked contrast to the lack of effect of any pairwise combination of mPER:mPER or mPER:mTIM interactions to translocate mPER1 and mPER2 to the nucleus, each mCRY protein profoundly changes the location of all three mPER proteins in NIH3T3 and COS7 cells. This is most apparent for mPER1 and mPER2, which are almost entirely nuclear after cotransfection with either mCRY1 or mCRY2. Curiously, each mCRY protein changes mPER3 from mainly cytoplasm only (>80%) to both cytoplasm and nucleus (>70%) to a degree similar to that induced by cotransfection of mPER3 with mPER1. However, when mPER3 is cotransfected with mPER1 and either mCRY1 or mCRY2, each of the three protein combinations change mPER3's location from 13%-20% exclusively nuclear location to an exclusively nuclear location in a majority of cells (Kume, 1999).

These data indicate that the mCRY proteins can heterodimerize with the mPER proteins and mTIM and that mCRY:mPER interactions mimic the in vivo situation, that is, the almost complete translocation of mPER1 and mPER2 to the nucleus. Moreover, trimeric interactions among the mPER and mCRY proteins appear necessary for complete nuclear translocation of mPER3. The data also suggest that the nuclear translocation of the mPER proteins is dependent on mCRY1 and mCRY2. The mCRY proteins, however, appear to be able to translocate to the nucleus independent of the mPERs. Even with massive overexpression of mCRY proteins in cell culture, they are always greater than 90% nuclear. If a PER partner were required for CRY nuclear translocation, a high CRY:PER ratio should result in cytoplasmic trapping of CRY. This was not observed (Kume, 1999).

The discovery of the functions of mCRY1 and mCRY2 within the clock feedback loop provides a sharper view of the molecular working of the mammalian clock. The cloning of a family of three mPer genes over the past 2 years has added to understanding of the negative limb of a mammalian clock feedback loop. But close examination of these putative clock elements and mTim has shown that they alone cannot fully explain the negative limb of the feedback loop. It thus seemed likely that other factors were involved. mCRY1 and mCRY2 have been shown to be major players in the negative limb of the clock feedback loop. These data also explain the strong loss-of-function phenotype of mCry1-/-mCry2-/- mice (Kume, 1999).

The cell culture data show that the mCRY proteins function as dimeric and potentially trimeric partners for the mPER proteins and that these interactions lead to the nuclear translocation and/or retention of the mPER proteins. This is in marked contrast to the inability of mTIM to translocate the three mPER proteins to the nucleus in cell culture and the invariant nature of endogenous mTIM levels in the nuclei of SCN neurons; mTIM immunoreactivity is present in the nucleus of most SCN neurons at all times throughout the circadian cycle. Thus, the mCRY proteins appear to function as nuclear translocators of the mPERs. In addition, mCRY nuclear translocation does not appear to be dependent on mPER:mCRY interactions. This is different from the situation in the fly in which Per:Tim heterodimers appear essential for the translocation of both Per and Tim to the nucleus (Kume, 1999).

The role of mTIM in the mammalian clock remains enigmatic. Even though mTIM does not appear to be important for the nuclear translocation of the mPER proteins, mTIM is localized to the nucleus in vivo, and it does cause a modest inhibition of CLOCK:BMAL1- and MOP4:BMAL1-mediated transcription in cell culture. In addition, mCRY1 and mCRY2 each appear capable of forming heterodimeric complexes with mTIM. Once in the nucleus, mTIM could therefore still have a role in modulating negative feedback of the mPER and/or mCRY1 rhythms. Another observation from these studies is the finding that the mCry1 gene forms its own interacting loop within the collective mammalian clock feedback mechanism. Evidence for this contention is substantial. mCry1 RNA and protein levels exhibit a circadian rhythm in the SCN; the RNA rhythm is dependent on a functional CLOCK protein, and the mCry1 promoter region contains a functional CACGTG E box. In fact, it is entirely possible that the mCry1 rhythm is the dominant oscillation in the mammalian clock feedback loop. This might explain the dominant circadian function of the mCry1 gene over mCry2, whose RNA levels do not oscillate. One normal mCry1 allele sustains normal circadian rhythms in behavior, while one mCry2 allele leads to arrhythmicity with increasing time in constant darkness. It is not known precisely how the mPER and mCRY proteins inhibit CLOCK:BMAL1-mediated transcription, but the data suggest differential sites of action. In the fly, multimeric complexes involving Per, Tim, and Clock appear to be important. It is thus possible that the mPER proteins, mTIM, and the mCRY proteins are all complexed with CLOCK. In addition, mCRY1 and mCRY2 appear to be capable of inhibiting E box-mediated transcription independent of CLOCK. This suggests that the mammalian cryptochromes also interact directly with either BMAL1 or the E box itself. Indeed, mCRY1 can bind tightly to dsDNA Sepharose. Even though the major components of the loop have been identified, the way in which a 24 hr time constant is incorporated into the mammalian clock loop has not been elucidated. Based on studies in Drosophila, posttranslational processes such as phosphorylation, proteosomal proteolysis, and gated nuclear entry are likely to contribute to the time delay. It is not yet known which component(s) of the loop is affected by these processes (Kume, 1999 and references).

In summary, the data show that mCRY1 and mCRY2 are redundant but still essential components of the negative limb of the clock feedback loop. The redundant function of these proteins explains the maintenance of circadian rhythmicity when either gene is deleted and explains the strong arrhythmic phenotype of double knockout mice. The different direction of period change in mCry1-/- versus mCry2-/- mice may result from differing affinities of these proteins for the mPER proteins or other clock components, and/or different levels of protein expression. It is predicted that the SCN of mCry1-/-mCry2-/- animals will show disrupted mPer RNA and protein rhythms with the mPER proteins stuck in the cytoplasm and mPer RNA levels at constant high values because of the absence of negative feedback. Placing the mammalian cryptochromes in the negative limb of the clock feedback loop sets forth a number of new hypotheses that can now be tested (Kume, 1999 and references).

Cryptochrome (Cry), a photoreceptor for the circadian clock in Drosophila, binds to the clock component TIM in a light-dependent fashion and blocks its function. In mammals, genetic evidence suggests a role for Crys within the biological clock, distinct from hypothetical photoreceptor functions. Mammalian CRY1 and CRY2 have been shown to act as light-independent inhibitors of CLOCK-BMAL1, the activator driving Per1 transcription. CRY1 or CRY2 (or both) show light-independent interactions with CLOCK and BMAL1, as well as with PER1, PER2, and TIM. No systematic or substantive differences have been observed in the ability of hCRY1 or hCRY2 to inhibit CLOCK-BMAL1 in light and dark conditions. Thus, mammalian CRY proteins act as light-independent components of the circadian clock and probably regulate Per1 transcriptional cycling by contacting both the activator and its feedback inhibitors. The light-independent role of mammalian Cry proteins in circadian clock negative feedback contrasts sharply with that of Drosophila Cry, which has been demonstrated to function directly as a photoreceptor that regulates the action of the Per-Tim complex. It is suggested that Drosophila Cry exemplifies the ancestral role of a photoreceptor acting as a light-dependent regulator of the circadian feedback loop, whereas mammalian Crys have preserved a role within the circadian feedback loop but shed their direct photoreceptor function. The possibility cannot be excluded that mammalian Cry proteins act as photoreceptors for other possible functions, circadian or otherwise, not detected in these assays (Griffin, 1999).

In the mouse, the core mechanism for the master circadian clock consists of interacting positive and negative transcription and translation feedback loops. Analysis of Clock/Clock mutant mice, homozygous Period2Brdm1 mutants, and Cryptochrome-deficient mice reveals substantially altered Bmal1 rhythms, consistent with the dominant role of Period2 in the positive regulation of the Bmal1 loop. In vitro analysis of Cryptochrome inhibition of Clock:BMAL1 heterodimer-mediated transcription shows that the inhibition is through direct protein:protein interactions, independent of the Period and Timeless proteins. Period2 is a positive regulator of the Bmal1 loop, and Cryptochromes are the negative regulators of the Period and Cryptochrome cycles (Shearman, 2000).

Bmal1 mRNA rhythm has been documented in mouse SCN using in situ hybridization with an antisense riboprobe to the two major Bmal1 transcripts found in the SCN. Wild-type mice exhibit a robust circadian rhythm in Bmal1 RNA levels, with low levels from circadian time (CT) 6 to 9 and peak levels from CT 15 to 18. The phase of the Bmal1 rhythm is opposite that of the mPer1, mPer2, and mPer3 RNA rhythms. In addition to driving rhythmic transcription of the mPer and mCry genes, it seems possible that CLOCK:BMAL1 heterodimers might simultaneously negatively regulate Bmal1 gene expression, as proposed for Clk regulation in Drosophila. If CLOCK:BMAL1 heterodimers negatively regulate Bmal1 gene expression and if the mutant CLOCK protein is ineffective in this activity, then Bmal1 RNA levels should be elevated and less rhythmic in homozygous Clock mutant (Clk/Clk) mice. Compared with wild types, however, Clk/Clk animals express a severely dampened circadian rhythm of Bmal1 RNA levels in the SCN. Trough Bmal1 RNA levels do not differ between Clk/Clk mice and wild types. The peak level of the RNA rhythm in homozygous Clk mutant mice is only ~30% of the peak value in wild types (Shearman, 2000).

The temporal profile of Clk RNA levels in the SCN of Clk/Clk mutant animals was examined, because Clk RNA levels have been shown to be decreased in the eye and hypothalamus of Clk/Clk mutant mice. Clk RNA levels do not manifest a circadian oscillation in mouse SCN. Clk RNA levels in the SCN of Clk/Clk mutant mice are not significantly different from those in the SCN of wild-type animals. Thus, the Clk mutation appears to alter regulation of Bmal1 gene expression in SCN, but not the regulation of the Clk gene itself. Clk expression may be decreased in other hypothalamic regions (Shearman, 2000).

The low levels of Bmal1 RNA in the SCN of homozygous Clk mutant animals show that CLOCK is not required for the negative regulation of Bmal1. Instead, these data indicate that CLOCK is actually necessary for the positive regulation of Bmal1. The positive effect of CLOCK on Bmal1 levels is probably indirect and may occur through the mPER and/or mCRY proteins, which are expressed in the nucleus of SCN neurons at the appropriate circadian time to enhance Bmal1 gene expression. In addition, the mPer1, mPer2, mPer3, mCry1, and mCry2 RNA oscillations are all down-regulated in Clk/Clk mutant mice. Reduced levels of the protein products of one or more of these genes may lead to the reduced levels of Bmal1 in the mutant mice, through loss of a positive drive on Bmal1 transcription (Shearman, 2000).

Homozygous mPer2Brdm1 mutant animals have depressed mPer1 and mPer2 RNA rhythms. The Bmal1 rhythm was examined in homozygous mPer2Brdm1 mutants to determine whether the positive drive on the Bmal1 feedback loop might come from the mPER2 protein. The effects of this mutation on the mCry1 RNA rhythm were also examined. The temporal profiles of gene expression were analyzed at six time points over the first day in DD in homozygous mPer2Brdm1 mutant mice and wild-type littermates. The Bmal1 RNA rhythm in the SCN of wild-type animals is substantially different from that of mutant mice. Trough RNA levels do not differ between wild-type and mutant animals, but the increase in Bmal1 RNA levels is advanced and truncated in the mutants, compared with the wild-type rhythm (Shearman, 2000).

The mCry1 RNA rhythm is also significantly altered. In the SCN of mPer2Brdm1 mutant mice, the peak levels of the mCry1 RNA rhythm are suppressed by ~50%, as reported for mPer1 and mPer2 RNA rhythms in this mouse line. These data suggest that maintenance of a normal Bmal1 RNA rhythm is important for the positive transcriptional regulation of the mPer and mCry feedback loops. Thus, rhythmic Bmal1 RNA levels may drive rhythmic BMAL1 levels, which, in turn, regulate CLOCK:BMAL1-mediated transcriptional enhancement in the master clock. Indeed, mPer1, mPer2, and mCry1 RNA rhythms are all blunted in the SCN of mPer2Brdm1 mutant mice, in which the Bmal1 rhythm is also blunted. In addition, the homozygous mPer2Brdm1 mutation is associated with a shortened circadian period and ensuing arrhythmicity in DD (Shearman, 2000).

These data, along with the fact that Clk RNA levels are unaltered in the SCN of homozygous mPer2Brdm1 mutants, also provide evidence that mPER2 is a positive regulator of the Bmal1 RNA rhythm. This effect may be unique to mPER2. For example, the diurnal oscillation in mPer2 RNA is not altered in the SCN of mPer1-deficient mice, and mPer1, mPer2, and Bmal1 RNA circadian rhythms are not altered in the SCN of mPer3-deficient mice. Moreover, circadian rhythms in behavior are sustained in mice deficient in either mPer1 or mPer3 (Shearman, 2000).

There are at least two ways that the mPer2Brdm1 mutation could alter the positive drive of the clock feedback loops. The mutation could disrupt mPER:mCRY interactions important for the synchronous oscillations of their nuclear localization and/or alter the protein's ability to interact with other proteins (e.g., transcription factors). Experiments show that functional mPER2:mCRY interactions are not mediated through the PAS domain. Similarly, the PAS domain is not important for the mCRY-mediated nuclear translocation of mPER1 in COS-7 cells (Shearman, 2000).

Because the mPER:mCRY interactions necessary for nuclear transport occur outside the PAS region, the PAS domain of an mPER2:mCRY heterodimer might be free to bind to an activator (e.g., transcription factor) and shuttle it into the nucleus to activate Bmal1 transcription. Alternatively, once in the nucleus, mPER2:mCRY heterodimers or mPER2 monomers could coactivate Bmal1 transcription through a PAS-mediated interaction with a transcription factor. mPER2 itself does not have a DNA-binding motif (Shearman, 2000).

The tonic mid-to-high mPer1 and mPer2 RNA levels in mCry-deficient mice suggest that CLOCK:BMAL1 heterodimers might be constantly driving mPer1 and mPer2 gene expression in the absence of transcriptional inhibition by the mCRY proteins. To examine whether Bmal1 RNA levels would also be modestly elevated, Bmal1 RNA levels in the SCN of mCry-deficient mice were compared with those in the SCN of wild-type mice of the same genetic background at CT 6 and at CT 18. Clk RNA levels were also examined in these animals. In wild-type animals, the typical circadian variation in Bmal1 RNA levels is apparent with high levels at CT 18 and low levels at CT 6. In contrast, inmCry-deficient mice, Bmal1 RNA levels are low at both circadian times. It is suggested that Clk RNA levels do not differ as a function of circadian time or genotype (Shearman, 2000).

The unexpected low Bmal1 gene expression in the SCN of mCry-deficient mice suggests that the Bmal1 feedback loop is disrupted in the mutant animals, with a resultant nonfunctional circadian clock. Nevertheless, enough Bmal1 gene expression and protein synthesis occurs for heterodimerization with CLOCK so that, without the strong negative feedback normally exerted by the mCRY proteins, mPer1 and mPer2 gene expression is driven sufficiently by the heterodimer to give intermediate to high RNA values (depending on RNA stability) (Shearman, 2000).

The mid-to-high mPer1 and mPer2 RNA levels in the SCN of mCry-deficient mice and simultaneous low Bmal1 levels suggest that mPER1 and mPER2 proteins may not be exerting much positive or negative influence on the core feedback loops. To test this, it had to be determined whether mPER1 and mPER2 are tonically expressed in the nuclei of SCN cells in mCry-deficient mice, because nuclear location appears necessary for action on transcription. mPER1 immunoreactivity exhibits a robust rhythm of nuclear staining in the SCN of wild-type mice, with high values at CT 12 and significantly lower values at CT 24 (Shearman, 2000). In mCry-deficient mice, however, mPER1 immunoreactivity is detected in the nucleus of a similar number of SCN neurons at each of the two circadian times (CT 12 and CT 24), and the values at each time were at ~40% of those seen at peak (CT 12) in wild-type animals (Shearman, 2000).

The double mCry mutation also alters the subcellular distribution of mPER1 staining in the SCN. In wild-type mice, mPER1 staining viewed under contrast interference is nuclear with a very condensed immunoreaction and a clear nucleolus. The neuropil of the SCN in wild types is devoid of mPER1 immunoreactivity. In the SCN of mCry-deficient animals, mPER1 staining is nuclear, but the nuclear profiles are less well defined and less intensely stained: perinuclear, cytoplasmic immunoreaction can be observed. In addition, the neuropil staining for mPER1 is higher in mCry-deficient mice, although dendritic profiles are not discernible. In the same brains, the constitutive nuclear staining for mPER1 normally seen in the piriform cortex is not altered in mCry-deficient animals (Shearman, 2000).

mPER2 immunoreactivity also exhibits a robust rhythm of nuclear staining in the SCN of wild-type mice, with high values at CT 12 and significantly lower counts at CT 24. In contrast, the pattern of mPER2 immunoreactivity in the SCN of mCry-deficient mice is markedly altered, with few mPER2 immunoreactive cells in the SCN of mCry-deficient animals at either circadian time (CT 12 or CT 24) (Shearman, 2000).

In the wild-type mice, the mPER2 staining profiles are nuclear, with well-defined outlines and nucleoli devoid of reaction product. In the few mPER2 immunoreactive cells in the SCN of mCry-deficient mice, low-level mPER2 staining is observed in the nucleus, but the profiles are poorly defined and low-intensity perinuclear staining can also be observed. As for mPER1, genotype has no discernible effect on nuclear mPER2 immunoreactivity in the piriform cortex, although there is evidence of a low level of perinuclear immunoreactivity for mPER2 in piriform cortex of mCry-deficient mice. The marked reduction of mPER2 staining in the SCN of mCry-deficient animals suggests that the mCRY proteins are either directly or indirectly important for mPER2 stability, because mPer2 RNA levels are at tonic intermediate to high levels in mCry-deficient mice, similar to those found for mPer1 RNA levels. The low levels of mPER2 immunoreactivity in the SCN of mCry-deficient mice, in conjunction with tonically low Bmal1 RNA levels, are consistent with an important role of mPER2 in the positive regulation of the Bmal1 loop. Because mPER1 is present in SCN nuclei in mCry-deficient mice, yet Bmal1 RNA is low, mPER1 likely has little effect on the positive regulation of the Bmal1 feedback loop or negative regulation of the mPer1, mPer2, and mPer3 cycles (Shearman, 2000).

mPER1 and mPER2 can each enter the nucleus even in the absence of mCRY:mPER interactions. mPER1 is expressed in the nucleus of SCN neurons from mCry-deficient mice, and both mPER1 and mPER2 are constitutively expressed in the nucleus of cells in the piriform cortex of mCry-deficient animals. The phosphorylation state of mPER1 dictates its cellular location in the absence of mPER1:mCRY interactions, because its phosphorylation by casein kinase I epsilon leads to cytoplasmic retention in vitro. Thus, the nuclear location of both mPER1 and mPER2 in vivo may depend on several factors, including interactions with mCRY and other proteins and their phosphorylation (Shearman, 2000).

The intermediate to high levels of mPer1 and mPer2 gene expression throughout the circadian day in mCry-deficient mice are consistent with a prominent role of the mCRY proteins in negatively regulating CLOCK:BMAL1-mediated transcription. When cotransfected, mouse (m)CLOCK and syrian hamster (sh)BMAL1 heterodimers induce a large increase in transcriptional activity (1744-fold) that is reduced by >90% by mCRY1 or mCRY2. The mCLOCK: shBMAL1- and hMOP4:shBMAL1-induced transcription in S2 cells is dependent on an intact CACGTG E box, because neither heterodimer causes an increase in transcription when a mutated E box reporter is used in the transcriptional assay. Immunofluorescence of epitope-tagged mCRY1 or mCRY2 expressed in S2 cells shows that each is >90% nuclear in location, as in mammalian cells (Shearman, 2000).

These data indicate that mCRY1 and mCRY2 are nuclear proteins that can each inhibit mCLOCK:shBMAL1-induced transcription independent of the mPER and mTIM proteins and of each other. The results also show that the inhibitory effect is not mediated by the interaction of either mCRY1 or mCRY2 with the E box itself, because E box-mediated transcription is not blocked by the mCRY proteins when transcription is activated by dCLOCK:CYC heterodimers. It thus appears that the mCRY proteins inhibit mCLOCK: shBMAL1-mediated transcription by interacting with either or both of the transcription factors, because a similar inhibition is found with hMOP4:shBMAL1-induced transcription. Yeast two-hybrid assays have revealed strong interactions of each mCRY protein with mCLOCK and shBMAL1. Weaker interactions have been detected between each mCRY protein and hMOP4. This is further evidence of functionally relevant associations of each mCRY protein with each of the three transcription factors. Attempts were made to determine whether the mCRY-induced inhibition of transcription is through interaction with CLOCK and/or BMAL1. mCRY inhibits mCLOCK:shBMAL1-induced transcription through interaction with either mCLOCK alone or through an association with both mCLOCK and BMAL1 in a multiprotein complex (Shearman, 2000).

A working model of the SCN clockwork proposes three types of interacting molecular loops. The mCry genes comprise one loop, with true autoregulatory, negative feedback, in which the protein products feed back to turn off their own transcription. The second loop is that manifested by each of the mPer genes and some clock controlled output genes (CCGs: for example, vasopressin prepropressophysin). This loop type is driven by the same positive elements (CLOCK:BMAL1) as the mCry loop, but transcription is not turned off by the respective gene products. Instead, the mCRY protein acts as a negative regulator, leaving the protein products free for other actions. Thus, mPER2 positively drives transcription of the Bmal1 gene, mPER1 may function to stabilize protein components of the loop, and CCG products (which might include mPER3) function as output signals. The rhythmic regulation of Bmal1 comprises the third loop with rhythmicity controlled by the cycling presence and absence of a positive element dependent on mPER2. This positive feedback loop augments the positive regulation of the first two loops (Shearman, 2000).

This model of interacting loops proposes that at the start of the circadian day, mPer and mCry transcription are driven by accumulating CLOCK:BMAL1 heterodimers acting through E box enhancers. After a delay, the mPER and mCRY proteins are synchronously expressed in the nucleus where the mCRY proteins shut off CLOCK:BMAL1-mediated transcription by directly interacting with these transcription factors. At the same time that the mCRY proteins are inhibiting CLOCK:BMAL1-mediated transcription, mPER2 either shuttles a transcriptional activator into the nucleus or coactivates a transcriptional complex to enhance Bmal1 transcription. The importance of the Bmal1 RNA rhythm is to drive a BMAL1 rhythm after a 4- to 6-hour delay. This delay in the protein rhythm would provide increasingly available CLOCK:BMAL1 heterodimers at the appropriate circadian time to drive mPer/mCry transcription, thereby restarting the cycle. It is thus predicted that BMAL1 availability is rate limiting for heterodimer formation and critical for restarting the loops. Delineating factors that regulate clock protein stability and interactions (phosphorylation and proteolysis) are important next steps for defining how a 24-hour time constant is built into the clockwork (Shearman, 2000).

Zebrafish tissues and cells have the unusual feature of not only containing a circadian clock, but also being directly light-responsive. Several zebrafish genes are induced by light, but little is known about their role in clock resetting or the mechanism by which this might occur. This study shows that Cryptochrome 1a (Cry1a) plays a key role in light entrainment of the zebrafish clock. Intensity and phase response curves reveal a strong correlation between light induction of Cry1a and clock resetting. Overexpression studies show that Cry1a acts as a potent repressor of clock function and mimics the effect of constant light to 'stop' the circadian oscillator. Yeast two-hybrid analysis demonstrates that the Cry1a protein interacts directly with specific regions of core clock components, CLOCK and BMAL, blocking their ability to fully dimerize and transactivate downstream targets, providing a likely mechanism for clock resetting. A comparison of entrainment of zebrafish cells to complete versus skeleton photoperiods reveals that clock phase is identical under these two conditions. However, the amplitude of the core clock oscillation is much higher on a complete photoperiod, as are the levels of light-induced Cry1a. It is believed that Cry1a acts on the core clock machinery in both a continuous and discrete fashion, leading not only to entrainment, but also to the establishment of a high-amplitude rhythm and even stopping of the clock under long photoperiods (Tamai, 2007).

Cryptochrome domain structure

Nuclear entry of circadian oscillatory gene products is a key step for the generation of a 24-hr cycle of the biological clock. Nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchronous clock in cultured cells, have been examined. mCRY1 and mCRY2 have been shown to complex with PER proteins leading to nuclear import. Nuclear translocation of mPER1 and mPER2 (1) involves physical interactions with mPER3; (2) is accelerated by serum treatment, and (3) still occurs in mCry1/mCry2 double-deficient cells lacking a functional biological clock. Moreover, nuclear localization of endogenous mPER1 is observed in cultured mCry1/mCry2 double-deficient cells as well as in the liver and the suprachiasmatic nuclei (SCN) of mCry1/mCry2 double-mutant mice. This indicates that nuclear translocation of at least mPER1 also can occur under physiological conditions (i.e., in the intact mouse) in the absence of any CRY protein. The mPER3 amino acid sequence predicts the presence of a cytoplasmic localization domain (CLD) and a nuclear localization signal (NLS). Deletion analysis suggests that the interplay of the CLD and NLS proposed to regulate nuclear entry of PER in Drosophila is conserved in mammals, but with the novel twist that mPER3 can act as the dimerizing partner (Yagita, 2000).

Mouse mCRY1 and zebrafish zCRY1a and zCRY3 belong to the DNA photolyase/Cryptochrome family. mCRY1 and zCRY1a repress CLOCK:BMAL1-mediated transcription, whereas zCRY3 does not. Reciprocal chimeras between zCRY1a and zCRY3 were generated to determine the zCRY1a regions responsible for nuclear translocation, interaction with the CLOCK:BMAL1 heterodimer, and repression of CLOCK:BMAL1-mediated transcription. Three regions, RD-2a-(126-196), RD-1-(197-263), and RD-2b-(264-293), were identified. Proteins in this family consist of an N-terminal alpha/beta domain and a C-terminal helical domain connected by an interdomain loop. RD-2a is within this loop, RD-1 is at the N-terminal 50 amino acids, and RD-2b at the following 31 amino acid residues of the helical domain. Either RD-2a or RD-1 is required for interaction with the CLOCK: BMAL1 heterodimer, and either RD-1 or RD-2b is required for the nuclear translocation of CRY. Both of these functions are prerequisites for the transcriptional repressor activity. The functional nuclear localizing signal in the RD-2b region also was identified. The sequence is well conserved among repressor-type CRYs, including mCRY1. Mutations in the nuclear localizing signal of mCRY1 reduce the extent of its nuclear localization. These findings show that both nuclear localization and interaction with the CLOCK:BMAL heterodimer are essential for transcriptional repression by CRY (Hirayama, 2003).

Circadian rhythms are driven by molecular clocks composed of interlocking transcription/translation feedback loops. Cryptochrome proteins are critical components of these clocks and repress the activity of the transcription factor heterodimer CLOCK/BMAL1. Unlike the homologous DNA repair enzyme 6-4 PHOTOLYASE, Cryptochromes have extended carboxyl-terminal tails and cannot repair DNA damage. Unlike mammals, Xenopus laevis contains both Cryptochromes (xCRYs) and 6-4 PHOTOLYASE (xPHOTOLYASE), providing an excellent comparative tool to study Cry repressive function. Findings can be extended to CRYs in general because xCRYs share high sequence homology with mammalian CRYs. Deletion of xCRYs' C-terminal domain produces proteins that are, like xPHOTOLYASE, unable to suppress CLOCK/BMAL1 activation. However, these truncations also cause the proteins to be cytoplasmically localized. A heterologous nuclear localization signal (NLS) restores the truncation mutants' nuclear localization and repressive activity. These results demonstrate that the CRYs' C termini are essential for nuclear localization but not necessary for the suppression of CLOCK/BMAL1 activation; this finding indicates that these two functions reside in separable domains. Furthermore, the functional differences between CRYs and PHOTOLYASE can be attributed to the few amino acid changes in the conserved portions of these proteins (Zhu, 2003).

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. A molecular genetic screen was developed in mammalian cells to identify mutants of the circadian transcriptional activators Clock and BMAL1, which were uncoupled from Cryptochrome-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of Clock and the C terminus of BMAL1 result in synergistic insensitivity through reduced physical interactions with Cry. Coexpression of these mutant proteins in cultured fibroblasts causes arrhythmic phenotypes in population and single-cell assays. These data demonstrate that Cry-mediated repression of the Clock/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function (Sato, 2006).

Circadian clock-controlled regulation of cGMP-protein kinase G

Circadian clocks comprise a cyclic series of dynamic cellular states, characterized by the changing availability of substrates that alter clock time when activated. To determine whether circadian clocks, like the cell cycle, exhibit regulation by key phosphorylation events, endogenous kinase regulation of timekeeping was examined in the mammalian suprachiasmatic nucleus (SCN). Short-term inhibition of PKG-II (see Drosophila Foraging) but not PKG-Ibeta using antisense oligodeoxynucleotides delayed rhythms of electrical activity and Bmal1 mRNA. Phase resetting was rapid and dynamic; inhibition of PKG-II forced repetition of the last 3.5 hr of the cycle. Chronic inhibition of PKG-II disrupted electrical activity rhythms and tonically increased Bmal1 mRNA. PKG-II-like immunoreactivity was detected after coimmunoprecipitation with CLOCK, and CLOCK is phosphorylated in the presence of active PKG-II. PKG-II activation may define a critical control point for temporal progression into the daytime domain by acting on the positive arm of the transcriptional/translational feedback loop (Tischkau, 2004).

Signaling mediated by the dopamine D2 receptor potentiates circadian regulation by CLOCK:BMAL1

Environmental cues modulate a variety of intracellular pathways whose signaling is integrated by the molecular mechanism that constitutes the circadian clock. Although the essential gears of the circadian machinery have been elucidated, very little is known about the signaling systems regulating it. Signaling mediated by the dopamine D2 receptor (D2R) enhances the transcriptional capacity of the CLOCK:BMAL1 complex. This effect involves the mitogen-activated protein kinase transduction cascade and is associated with a D2R-induced increase in the recruiting and phosphorylation of the transcriptional coactivator cAMP-responsive element-binding protein (CREB) binding protein. Importantly, CLOCK:BMAL1-dependent activation and light-inducibility of mPer1 gene transcription is drastically dampened in retinas of D2R-null mice. Because dopamine is the major catecholamine in the retina, central for the neural adaptation to light, these findings establish a physiological link among photic input, dopamine signaling, and the molecular clock machinery (Yujnovsky, 2006).

Pacemaker function of Clock positive cells

A brain pacemaker driving circadian rhythms in mammals resides in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. The SCN senses changes in ambient lighting through both direct and indirect retina-to-SCN neural pathways. In this way, light synchronizes (entrains) the SCN and its driven output rhythms to the 24 hr day. An important feature of SCN organization is that it is a multioscillatory system with the entire clockwork residing in single neurons. Individual neurons dissociated from rat suprachiasmatic nucleus have been shown to express independently phased circadian firing rhythms. Interactions among these "clock cells" in the whole SCN serve to synchronize individual circadian clocks to generate coordinated circadian outputs. These outputs ultimately control a vast array of circadian rhythms in physiology and behavior (Jin, 1999 and references).

It is of interest to determine if the visual phototransduction cascade plays a role in light entrainment of photoreceptor circadian oscillators. mRNA levels of iodopsin and the chicken homolog of Clock (cClock) were compared in the retinas of normal and rd (retinal degeneration) chickens that lack functional rod and cone phototransduction cascades. Iodopsin is a circadian-regulated, photoreceptor-specific gene expressed in chicken retina, and Clock is a transcription factor that has been shown to play a role in the circadian clock mechanism in mouse and Drosophila. cClock and iodopsin transcript levels undergo daily oscillations in retinas of normal animals housed under 12 h light:12 h dark (12L:12D) conditions, and these oscillations are maintained in the absence of light. Levels of these transcripts in the retinas of rd/rd chickens housed under cyclic light conditions do not change significantly over the course of a 12L:12D cycle; however, there is evidence that the photoreceptor oscillators are entrained in these animals. Comparisons of normal and rd/rd data suggest that there are at least two light entrainment pathways that impinge on the oscillators found in photoreceptor cells, one of which is effectively disabled by the GC1 null mutation carried by the rd chicken (Larkin, 1999).

The Clock mutation lengthens periodicity and reduces amplitude of circadian rhythms in mice. The effects of Clock are cell intrinsic and can be observed at the level of single neurons in the suprachiasmatic nucleus. To address how cells of contrasting genotype functionally interact in vivo to control circadian behavior, a series of Clock mutant mouse aggregation chimeras have been analyzed. Circadian behavior in Clock/Clock/wild-type chimeric individuals was determined by the proportion of mutant versus normal cells. Significantly, a number of intermediate phenotypes, including Clock/+ phenocopies and novel combinations of the parental behavioral characteristics, were seen in balanced chimeras. Multivariate statistical techniques were used to quantitatively analyze relationships among circadian period, amplitude, and suprachiasmatic nucleus composition. Together, these results demonstrate that complex integration of cellular phenotypes determines the generation and expression of coherent circadian rhythms at the level of the organism (Low-Zeddies, 2001).

In Clock/Clock chimeras, both WT and Clock mutant cells are capable of influencing circadian behavior. This was made evident by the representation among chimeras of both component-strain phenotypes. That the component cellular genotypes could jointly influence circadian behavior is shown in the incidence of intermediate and novel mixed phenotypic profiles. A phenotypic gradient across this series of chimeras reflects an incremental dissection of the effects of Clock mutant cell dosage on circadian behavior. A majority of either WT or mutant cells is required to dominate whole-animal behavior. That circadian behavior is generally representative of SCN composition as a whole contrasts with the dynamics of other rhythmic cellular networks like the myogenic pacemaker, in which the fastest cell sets the heart rate. Populations of Clock/Clock and Clock/+ chimeras collectively contain equivalent numbers of WT cells -- that Clock/Clock chimeras produce a greater range of mutant severity than Clock/+ chimeras indicates that it is not simply the number of WT SCN cells in a chimera that determines its circadian behavior. Instead, in chimeras, Clock mutant cells play an active role in lengthening the period and reducing the amplitude of the overt behavioral rhythm. Further, the difference between Clock/Clock and Clock/+ chimeras demonstrates a dosage effect of mutant alleles on circadian cellular physiology in the intact animal (Low-Zeddies, 2001).

The effects of the Clock mutation on circadian period, amplitude, and phase shifts did not necessarily covary in Clock chimeras. In particular, principal components analyses indicate that period and amplitude largely vary independently. These observations support the idea that the circadian clock comprises separable functional units, and suggest that different sets of cells may be primary determinants of the period and amplitude of circadian behavioral rhythms. Phase-shifting behavior in chimeras is not reliably predicted by prior circadian period or amplitude, suggesting that phase shifts are not determined by the same complements of cells as those that determine period and amplitude. This may reflect an effect of Clock on cells on the light input pathway, and/or on a set of pacemaker cells that are responsive to light. Possible effects of the Clock mutation on tissues extrinsic to the SCN that influence the overt rhythm of activity cannot be ruled out, although in constant daylight it is expected that extra pacemaker influences would be minimal. Although Clock is expressed in tissues throughout the body, pleiotropic effects of the Clock mutation are not readily apparent. It is imagined that the specific distribution of WT versus mutant cells in each chimera determine which aspects of the mutant phenotype it expresses. In the search for 'essential' SCN pacemaker cells, which by definition determine properties of period, phase, and amplitude, many studies have been interpreted as indicating equipotentiality rather than localization of function. Might this be because the tissue substrate mediating these properties is diffuse? The analyses carried out in this study have indicated that the foci for different properties of circadian behavior may be spatially separated in the SCN (Low-Zeddies, 2001).

Clock and melatonin rhythmicity

Xenopus laevis retinas, like retinas from all vertebrate classes, have endogenous circadian clocks that control many aspects of normal retinal physiology occurring in cells throughout all layers of the retina. The localization of the clock(s) that controls these various rhythms remains unclear. One of the best studied rhythmic events is the nocturnal release of melatonin. Photoreceptor layers can synthesize rhythmic melatonin when these cells are in isolation. However, within the intact retina, melatonin is controlled in a complex way, indicating that signals from many parts of the retina may contribute to the production of melatonin rhythmicity. To test this hypothesis, transgenic tadpoles were generated that express different levels of a dominant negative Xenopus CLOCK specifically in the retinal photoreceptors. Eyes from these tadpoles continue to produce melatonin at normal levels, but with greatly disrupted rhythmicity, the severity of which correlated with the transgene expression level. These results demonstrate that although many things contribute to melatonin production in vivo, the circadian clock localized in the retinal photoreceptors is necessary for its rhythmicity. Furthermore, these data show that the control of the level of melatonin synthesis is separable from the control of its rhythmicity and may be controlled by different molecular machinery. This type of specific 'molecular lesion' allows perturbation of the clock in intact tissues and is valuable for dissection of clock control of tissue-level processes in this and other complex systems (Hayasaka, 2002).

This study raises the question of whether the circadian clock controlling melatonin release rhythms is localized either in rods or cones or in both photoreceptor cell types. In Xenopus eyes, all of the clock gene homologs identified thus far are expressed in both rod and cone photoreceptor cells, implying that both cell types have a circadian oscillator(s). Another interesting issue is whether the circadian clock that controls rhythmic melatonin expression regulates other outputs as well. Many physiological rhythms are under the control of the ocular clock(s), although it is not clear whether one circadian oscillator in the photoreceptor cells directs the many different rhythms in the retina, or whether more than one oscillator, located in different retinal cell types, controls different circadian processes (Hayasaka, 2002).

Developmental role for Clock

The circadian cycle is a simple, universal molecular mechanism for imposing cyclical control on cellular processes. The regulation of one of the key circadian genes, Clock, was examined in early Xenopus development. Xclk expression is found initially in the organizer region and overlying ectoderm, coincident with the neural inducer noggin. Further, noggin can induce Xclk expression in ectodermal explants along with markers of neural plate, including the cell adhesion molecule NCAM. Interestingly, NCAM is required for photic resetting of the circadian clock in mice; a mutation in NCAM that blocks its conjugation to polysialic acid results in the gradual running down of the circadian cycle in the SCN. The expression of Clock is dependent on developmental stage, not on time per se, and is mostly restricted to the anterior neural plate. It's expression can be induced by the secreted polypeptide noggin, and subsequently upregulated by Otx2, a transcription factor required for the determination of anterior fate (Green, 2001).

Clock: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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