The Circadian Clock Protein CRY1 Is a Negative Regulator of HIF-1α.

The circadian clock and the hypoxia-signaling pathway are regulated by an integrated interplay of positive and negative feedback limbs that incorporate energy homeostasis and carcinogenesis. We show that the negative circadian regulator CRY1 is also a negative regulator of hypoxia-inducible factor (HIF). Mechanistically, CRY1 interacts with the basic-helix-loop-helix domain of HIF-1α via its tail region. Subsequently, CRY1 reduces HIF-1α half-life and binding of HIFs to target gene promoters. This appeared to be CRY1 specific because genetic disruption of CRY1, but not CRY2, affected the hypoxia response. Furthermore, CRY1 deficiency could induce cellular HIF levels, proliferation, and migration, which could be reversed by CRISPR/Cas9- or short hairpin RNA-mediated HIF knockout. Altogether, our study provides a mechanistic explanation for genetic association studies linking a disruption of the circadian clock with hypoxia-associated processes such as carcinogenesis.

Critical cholangiocarcinogenesis control by cryptochrome clock genes.

A coordinated network of molecular circadian clocks in individual cells generates 24-hr rhythms in liver metabolism and proliferation. Circadian disruption through chronic jet lag or Per2 clock gene mutation was shown to accelerate hepatocarcinoma development in mice. As divergent effects were reported for clock genes Per and Cry regarding xenobiotic toxicity, we questioned the role of Cry1 and Cry2 in liver carcinogenesis. Male WT and Cry1-/- Cry2-/- mice (C57Bl/6 background) were chronically exposed to diethylnitrosamine (DEN) at ZT11. Rest-activity and body temperature rhythms were monitored using an implanted radiotransmitter. Serum aspartate and alanine aminotransferases (AST and ALT) were determined on four occasions during the progression stage. After 7 months, serum alkaline phosphatases (ALP) were determined, and livers were sampled for microscopic tumor nodule counting and histopathology. Five months after initiation of DEN treatment, we found that Cry1-/- Cry2-/- mice developed severe liver dysplasia, as evident from the increased AST, ALT and ALP levels, as compared to WT mice. DEN exposure induced primary liver cancers in nearly fivefold as many Cry1-/- Cry2-/- mice as compared to WT mice (p = 0.01). Microscopic study revealed no difference in the average number of hepatocarcinomas and a nearly eightfold increase in the average number of cholangiocarcinomas in Cry1-/- Cry2-/- mice, as compared to WT mice. This study validated the hypothesis that molecular circadian clock disruption dramatically increased chemically induced liver carcinogenesis. In addition, the pronounced shift toward cholangiocarcinoma in DEN exposed Cry1-/- Cry2-/- mice revealed a critical role of the Cry clock genes in bile duct carcinogenesis.

Colorectal liver metastases with a disrupted circadian rhythm phase shift the peripheral clock in liver and kidney.

Circadian clock genes regulate 10-15% of the transcriptome, and might function as tumor suppressor genes. Relatively little is known about the circadian clock in tumors and its effect on surrounding healthy tissues. Therefore, we compared the 24-hr expression levels of key circadian clock genes in liver and kidney of healthy control mice with those of mice bearing C26 colorectal tumor metastases in the liver. Metastases were induced by injection of C26 colorectal carcinoma cells into the spleen. Subsequently, tumor, liver and kidney tissue was collected around the clock to compare circadian rhythmicity. Expression levels of five clock genes (Rev-Erbα, Per1, Per2, Bmal1 and Cry1) and three clock-controlled genes (CCGs; Dbp, p21 and Wee1) were determined by qRT-PCR. Liver and kidney tissue of healthy control mice showed normal 24-hr oscillations of all clock genes and CCGs, consistent with normal circadian rhythmicity. In colorectal liver metastases, however, 24-hr oscillations were completely absent for all clock genes and CCGs except Cry1. Liver and kidney tissue of tumor-bearing mice showed a shift in clock periodicity relative to control mice. In the liver we observed a phase advance, whereas in the kidney the phase was delayed. These data show that hepatic metastases of C26 colon carcinoma with a disrupted circadian rhythm phase shift liver and kidney tissue clocks, which strongly suggests a systemic effect on peripheral clocks. The possibility that tumors may modify peripheral clocks to escape from ordinary circadian rhythms and in this way contribute to fatigue and sleep disorders in cancer patients is discussed.

Histone methyltransferase MLL3 contributes to genome-scale circadian transcription.

Daily cyclical expression of thousands of genes in tissues such as the liver is orchestrated by the molecular circadian clock, the disruption of which is implicated in metabolic disorders and cancer. Although we understand much about the circadian transcription factors that can switch gene expression on and off, it is still unclear how global changes in rhythmic transcription are controlled at the genomic level. Here, we demonstrate circadian modification of an activating histone mark at a significant proportion of gene loci that undergo daily transcription, implicating widespread epigenetic modification as a key node regulated by the clockwork. Furthermore, we identify the histone-remodelling enzyme mixed lineage leukemia (MLL)3 as a clock-controlled factor that is able to directly and indirectly modulate over a hundred epigenetically targeted circadian "output" genes in the liver. Importantly, catalytic inactivation of the histone methyltransferase activity of MLL3 also severely compromises the oscillation of "core" clock gene promoters, including Bmal1, mCry1, mPer2, and Rev-erbα, suggesting that rhythmic histone methylation is vital for robust transcriptional oscillator function. This highlights a pathway by which the clockwork exerts genome-wide control over transcription, which is critical for sustaining temporal programming of tissue physiology.

Altered phase-relationship between peripheral oscillators and environmental time in Cry1 or Cry2 deficient mouse models for early and late chronotypes.

The mammalian circadian system is composed of a light-entrainable central clock in the suprachiasmatic nuclei (SCN) of the brain and peripheral clocks in virtually any other tissue. It allows the organism to optimally adjust metabolic, physiological and behavioral functions to the physiological needs it will have at specific time of the day. According to the resonance theory, such rhythms are only advantageous to an organism when in tune with the environment, which is illustrated by the adverse health effects originating from chronic circadian disruption by jetlag and shift work. Using short-period Cry1 and long-period Cry2 deficient mice as models for morningness and eveningness, respectively, we explored the effect of chronotype on the phase relationship between the central SCN clock and peripheral clocks in other organs. Whereas the behavioral activity patterns and circadian gene expression in the SCN of light-entrained Cry1(-/-) and Cry2(-/-) mice largely overlapped with that of wild type mice, expression of clock and clock controlled genes in liver, kidney, small intestine, and skin was shown to be markedly phase-advanced or phase-delayed, respectively. Likewise, circadian rhythms in urinary corticosterone were shown to display a significantly altered phase relationship similar to that of gene expression in peripheral tissues. We show that the daily dissonance between peripheral clocks and the environment did not affect the lifespan of Cry1(-/-) or Cry2(-/-) mice. Nonetheless, the phase-shifted peripheral clocks in light-entrained mice with morningness and eveningness-like phenotypes may have implications for personalized preventive and therapeutic (i.e. chronomodulation-based) health care for people with early and late chronotypes.

Mammalian cryptochromes impinge on cell cycle progression in a circadian clock-independent manner.

By gating cell cycle progression to specific times of the day, the intracellular circadian clock is thought to reduce the exposure of replicating cells to potentially hazardous environmental and endogenous genotoxic compounds. Although core clock gene defects that eradicate circadian rhythmicity can cause an altered in vivo genotoxic stress response and aberrant proliferation rate, it remains to be determined to what extent these cell cycle related phenotypes are due to a cell-autonomous lack of circadian oscillations. We investigated the DNA damage sensitivity and proliferative capacity of cultured primary Cry1(-/- )|Cry2(-/-) fibroblasts. Contrasting previous in vivo studies, we show that the absence of CRY proteins does not affect the cell-autonomous DNA damage response upon exposure of primary cells in vitro to genotoxic agents, but causes cells to proliferate faster. By comparing primary wild-type, Cry1(-/-) |Cry2(-/-), Cry1(+/-)|Cry2(-/-) and Cry1(-/-)|Cry2(+/-) fibroblasts, we provide evidence that CRY proteins influence cell cycle progression in a cell-autonomous, but circadian clock-independent manner and that the accelerated cell cycle progression of Cry-deficient cells is caused by global dysregulation of Bmal1-dependent gene expression. These results suggest that the inconsistency between in vivo and in vitro observations might be attributed to systemic circadian control rather than a direct cell-autonomous control.

Impact of the circadian clock on in vitro genotoxic risk assessment assays.

Our society expects safety assessment for drugs, chemicals, cosmetics, and foods, which to date cannot be achieved without the use of laboratory animals. At the same time, society aims at refining, reducing, and (ultimately) replacing animal testing. As a consequence, much effort is taken to establish alternatives, such as toxicogenomics-based risk assessment assays on cultured cells and tissues. Evidently, the properties of cells in vitro will considerably differ from the in vivo situation. This review will discuss the impact of the circadian clock, an internal time keeping system that drives 24-h rhythms in metabolism, physiology and behavior, on in vitro genotoxic risk assessment. Our recent observation that DNA damaging agents can synchronize the circadian clock of individual cells in culture (and as a consequence the cyclic expression of clock-controlled genes, comprising up to 10% of the transcriptome) implies that the circadian clock should not be neglected when developing cell or tissue-based alternatives for chronic rodent toxicity assays.

Photic sensitivity ranges of hamster pupillary and circadian phase responses do not overlap.

Mammalian retinal photoreceptors form an irradiance detection system that drives many nonvisual responses to light such as pupil reflex and resetting of the circadian clock. To understand the role of pupil size in circadian light responses, pupil diameter was pharmacologically manipulated and the effect on behavioral phase shifts at different irradiance levels was studied in the Syrian hamster. Dose-response curves for steady-state pupil size and for behavioral phase shifts were constructed for 3 pupil conditions (dilated, constricted, and control). Retinal irradiance was calculated from corneal irradiance, pupil size, retinal surface area, and absorption of ocular media. The sensitivity of photic responses to retinal irradiance is approximately 1.5 log units higher than to corneal irradiance. When plotted against corneal irradiance, pharmacological pupil constriction reduces the light sensitivity of the circadian system, but pupil dilation has no effect. As expected, when plotted against retinal irradiance all dose-response curves superimposed, confirming that the circadian system responds to photon flux on the retina. Pupil dilation does not increase the circadian response to increasing irradiance, since the response of the circadian system attains saturation at irradiance levels lower than those required to induce pupil constriction. The main finding shows that due to the different response sensitivities, the effect of pupil constriction on the light sensitivity of the circadian system in the hamster under natural conditions is virtually negligible. We further suggest the existence of distinct modulating mechanisms for the differential retinal irradiance sensitivity of the pupil system and the circadian system, which enables the different responses to be tuned to their specific tasks while using similar photoreceptive input.

Phase resetting of the mammalian circadian clock by DNA damage.

To anticipate the momentum of the day, most organisms have developed an internal clock that drives circadian rhythms in metabolism, physiology, and behavior [1]. Recent studies indicate that cell-cycle progression and DNA-damage-response pathways are under circadian control [2-4]. Because circadian output processes can feed back into the clock, we investigated whether DNA damage affects the mammalian circadian clock. By using Rat-1 fibroblasts expressing an mPer2 promoter-driven luciferase reporter, we show that ionizing radiation exclusively phase advances circadian rhythms in a dose- and time-dependent manner. Notably, this in vitro finding translates to the living animal, because ionizing radiation also phase advanced behavioral rhythms in mice. The underlying mechanism involves ATM-mediated damage signaling as radiation-induced phase shifting was suppressed in fibroblasts from cancer-predisposed ataxia telangiectasia and Nijmegen breakage syndrome patients. Ionizing radiation-induced phase shifting depends on neither upregulation or downregulation of clock gene expression nor on de novo protein synthesis and, thus, differs mechanistically from dexamethasone- and forskolin-provoked clock resetting [5]. Interestingly, ultraviolet light and tert-butyl hydroperoxide also elicited a phase-advancing effect. Taken together, our data provide evidence that the mammalian circadian clock, like that of the lower eukaryote Neurospora[6], responds to DNA damage and suggest that clock resetting is a universal property of DNA damage.

The precision of circadian clocks: assessment and analysis in Syrian hamsters.

Locomotor activity recordings of Syrian hamsters were systematically analyzed to estimate the precision of the overt circadian activity rhythm in constant darkness. Phase variation, i.e., the standard deviation of phase markers around the regression line, varied with the definition of phase. Smallest phase variation was found in the onset of wheel running activity defined by 1h running means of the raw data. Both lower and higher degrees of smoothing lead to decreased precision measured in the overt rhythm. With passive infrared recordings, the midpoint of activity defined by 3h running means was the least variable. This demonstrates that the choice of phase marker should vary between recording methods. Phase variation decreased with increasing activity and was larger in females than in males. By calculating the average cycle variation and serial covariance of consecutive cycles, we estimated the contribution of 'clock' and 'non-clock' related processes to the overt rhythm variability. Variance in precision between phase markers could be shown to be attributable mainly to nonclock processes. Variance in pacemaker cycle length appeared reduced in wheel running activity records compared with passive infrared sensing records, suggesting feedback from running activity onto pacemaker function.

Enhanced longevity in tau mutant Syrian hamsters, Mesocricetus auratus.

The single-gene mutation tau in the Syrian hamster shortens the circadian period by about 20% in the homozygous mutant and simultaneously increases the mass-specific metabolic rate by about 20%. Both effects might be expected to lead to a change in longevity. To test such expectations, the life span of male and female hamsters from three genotypes (wild-type, heterozygous, and homozygous tau mutants, all derived from heterozygote crosses to randomize the genetic background) was recorded in constant darkness. Male hamsters lived significantly longer than females: the overall average life span was 96.9 weeks (SE = 2.5, n = 118) for males and 82.0 weeks (SE = 2.1, n = 99) for females. To our surprise, male and female homozygous mutant hamsters lived significantly longer rather than shorter compared to wild-types. For males, the difference between the two genotypes was on average 14%; for females, the difference was 16%. The mortality rate of wild-type males was significantly different from that of homozygous tau males but not different from that of heterozygotes. Overall, survival of wild-type females was statistically distinguishable from both heterozygous and homozygous mutant females. Male and female wild-type hamsters were heavier than homozygote mutants throughout the entire life span, and heterozygous mutants had intermediate weights. There was no correlation between body mass and life span, and the causes of the extended life span in tau mutant hamsters remain unresolved.

Vasopressin immunoreactivity and release in the suprachiasmatic nucleus of wild-type and tau mutant Syrian hamsters.

Despite the prominent role of the Syrian hamster (Mesocricetus auratus) in studies of circadian rhythms, there are no data available on the temporal dynamics of the neuropeptide vasopressin (AVP), a major output system of the suprachiasmatic nucleus (SCN). We studied the hamster SCN-AVP system in vivo across the light period and in vitro using long-term organotypic SCN cultures. Additionally, we compared wild-type and tau mutant hamsters with an endogenous circadian period of approximately 24 h and approximately 20 h, respectively. The in vivo study revealed no differences in the number of SCN-AVP neurons between the two genotypes of hamsters studied at three time points across the light period of the circadian cycle. A significantly higher level of AVP-immunoreactivity, however, was found in the SCN of wild-type compared to tau mutant hamsters at the beginning and in the middle of the light period, but not at the end of the light period. SCN-AVP cell number and immunostaining decreased significantly across the light period in wild-type hamsters, but not in tau mutants. The in vitro study revealed a significantly higher rate of AVP release per 24 h from the tau mutant SCN compared to the wild-type SCN. Robust circadian oscillations in AVP release were not found in either type of hamster. These results may suggest that the SCN-AVP system of hamsters, irrespective of genotype, is relatively weak compared to other species. Moreover, the tau mutation seems to influence the SCN-AVP system by enhancing the rate of AVP release and by reducing AVP content and its daily fluctuation.