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 chron

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

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-1a via its tail region. Subsequently, CRY1 reduces HIF-1a 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

Characteristic of survival (in days) in male and female wild type (Wt), <i>Cry1</i> deficient (<i>Cry1^-/-</i>), and <i>Cry2</i> deficient (<i>Cry2^-/-</i>) mice.

<p>Characteristic of survival (in days) in male and female wild type (Wt), <i>Cry1</i> deficient (<i>Cry1^-/-</i>), and <i>Cry2</i> deficient (<i>Cry2^-/-</i>) mice.</p

Altered Phase-Relationship between Peripheral Oscillators and Environmental Time in <i>Cry1</i> or <i>Cry2</i> Deficient Mouse Models for Early and Late Chronotypes

<div><p>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 <i>Cry1</i> and long-period <i>Cry2</i> 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 <i>Cry1^-/-</i> and <i>Cry2^-/-</i> 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 <i>Cry1^-/-</i> or <i>Cry2^-/-</i> 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.</p></div

Circadian gene expression in the SCN of <i>Cry1^-/-</i> and <i>Cry2^-/-</i> mice.

<p>Relative mRNA expression profiles of core clock genes <i>Bmal1</i> (A) and <i>Per2</i> (B) and the clock-controlled genes <i>Dbp</i> (C) and <i>Pk2</i> (D) in the SCN of light entrained (LD) wild type (black), <i>Cry1^-/-</i> (green) and <i>Cry2^-/-</i> (red) mice, as measured by RT-qPCR. For each genotype, mRNA levels (mean ± s.e.m) are expressed relative to the peak of expression (set at 100%) and fitted with a wave curve. The two-way ANOVA test revealed a significant effect of genotype and time interaction (*p<0.05). Data at ZT0 and ZT24 are double-plotted for easier visualization of daily expression. The bar above panels indicates time of light on and off.</p