Molecular components of the circadian clock in mammals.

The circadian clock mechanism in animals involves a transcriptional feedback loop in which the bHLH-PAS proteins CLOCK and BMAL1 form a transcriptional activator complex to activate the transcription of the Period and Cryptochrome genes, which in turn feed back to repress their own transcription. In the mouse liver, CLOCK and BMAL1 interact with the regulatory regions of thousands of genes, which are both cyclically and constitutively expressed. The circadian transcription in the liver is clustered in phase and this is accompanied by circadian occupancy of RNA polymerase II recruitment and initiation. These changes also lead to circadian fluctuations in histone H3 lysine4 trimethylation (H3K4me3) as well as H3 lysine9 acetylation (H3K9ac) and H3 lysine27 acetylation (H3K27ac). Thus, the circadian clock regulates global transcriptional poise and chromatin state by regulation of RNA polymerase II.

Depressive-like behavior and stress reactivity are independent traits in a Wistar Kyoto x Fisher 344 cross.

Depression is a heritable disorder that is often precipitated by stress. Abnormalities of the stress-reactive hypothalamic-pituitary-adrenal (HPA) axis are also common in depressed patients. In animal models, the forced swim test (FST) is the most frequently used test of depressive-like behavior. We have used a proposed animal model of depression, the Wistar Kyoto (WKY) rat, to investigate the relationship as well as the mode of inheritance of FST behaviors and HPA measures. Through reciprocal breeding of WKY and F344 parent strains and brother-sister breeding of the F1 generation, we obtained 486 F2 animals. Parent, F1 and F2 animals were tested in the FST. Blood samples were collected for determination of basal and stress (10-min restraint) plasma corticosterone (CORT) levels, and adrenal weights were measured. We found that all measures were heritable to some extent and that this heritability was highly sex dependent. Both correlation and factor analyses of the F2 generation data demonstrate that FST behavior and HPA axis measures are not directly related. Thus, the underlying genetic components of depressive-like behavior and HPA axis abnormalities are likely to be disparate in the segregating F2 generation of a WKY x F344 cross.

Overview of circadian rhythms.

The daily light-dark cycle governs rhythmic changes in the behavior and/or physiology of most species. Studies have found that these changes are governed by a biological clock, which in mammals is located in two brain areas called the suprachiasmatic nuclei. The circadian cycles established by this clock occur throughout nature and have a period of approximately 24 hours. In addition, these circadian cycles can be synchronized to external time signals but also can persist in the absence of such signals. Studies have found that the internal clock consists of an array of genes and the protein products they encode, which regulate various physiological processes throughout the body. Disruptions of the biological rhythms can impair the health and well-being of the organism.

Stopping time: the genetics of fly and mouse circadian clocks.

Forward genetic analyses in flies and mice have uncovered conserved transcriptional feedback loops at the heart of circadian pacemakers. Conserved mechanisms of posttranslational regulation, most notably phosphorylation, appear to be important for timing feedback. Transcript analyses have indicated that circadian clocks are not restricted to neurons but are found in several tissues. Comparisons between flies and mice highlight important differences in molecular circuitry and circadian organization. Future studies of pacemaker mechanisms and their control of physiology and behavior will likely continue to rely on forward genetics.

Familial advanced sleep phase syndrome.

BACKGROUND: The circadian rhythms of sleep propensity and melatonin secretion are regulated by a central circadian clock, the suprachiasmatic nucleus of the hypothalamus. The most common types of sleep disorders attributed to an alteration of the circadian clock system are the sleep/wake cycle phase disorders, such as delayed sleep phase syndrome and advanced sleep phase syndrome (ASPS). Advanced sleep phase syndrome is characterized by the complaint of persistent early evening sleep onset and early morning awakening. Although the complaint of awakening earlier than desired is relatively common, particularly in older adults, extreme advance of sleep phase is rare. OBJECTIVE: To phenotypically characterize a familial case of ASPS. METHODS: We identified a large family with ASPS; 32 members of this family gave informed consent to participate in this study. Measures of sleep onset and offset, dim light melatonin onset, the Horne-Ostberg morningness-eveningness questionnaire, and clinical interviews were used to characterize family members as affected or unaffected with ASPS. RESULTS: Affected members rated themselves as "morning types" and had a significant advance in the phase of sleep onset (P<.001) and offset (P =.006) times. The mean sleep onset was 2121 hours for the affected family members and 0025 hours for the unaffected family members. The mean sleep offset was 0507 hours for the affected members and 0828 hours for the unaffected members. (Times are given in military form.) In addition, the phase of the circadian rhythm of melatonin onset for the affected family members was on average 3-1/2 hours earlier than for the unaffected members. CONCLUSIONS: The ASPS trait segregates with an autosomal dominant mode of inheritance. The occurrence of familial ASPS indicates that human circadian rhythms, similar to those in animals, are under genetic regulation. Genetic analysis of familial sleep and circadian rhythm disorders is important for identifying a specific gene(s) responsible for the regulation of sleep and circadian rhythms in humans.

Genome-wide epistatic interaction analysis reveals complex genetic determinants of circadian behavior in mice.

Genetic heterogeneity underlies many phenotypic variations observed in circadian rhythmicity. Continuous distributions in measures of circadian behavior observed among multiple inbred strains of mice suggest that the inherent contributions to variability are polygenic in nature. To identify genetic loci that underlie this complex behavior, we have carried out a genome-wide complex trait analysis in 196 (C57BL/6J X BALB/cJ)F(2) hybrid mice. We have characterized variation in this panel of F(2) mice among five circadian phenotypes: free-running circadian period, phase angle of entrainment, amplitude of the circadian rhythm, circadian activity level, and dissociation of rhythmicity. Our genetic analyses of these phenotypes have led to the identification of 14 loci having significant effects on this behavior, including significant main effect loci that contribute to three of these phenotypic measures: period, phase, and amplitude. We describe an additional locus detection method, genome-wide genetic interaction analysis, developed to identify locus pairs that may interact epistatically to significantly affect phenotype. Using this analysis, we identified two additional pairs of loci that have significant effects on dissociation and activity level; we also detected interaction effects in loci contributing to differences of period, phase, and amplitude. Although single gene mutations can affect circadian rhythms, the analysis of interstrain variants demonstrates that significant genetic complexity underlies this behavior. Importantly, most of the loci that we have detected by these methods map to locations that differ from the nine known clock genes, indicating the presence of additional clock-relevant genes in the mammalian circadian system. These data demonstrate the analytical value of both genome-wide complex trait and epistatic interaction analyses in further understanding complex phenotypes, and point to promising approaches for genetic analysis of such phenotypes in other mammals, including humans.

Chimera analysis of the Clock mutation in mice shows that complex cellular integration determines circadian behavior.

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, we have analyzed a series of Clock mutant mouse aggregation chimeras. 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, our results demonstrate that complex integration of cellular phenotypes determines the generation and expression of coherent circadian rhythms at the organismal level.

The mouse Clock locus: sequence and comparative analysis of 204 kb from mouse chromosome 5.

The Clock gene encodes a basic helix-loop-helix (bHLH)-PAS transcription factor that regulates circadian rhythms in mice. We previously cloned Clock in mouse and human using a battery of behavioral and molecular techniques, including shotgun sequencing of two bacterial artificial chromosome (BAC) clones. Here we report the finished sequence of a 204-kb region from mouse chromosome 5. This region contains the complete loci for the Clock and Tpardl (pFT27) genes, as well as the 3' partial locus of the Neuromedin U gene; sequence analysis also suggests the presence of two previously unidentified genes. In addition, we provide a comparative genomic sequence analysis with the syntenic region from human chromosome 4. Finally, a new BAC transgenic line indicates that the genomic region that is sufficient for rescue of the Clock mutant phenotype is no greater than 120 kb and tightly flanks the 3' end of the Clock gene.

Genetics of the mammalian circadian system: Photic entrainment, circadian pacemaker mechanisms, and posttranslational regulation.

During the past four years, significant progress has been made in identifying the molecular components of the mammalian circadian clock system. An autoregulatory transcriptional feedback loop similar to that described in Drosophila appears to form the core circadian rhythm generating mechanism in mammals. Two basic helix-loop-helix (bHLH) PAS (PER-ARNT-SIM) transcription factors, CLOCK and BMAL1, form the positive elements of the system and drive transcription of three Period and two Cryptochrome genes. The protein products of these genes are components of a negative feedback complex that inhibits CLOCK and BMAL1 to close the circadian loop. In this review, we focus on three aspects of the circadian story in mammals: the genetics of the photic entrainment pathway; the molecular components of the circadian pacemaker in the hypothalamic suprachiasmatic nucleus; and the role of posttranslational regulation of circadian elements. A molecular description of the mammalian circadian system has revealed that circadian oscillations may be a fundamental property of many cells in the body and that a circadian hierarchy underlies the temporal organization of animals.

The circadian clock mutation alters sleep homeostasis in the mouse.

The onset and duration of sleep are thought to be primarily under the control of a homeostatic mechanism affected by previous periods of wake and sleep and a circadian timing mechanism that partitions wake and sleep into different portions of the day and night. The mouse Clock mutation induces pronounced changes in overall circadian organization. We sought to determine whether this genetic disruption of circadian timing would affect sleep homeostasis. The Clock mutation affected a number of sleep parameters during entrainment to a 12 hr light/dark (LD 12:12) cycle, when animals were free-running in constant darkness (DD), and during recovery from 6 hr of sleep deprivation in LD 12:12. In particular, in LD 12:12, heterozygous and homozygous Clock mutants slept, respectively, approximately 1 and approximately 2 hr less than wild-type mice, and they had 25 and 51% smaller increases in rapid eye movement (REM) sleep during 24 hr recovery, respectively, than wild-type mice. The effects of the mutation on sleep are not readily attributable to differential entrainment to LD 12:12 because the baseline sleep differences between genotypes were also present when animals were free-running in DD. These results indicate that genetic alterations of the circadian clock system and/or its regulatory genes are likely to have widespread effects on a variety of sleep and wake parameters, including the homeostatic regulation of sleep.

The basic helix-loop-helix-PAS protein MOP9 is a brain-specific heterodimeric partner of circadian and hypoxia factors.

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, we have characterized a novel member of the PAS superfamily, MOP9 (member of PAS superfamily), that maps to human chromosome 12p11.22-11.23. 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.

Molecular genetics of circadian rhythms in mammals.

Recent gene discovery approaches have led to a new era in our understanding of the molecular basis of circadian oscillators in animals. A conserved set of genes in Drosophila and mammals (Clock, Bmal1, Period, and Timeless) provide a molecular framework for the circadian mechanism. These genes define a transcription-translation-based negative autoregulatory feedback loop that comprises the core elements generating circadian rhythmicity. This circadian core provides a focal point for understanding how circadian rhythms arise, how environmental inputs entrain the oscillatory system, and how the circadian system regulates its outputs. The addition of molecular genetic approaches to the existing physiological understanding of the mammalian circadian system provides new opportunities for understanding this basic life process.

Locomotor response to an open field during C57BL/6J active and inactive phases: differences dependent on conditions of illumination.

Time of day has proven to be a source of variability in diverse behavioral measures. Knowledge of the pattern of this temporal effect as well as its origin (exogenous or endogenous) is essential for a precise description of any behavior. This study analyzed the effect of the external light-dark cycle and the internal rest-activity rhythm on the response of C57BL/6J mice to a novel environment. In a first experiment, animals maintained in a 12:12-h light-dark cycle were tested in an open field at six different times of day. A diurnal rhythm of ambulation in the open field was observed with greater levels of activity exhibited by those groups tested at night. Long-term and short-term behavioral habituation to spatial novelty were also affected by phase of the light-dark cycle. A second experiment was designed to control for any direct effect of the light-dark cycle by keeping the animals in dim green light where entrainment was maintained by a skeleton photoperiod (two 15-min bright-light pulses separated by 12 hours of green, dim light). This second group of animals was tested at two different circadian phases under the same conditions of illumination. One group was tested during the subjective night and another group during the subjective day, i.e., 2 or 14 h after the onset of the active phase, as assessed by wheel-running behavior. No effect of circadian phase on ambulation or habituation of this response to the open field was observed in these animals. Taken together, these results suggest that spatial novelty is equally arousing regardless of circadian phase and that the conditions of illumination can dramatically alter the response to a novel environment.

Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau.

The tau mutation is a semidominant autosomal allele that dramatically shortens period length of circadian rhythms in Syrian hamsters. We report the molecular identification of the tau locus using genetically directed representational difference analysis to define a region of conserved synteny in hamsters with both the mouse and human genomes. The tau locus is encoded by casein kinase I epsilon (CKIepsilon), a homolog of the Drosophila circadian gene double-time. In vitro expression and functional studies of wild-type and tau mutant CKIepsilon enzyme reveal that the mutant enzyme has a markedly reduced maximal velocity and autophosphorylation state. In addition, in vitro CKIepsilon can interact with mammalian PERIOD proteins, and the mutant enzyme is deficient in its ability to phosphorylate PERIOD. We conclude that tau is an allele of hamster CKIepsilon and propose a mechanism by which the mutation leads to the observed aberrant circadian phenotype in mutant animals.

Nonphotic phase-shifting in clock mutant mice.

Nonphotic phase-shifting was studied in mice bearing the Clock mutation. First, free-running mice heterozygous for Clock and wild-type mice were induced to become active through a 4-h confinement to a novel running over 3 days. Second, mice exposed to light-dark cycle received daily hypocaloric food during 2 weeks, before being transferred to constant darkness and fed ad libitum. Behavioral activation during the mid-subjective day induced 40-min phase advances in the locomotor activity rhythm of wild-type mice, whereas it produced 50-min phase delays in the circadian behavior of Clock/+ mice. Calorie restriction phase-advanced by 80 min the locomotor activity rhythm in wild-type mice, but not in Clock/+ mice. Therefore, the response of the Clock/+ mice to nonphotic phase shifting differs from that of wild-type mice.

The Xenopus clock gene is constitutively expressed in retinal photoreceptors.

Many aspects of normal retinal physiology are controlled by a retinal circadian clock. In Xenopus laevis, the photoreceptor cells within the retina contain a circadian clock that controls melatonin release. In this report we present the cloning and characterization of the Xenopus homolog of the Clock gene, known to be critical for normal circadian behavioral rhythms in the mouse. The Xenopus Clock gene is expressed primarily in photoreceptors within the eye and is expressed at constant levels throughout the day. Analysis of other tissues revealed that, as in other species, the Xenopus Clock gene is widely expressed. This characterization of the Clock gene provides a useful tool for further exploration of the role of the circadian clock in normal retinal function.

Mop3 is an essential component of the master circadian pacemaker in mammals.

Circadian oscillations in mammalian physiology and behavior are regulated by an endogenous biological clock. Here we show that 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.

Integration and saturation within the circadian photic entrainment pathway of hamsters.

The sensitivity of the visual pathway that subserves circadian entrainment was measured in hamsters after prior stimulation and using trains of multiple pulses. Immediately after subsaturating stimulation in the late subjective night, there was a significant decrease in responsiveness that persisted for at least 1 h. The reduced responsiveness was not due to light adaptation (shifting of the stimulus-response curve) but rather to response saturation, which appeared to reduce the sensitivity to subsequent stimulation and limit the maximum response of the pacemaker. The system, therefore, integrates the total number of photons delivered in two light stimuli separated in time by up to 1 h. The responsiveness was also measured using stimulus trains containing 10-1,000 individual pulses of equal irradiance and equal total photons. Results suggest that this pathway is responsive to the total photons delivered in all of the stimuli and is not responsive to light onsets or offsets associated with individual stimuli. These data outline several fundamental characteristics of phase shifting for the circadian photic entrainment pathway in hamsters. Knowledge of these characteristics is important for designing and interpreting results of future studies to dissect the cellular and molecular nature of the mammalian circadian clock and for understanding how visual information affects the cellular clock during entrainment.