Posttranscriptional regulation of mammalian circadian clock output.

Circadian clocks are present in many different cell types/tissues and control many aspects of physiology. This broad control is exerted, at least in part, by the circadian regulation of many genes, resulting in rhythmic expression patterns of 5-10% of the mRNAs in a given tissue. Although transcriptional regulation is certainly involved in this process, it is becoming clear that posttranscriptional mechanisms also have important roles in producing the appropriate rhythmic expression profiles. In this chapter, we review the available data about posttranscriptional regulation of circadian gene expression and highlight the potential role of Nocturnin (Noc) in such processes. NOC is a deadenylase-a ribonuclease that specifically removes poly(A) tails from mRNAs-that is expressed widely in the mouse with high-amplitude rhythmicity. Deadenylation affects the stability and translational properties of mRNAs. Mice lacking the Noc gene have metabolic defects including a resistance to diet-induced obesity, decreased fat storage, changes in lipid-related gene expression profiles in the liver, and altered glucose and insulin sensitivities. These findings suggest that NOC has a pivotal role downstream from the circadian clockwork in the post-transcriptional regulation genes involved in the circadian control of metabolism.

Molecular control of Xenopus retinal circadian rhythms.

Vertebrate retinas contain endogenous circadian clocks that control many aspects of retinal physiology. Our work has focused on studying the molecular mechanism of this clock and the way in which it controls the many cellular rhythms within the retina. These studies focus on the retina of Xenopus laevis, a well-established model system extensively used for the study of both retinal physiology and circadian function. We have cloned Xenopus homologues of the genes thought to be critical for vertebrate clock function, including Clock, Bmal1, cryptochromes and period, as well as other rhythmic genes such as nocturnin. We have used these genes to manipulate the clock within different subsets of retinal photoreceptors via cell-specific promoters, in order to study the location of the clock within the retina. These in vivo experiments have shown that photoreceptor cells contain clocks that are necessary for the rhythmic production of melatonin. We have also used biochemical approaches to further investigate the molecular events that drive specific rhythmic outputs, such as circadian regulation of nocturnin gene transcription and control of post-transcriptional events within these clock-containing cells.

A putative flavin electron transport pathway is differentially utilized in Xenopus CRY1 and CRY2.

Xenopus laevis cryptochromes (xCRYs) can suppress xCLOCK/xBMAL1-mediated activation of a period E box-containing promoter. This suppression is a crucial part of the vertebrate circadian oscillator. Similar to CRYs in other species, as well as to the closely related photolyases, xCRYs have a conserved flavin binding domain. We show here that an intact flavin binding domain is required for normal function. However, it appears that each xCRY may utilize the bound flavin differently. Mutation in any of the three conserved tryptophan residues in the putative electron transport chain inhibits xCRY2b function, while only the mutation in the last of the three tryptophans significantly affects xCRY1 function. Although knockout studies in mice have suggested that CRY1 and CRY2 are not totally redundant, this is the first time that molecular/biochemical differences between CRY1 and CRY2 have been demonstrated. Both CRYs seem to require an intact flavin binding domain, suggesting that electron transport is important in their ability to suppress CLOCK/BMAL1 activation. However, only xCRY2b appears to depend on electron transport through the conserved tryptophan pathway.

Genetic control of susceptibility to experimental Lyme arthritis is polygenic and exhibits consistent linkage to multiple loci on chromosome 5 in four independent mouse crosses.

C3H/He mice infected with Borrelia burgdorferi develop severe arthritis and are high antibody responders, while infected C57BL/6 and BALB/c mice develop mild arthritis and less robust humoral responses. Genetic analysis using composite interval mapping (CIM) on reciprocal backcross populations derived from C3H/HeN and C57BL/6N or C3H/HeJ and BALB/cAnN mice identified 12 new quantitative trait loci (QTL) linked to 10 murine Lyme disease phenotypes. These QTL reside on chromosomes 1, 2, 4, 6, 7, 9, 10, 12, 14, 15, 16, and 17. A reanalysis of an F(2) intercross between C57BL/6N and C3H/HeN mice using CIM identified two new QTL on chromosomes 4 and 15 and confirmed the location of seven previously identified loci. Two or more experimental crosses independently verified six QTL controlling phenotypes after B. burgdorferi infection. Additionally, Bb2 on chromosome 5 was reproduced in four experimental populations and was linked to the candidate locus Cora1. Evidence of four distinct QTL residing within the 30-cM region of chromosome 5 encompassing the previously mapped Bb2 and Bb3 loci was shown by CIM. Interestingly, some alleles contributing to susceptibility to Lyme arthritis were derived from C57BL/6N and BALB/cAnN mice, showing that disease-resistant strains harbor susceptibility alleles.

Three cryptochromes are rhythmically expressed in Xenopus laevis retinal photoreceptors.

PURPOSE: To clone Xenopus laevis cryptochromes (crys) and to understand their role in the Xenopus retinal clock. METHODS: We designed degenerate PCR primers based on homology between mouse and human crys. DNA fragments generated from these PCR reactions were used to screen a Xenopus retinal cDNA library. Three independent clones were identified and sequenced. The temporal and spatial expression of these genes in retina were studied by Northern blot analysis and in situ hybridization. RESULTS: We cloned three cry homologs from Xenopus laevis retina. We named them xcry1, xcry2a, and xcry2b based on their high homology to the mouse crys. Sequence analysis shows that these Xenopus CRYs have more than 85% identity to mouse CRYs at the amino acid level. Northern blot analysis demonstrated that all three xcrys are rhythmically expressed in the retina with peaks at different times of the day. The xcrys are expressed in a variety of tissues. In retina, they are expressed predominantly in photoreceptor cells. CONCLUSIONS: Our finding of cry expression in Xenopus photoreceptor cells further supports the idea of independent circadian oscillators being present in these cells. The sequence similarities to mouse crys suggest similar functions in the circadian clock. However, their distinct temporal expression patterns suggest some unique role for xCRY in the Xenopus retina.

Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse.

BACKGROUND: Nocturnin was originally identified by differential display as a circadian clock regulated gene with high expression at night in photoreceptors of the African clawed frog, Xenopus laevis. Although encoding a novel protein, the nocturnin cDNA had strong sequence similarity with a C-terminal domain of the yeast transcription factor CCR4, and with mouse and human ESTs. Since its original identification others have cloned mouse and human homologues of nocturnin/CCR4, and we have cloned a full-length cDNA from mouse retina, along with partial cDNAs from human, cow and chicken. The goal of this study was to determine the temporal pattern of nocturnin mRNA expression in multiple tissues of the mouse. RESULTS: cDNA sequence analysis revealed a high degree of conservation among vertebrate nocturnin/CCR4 homologues along with a possible homologue in Drosophila. Northern analysis of mRNA in C3H/He and C57/Bl6 mice revealed that the mNoc gene is expressed in a broad range of tissues, with greatest abundance in liver, kidney and testis. mNoc is also expressed in multiple brain regions including suprachiasmatic nucleus and pineal gland. Furthermore, mNoc exhibits circadian rhythmicity of mRNA abundance with peak levels at the time of light offset in the retina, spleen, heart, kidney and liver. CONCLUSION: The widespread expression and rhythmicity of mNoc mRNA parallels the widespread expression of other circadian clock genes in mammalian tissues, and suggests that nocturnin plays an important role in clock function or as a circadian clock effector.

A novel promoter element, photoreceptor conserved element II, directs photoreceptor-specific expression of nocturnin in Xenopus laevis.

Nocturnin is a vertebrate circadian clock-regulated gene, and in Xenopus laevis its mRNA is specifically expressed in retinal photoreceptor cells. We have investigated the transcriptional regulatory mechanism that drives this precise spatial expression pattern of the nocturnin gene. A deletion series of the nocturnin 5'-flanking sequence driving the green fluorescence protein (GFP) reporter was used to generate transgenic Xenopus tadpoles. We found that a construct containing 2.6 kilobase pairs of 5'-flanking sequence targeted high level GFP reporter expression specifically to photoreceptor cells, in a pattern identical to endogenous nocturnin. This photoreceptor-specific expression pattern was maintained with several further deletions of 5'-upstream sequence, including a short 59-base pair fragment. Within this region of 59 base pairs, three perfect repeats of a novel protein binding site were identified by electrophoretic mobility shift assay. Competitions using varying oligonucleotide sequences demonstrated that the sequence required for protein binding is CAGACAGGCTATA, designated photoreceptor-conserved element II (PCE II). The protein complex that binds to this element is enriched in retinal extracts, and mutations of PCE II which fail to bind the protein complex also fail to direct GFP reporter expression to photoreceptors. These results indicate that the PCE II in the proximal promoter of the nocturnin gene is sufficient for driving the photoreceptor-specific expression of nocturnin.

The circadian gene Clock is restricted to the anterior neural plate early in development and is regulated by the neural inducer noggin and the transcription factor Otx2.

The circadian cycle is a simple, universal molecular mechanism for imposing cyclical control on cellular processes. Here we have examined the regulation of one of the key circadian genes, Clock, in early Xenopus development. We find that 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.

Symphony of rhythms in the Xenopus laevis retina.

The photoreceptor layer in the retina of Xenopus laevis harbors a circadian clock. Many molecular components known to drive the molecular clock in other organisms have been identified in Xenopus, such as XClock, Xper2, and Xcrys, demonstrating phylogenetic conservation. This model system displays a wide array of rhythms, including melatonin release, ERG rhythms, and retinomotor movements, suggesting that the ocular clock is important for proper retinal function. A flow-through culture system allows measurements of retinal rhythms such as melatonin release in vitro over time from a single eyecup. This system is suited for pharmacological perturbations of the clock, and has led to important observations regarding the circadian control of melatonin release, the roles of light and dopamine as entraining agents, and the circadian mechanisms regulating retinomotor movements. The development of a transgenic technique in Xenopus allows precise and reliable molecular perturbations. Since it is possible to follow rhythms in eyecups obtained from adults or tadpoles, the combination of the flow-through culture system and the transgenic technique leads to the fast generation of transgenic tadpoles to monitor the effects of molecular perturbations on the clock.

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.

Ontogeny of circadian and light regulation of melatonin release in Xenopus laevis embryos.

The retinal photoreceptors of Xenopus laevis contain a circadian clock that controls the synthesis and release of melatonin, resulting in high levels during the night and low levels during the day. Light is also an important regulator of melatonin synthesis and acts directly to acutely suppress melatonin synthesis during the day and indirectly to entrain the circadian clock. We examined the development of circadian and light regulation of melatonin release in Xenopus retinas and pineal glands. Pineal glands are capable of making measurable melatonin in culture soon after they evaginate from the diencephalon at stage 26. In cyclic light, the melatonin rhythms are robust, with higher overall levels and greater amplitudes than in constant darkness. However, the rhythm of melatonin release damps strongly and quickly toward baseline in constant darkness. Similar results are observed in older (stage 47) embryos, indicating that cyclic light has a positive effect on melatonin synthesis in this tissue. Optic vesicles dissected at stage 26 do not release melatonin in culture until the second or third day. It is weakly rhythmic in cyclic light, but in constant dark it is released at constitutively high levels throughout the day. By stage 41, the eyes release melatonin rhythmically in both cyclic light and constant darkness with similar amplitude. Our results show that Xenopus embryos develop a functional, photoresponsive circadian clock in the eye within the first few days of life and that rhythmic melatonin release from the pineal gland at comparable stages is highly dependent on a light-dark cycle.

How cells tell time.

Many physiological phenomena are rhythmic and coincide with a particular time of day. These 'circadian rhythms' are not dependent on external timing cues but are driven by internal circadian clocks that are ubiquitous features of living organisms. Although many of these rhythms manifest themselves as complex behavioural patterns, we now know that a circadian clock does not require a complex organism or an elaborate nervous system; it can be built from molecules within an individual cell. This review focuses on new advances in identifying and understanding the basic properties of cellular circadian clocks.

Lysis-sensitive targets stimulate an elevation of cAMP in human natural killer cells.

Natural killer (NK) cells are lymphocytes that are capable of destroying tumour cells and virally infected cells (cytolysis) without prior sensitization. When cAMP is artificially elevated in NK cells, it is a potent inhibitor of their cytolytic function. We investigated whether NK-cell cAMP levels are modulated in response to tumour target cells to determine the potential of cAMP as a physiological regulator of NK cytotoxic function. When NK cells are exposed to a range of lysis-sensitive (LS) tumour-target cells there is an increase in intracellular cAMP levels in the NK cells over a 60-min period. The peak increase in cAMP (200-400% above control) occurs at 30 min for all LS targets tested. There is no increase in NK-cell cAMP in response to lysis-resistant (LR) tumour-target cells. The cAMP elevation may be dependent on both LS-target-stimulated adenylyl cyclase (AC) activation and LS-target-stimulated phosphodiesterase (PDE) inhibition. When the NK cells are pretreated with the protein tyrosine kinase (PTK) inhibitor, genistein (30 micrograms/ml), the AC-activation component of the cAMP elevation is abolished. Thus, the AC-activation component appears to require PTK activation. When NK cells are pretreated with the protein kinase C (PKC) inhibitor, chelerythrine chloride (10 microM) the cAMP elevation in response to LS targets was not diminished. This indicates that neither the AC-activation component nor any PDE-inhibition component require PKC activation.

Transgene expression in Xenopus rods.

The photoreceptors of the vertebrate retina express a large number of proteins that are involved in the process of light transduction. These genes appear to be coordinately regulated at the level of transcription, with rod- and cone-specific isoforms (J. Hurley (1992) J. Bioenerg. Biomembr. 24, 219-226). The mechanisms that regulate gene expression in a rod/cone-specific fashion have been difficult to address using traditional approaches and remain unknown. Regulation of the phototransduction proteins is medically important, since mutations in several of them cause retinal degeneration (P. Rosenfeld and T. Dryja (1995) in: Molecular Genetics of Ocular Disease (J.L. Wiggs, Ed.), pp. 99-126, Wiley-Liss Inc.). An experimental system for rapidly producing retinas expressing a desired mutant would greatly facilitate investigations of retinal degeneration. We report here that transgenic frog embryos (K. Kroll and E. Amaya (1996) Development 122, 3173-3183) can be used to study cell-specific expression in the retina. We have used a 5.5 kb 5' upstream fragment from the Xenopus principal rod opsin gene (S. Batni et al. (1996) J. Biol. Chem. 271, 3179-3186) controlling a reporter gene, green fluorescent protein (GFP), to produce numerous independent transgenic Xenopus. We find that this construct drives expression only in the retina and pineal, which is apparent by 4 days post-nuclear injection. These are the first results using transgenic Xenopus for retinal promoter analysis and the potential for the expression in rod photoreceptors of proteins with dominant phenotypes.

Identification of a novel vertebrate circadian clock-regulated gene encoding the protein nocturnin.

Photoreceptors of the Xenopus laevis retina are the site of a circadian clock. As part of a differential display screen for rhythmic gene products in this system, we have identified a photoreceptor-specific mRNA expressed in peak abundance at night. cDNA cloning revealed an open reading frame encoding a putative 388 amino acid protein that we have named "nocturnin" (for night-factor). This protein has strong sequence similarity to the C-terminal domain of the yeast transcription factor, CCR4, as well as a leucine zipper-like dimerization motif. Nocturnin mRNA levels exhibit a high amplitude circadian rhythm and nuclear run-on analysis indicates that it is controlled by the retinal circadian clock at the level of transcription. Our observations suggest that nocturnin may function through protein-protein interaction either as a component of the circadian clock or as a downstream effector of clock function.

Tryptophan hydroxylase mRNA levels are regulated by the circadian clock, temperature, and cAMP in chick pineal cells.

Chick pineal cells contain a circadian oscillator that derives rhythmic synthesis and secretion of melatonin even in dispersed cell culture. Here, we demonstrate that the mRNA encoding tryptophan hydroxylase (TPH), the first enzyme in the melatonin synthetic pathway, is expressed rhythmically under the control of the circadian clock. TPH message levels doubled between early day and early night, under both cyclic lightning and constant lightning conditions. The amplitude of the TPH mRNA rhythm was increased to 4-fold by culturing the cells at 43.3 degrees C for 48 h instead of 36.7 degrees C. Addition of forskolin to the cultures in early day produced a modest increase (50%) in TPH message levels but had no effect at other times. Because TPH mRNA are regulated by the endogenous pineal circadian clock, this provides a valuable system in which the molecular mechanism of clock control of gene expression.

Use of a high stringency differential display screen for identification of retinal mRNAs that are regulated by a circadian clock.

We report here the initiation of a systematic screen to identify clock-controlled mRNAs from the retina of Xenopus laevis using mRNA differential display. Xenopus retina contains an endogenous circadian clock located within the photoreceptor layer. The retinal block controls many aspects of physiology, including gene transcription. This screen uses differential display, a PCR based procedure, to compare retinal mRNA populations at different times of day in constant darkness, for identification of messages that exhibit rhythmic expression. Out of approx. 2000 mRNAs that we have screened to date, we have identified four candidates for clock-controlled mRNAs. Initial characterization of one of these PCR products shows that it recognizes a pair of mRNA bands on Northern blots that exhibit high amplitude rhythms. This pair of messages is called RM1 and shows peak levels of expression in the subjective night. In situ hybridization shows that this clock-controlled message is specifically localized to the clock containing photoreceptor cell layer within the retina. Identification of new messages that are under the control of the circadian clock has broad relevance in retinal physiology and provides an opportunity to gain insight into the molecular mechanism of vertebrate circadian control.