All cryptochromes are currently classified as flavoproteins. In animals their best-described role is as components of the circadian clock. This circadian function is variable, and can be either light-dependent or -independent; the molecular origin of this difference is unknown. Type I animal cryptochromes are photoreceptors that entrain an organism's clock to its environment, whereas Type II (including mammals) regulate circadian timing in a light-independent manner. Here, we reveal that, in contrast to Type I, Type II animal cryptochromes lack the structural features to securely bind the photoactive flavin cofactor. We provide a molecular basis for the distinct circadian roles of different animal cryptochromes, which also has significant implications for the putative role of Type II cryptochromes in animal photomagnetoreception.

Early 2 factor (E2F) family transcription factors participate in myriad cell biological processes including: the cell cycle, DNA repair, apoptosis, development, differentiation, and metabolism. Circadian rhythms influence many of these phenomena. Here we find that a mammalian circadian rhythm component, Cryptochrome 2 (CRY2), regulates E2F family members. Furthermore, CRY1 and CRY2 cooperate with the E3 ligase complex SKP-CULLIN-FBXL3 (SCFFBXL3) to reduce E2F steady state protein levels. These findings reveal an unrecognized molecular connection between circadian clocks and cell cycle regulation and highlight another mechanism to maintain appropriate E2F protein levels for proper cell growth.

The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock-controlled protein expression, driven by daily cycles of CRYPTOCHROME-dependent transcriptional feedback repression. To test this, we used quantitative mass spectrometry to compare wild-type and CRY-deficient fibroblasts under constant conditions. In CRY-deficient cells, we found that temporal variation in protein, phosphopeptide, and K+ abundance was at least as great as wild-type controls. Most strikingly, the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY-deficient cells. This proteome imbalance in CRY-deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide-ranging phenotypes of CRY-deficient mice. Rather than generating large-scale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post-translational functions of CRY proteins ultimately act to maintain protein and osmotic homeostasis against daily perturbation.

Circadian rhythmicity is an endogenous biological cycle of about 24h, which exists in cyanobacteria and fungi, plants and animals. Circadian rhythms improve the adaptability of organisms in both constant and changing environments. The cryptochrome (CRY) is a key element of the circadian system in various animal groups including fishes. We studied evolution of cryptochromes in the phylogenetically and ecologically diverse fish taxa. The phylogenetic tree of fish Cry features two major clades: Cry1 and Cry2. Teleosts possess extra copies of Cry1 due to the genome duplication, which resulted in 3 main paralogous subfamilies (1A, 1B and 1C). Cry1 experienced further diversification through additional duplications in some taxa. 1A of Cry1 is more conserved than the other paralogs (dN=0.010 ± 0.003, π=0.119 ± 0.058). The analysis of selection indicated that, while the Cry homologs in fish evolved under the different levels of selection pressure, strong purifying selection (average ω=0.017) dominated in their evolution.

The cryptochrome 1 and 2 genes (cry1 and cry2) are necessary for the generation of circadian rhythms, as mice lacking both of these genes (cry1,2-/-) lack circadian rhythms. We studied sleep in cry1,2-/- mice under baseline conditions as well as under conditions of constant darkness and enforced wakefulness to determine whether cryptochromes influence sleep regulatory processes.

Plants are acutely sensitive of their light environment, adapting their growth habit and prioritizing developmental decisions to maximize fecundity. In addition to providing an energy source and directional information, light quality also contributes to entrainment of the circadian system, an endogenous timing mechanism that integrates endogenous and environmental signalling cues to promote growth. Whereas plants' perception of red and blue portions of the spectrum are well defined, green light sensitivity remains enigmatic. In this study, we show that low fluence rates of green light are sufficient to entrain and maintain circadian rhythms in Arabidopsis and that cryptochromes contribute to this response. Importantly, green light responses are distinguishable from low blue light-induced phenotypes. These data suggest a distinct signalling mechanism enables entrainment of the circadian system in green light-enriched environments, such as those found in undergrowth and in densely planted monoculture.

Cryptochromes are photolyase-like blue light receptors that are conserved in plants and animals. Although the light-dependent catalytic mechanism of photolyase is well studied, the photochemical mechanism of cryptochromes remains largely unknown. Lack of an appropriate protein expression system to obtain photochemically active cryptochrome holoproteins is a technical obstacle for the study of plant cryptochromes. We report here an easy-to-use method to express and study Arabidopsis cryptochrome in HEK293T cells. Our results indicate that Arabidopsis cryptochromes expressed in HEK293T are photochemically active. We envision a broad use of this method in the functional investigation of plant proteins, especially in the large-scale analyses of photochemical activities of cryptochromes such as blue light-dependent protein-protein interactions.

Mammalian metabolism is highly circadian and major hormonal circuits involving nuclear hormone receptors display interlinked diurnal cycling. However, mechanisms that logically explain the coordination of nuclear hormone receptors and the clock are poorly understood. Here we show that two circadian co-regulators, cryptochromes 1 and 2, interact with the glucocorticoid receptor in a ligand-dependent fashion and globally alter the transcriptional response to glucocorticoids in mouse embryonic fibroblasts: cryptochrome deficiency vastly decreases gene repression and approximately doubles the number of dexamethasone-induced genes, suggesting that cryptochromes broadly oppose glucocorticoid receptor activation and promote repression. In mice, genetic loss of cryptochrome 1 and/or 2 results in glucose intolerance and constitutively high levels of circulating corticosterone, suggesting reduced suppression of the hypothalamic-pituitary-adrenal axis coupled with increased glucocorticoid transactivation in the liver. Genomically, cryptochromes 1 and 2 associate with a glucocorticoid response element in the phosphoenolpyruvate carboxykinase 1 promoter in a hormone-dependent manner, and dexamethasone-induced transcription of the phosphoenolpyruvate carboxykinase 1 gene was strikingly increased in cryptochrome-deficient livers. These results reveal a specific mechanism through which cryptochromes couple the activity of clock and receptor target genes to complex genomic circuits underpinning normal metabolic homeostasis.

Mammalian circadian clocks are driven by a transcription/translation feedback loop composed of positive regulators (CLOCK/BMAL1) and repressors (CRYPTOCHROME 1/2 (CRY1/2) and PER1/2). To understand the structural principles of regulation, we used evolutionary sequence analysis to identify co-evolving residues within the CRY/PHL protein family. Here we report the identification of an ancestral secondary cofactor-binding pocket as an interface in repressive CRYs, mediating regulation through direct interaction with CLOCK and BMAL1. Mutations weakening binding between CLOCK/BMAL1 and CRY1 lead to acceleration of the clock, suggesting that subtle sequence divergences at this site can modulate clock function. Divergence between CRY1 and CRY2 at this site results in distinct periodic output. Weaker interactions between CRY2 and CLOCK/BMAL1 at this pocket are strengthened by co-expression of PER2, suggesting that PER expression limits the length of the repressive phase in CRY2-driven rhythms. Overall, this work provides a model for the mechanism and evolutionary variation of clock regulatory mechanisms.

Circadian rhythms are a pervasive property of mammalian cells, tissues and behaviour, ensuring physiological adaptation to solar time. Models of cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are therefore considered essential components of the cellular clock mechanism, supported by behavioural arrhythmicity of CRY-deficient (CKO) mice under constant conditions. Challenging this interpretation, we find locomotor rhythms in adult CKO mice under specific environmental conditions and circadian rhythms in cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have shorter periods than wild-type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and period determination by CK1δ/ε activity to be maintained. In the absence of CRY-mediated feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational.

Plant and non-plant species possess cryptochrome (CRY) photoreceptors to mediate blue light regulation of development or the circadian clock. The blue light-dependent homooligomerization of Arabidopsis CRY2 is a known early photoreaction necessary for its functions, but the photobiochemistry and function of light-dependent homooligomerization and heterooligomerization of cryptochromes, collectively referred to as CRY photooligomerization, have not been well established. Here, we show that photooligomerization is an evolutionarily conserved photoreaction characteristic of CRY photoreceptors in plants and some non-plant species. Our analyses of the kinetics of the forward and reverse reactions of photooligomerization of Arabidopsis CRY1 and CRY2 provide a previously unrecognized mechanism underlying the different photosensitivity and photoreactivity of these two closely related photoreceptors. We found that photooligomerization is necessary but not sufficient for the functions of CRY2, implying that CRY photooligomerization is presumably accompanied by additional function-empowering conformational changes. We further demonstrated that the CRY2-CRY1 heterooligomerization plays roles in regulating functions of Arabidopsis CRYs in vivo. Taken together, these results suggest that photooligomerization is an evolutionarily conserved mechanism determining the photosensitivity and photoreactivity of plant CRYs.

Cryptochromes 1 and 2 (CRY1/2) are key components of the negative limb of the mammalian circadian clock. Like many peripheral tissues, Cry1 and -2 are expressed in the retina, where they are thought to play a role in regulating rhythmic physiology. However, studies differ in consensus as to their localization and function, and CRY1 immunostaining has not been convincingly demonstrated in the retina. Here we describe the expression and function of CRY1 and -2 in the mouse retina in both sexes. Unexpectedly, we show that CRY1 is expressed throughout all retinal layers, whereas CRY2 is restricted to the photoreceptor layer. Retinal period 2::luciferase recordings from CRY1-deficient mice show reduced clock robustness and stability, while those from CRY2-deficient mice show normal, albeit long-period, rhythms. In functional studies, we then investigated well-defined rhythms in retinal physiology. Rhythms in the photopic electroretinogram, contrast sensitivity, and pupillary light response were all severely attenuated or abolished in CRY1-deficient mice. In contrast, these physiological rhythms are largely unaffected in mice lacking CRY2, and only photopic electroretinogram rhythms are affected. Together, our data suggest that CRY1 is an essential component of the mammalian retinal clock, whereas CRY2 has a more limited role.-Wong, J. C. Y., Smyllie, N. J., Banks, G. T., Pothecary, C. A., Barnard, A. R., Maywood, E. S., Jagannath, A., Hughes, S., van der Horst, G. T. J., MacLaren, R. E., Hankins, M. W., Hastings, M. H., Nolan, P. M., Foster, R. G., Peirson, S. N. Differential roles for cryptochromes in the mammalian retinal clock.

The cryptochrome/photolyase (CRY/PL) family is essential for life under sunlight because photolyases repair UV-damaged DNA and cryptochromes are normally part of the circadian clock that controls the activity-sleep cycle within the 24-h day. In this study, we aim to understand how the lineage and habitat of an insect affects its CRY/PL composition. To this end, we searched the large number of annotated protein sequences of 340 insect species already available in databases for CRY/PLs. Using phylogenetic tree and motif analyses, we identified four frequent CRY/PLs in insects: the photolyases 6-4 PL and CPDII PL, as well as the mammalian-type cryptochrome (MCRY) and Drosophila-type cryptochrome (DCRY). Assignment of CRY/PLs to the corresponding insects confirmed that light-exposed insects tend to have more CRY/PLs than insects with little light exposure. Nevertheless, even insects with greatly reduced CRY/PLs still possess MCRY, which can be regarded as the major insect cryptochrome. Only flies of the genus Schizophora, which includes Drosophila melanogaster, lost MCRY. Moreover, we found that MCRY and CPDII PL as well as DCRY and 6-4 PL occur very frequently together, suggesting an interaction between the two pairs.

Cryptochromes (CRYs) are blue-light photoreceptors with known or presumed functions in light-dependent and light-independent gene regulation in plants and animals. Although the photochemistry of plant CRYs has been studied in some detail, the photochemical behavior of animal cryptochromes remains poorly defined in part because it has been difficult to purify animal CRYs with their flavin cofactors. Here we describe the purification of type 4 CRYs of zebrafish and chicken as recombinant proteins with full flavin complement and compare the spectroscopic properties of type 4 and type 1 CRYs. In addition, we analyzed photoinduced proteolytic degradation of both types of CRYs in vivo in heterologous systems. We find that even though both types of CRYs contain stoichiometric flavin, type 1 CRY is proteolytically degraded by a light-initiated reaction in Drosophila S2, zebrafish Z3, and human HEK293T cell lines, but zebrafish CRY4 (type 4) is not. In vivo degradation of type 1 CRYs does not require continuous illumination, and a single light flash of 1 ms duration leads to degradation of about 80% of Drosophila CRY in 60 min. Finally, we demonstrate that in contrast to animal type 2 CRYs and Arabidopsis CRY1 neither insect type 1 nor type 4 CRYs have autokinase activities.

Cryptochromes (CRYs) and UV RESISTANCE LOCUS 8 (UVR8) photoreceptors perceive UV-A/blue (315-500 nm) and UV-B (280-315 nm) radiation in plants, respectively. While the roles of CRYs and UVR8 have been studied in separate controlled-environment experiments, little is known about the interaction between these photoreceptors. Here, Arabidopsis wild-type Ler, CRYs and UVR8 photoreceptor mutants (uvr8-2, cry1cry2 and cry1cry2uvr8-2), and a flavonoid biosynthesis-defective mutant (tt4) were grown in a sun simulator. Plants were exposed to filtered radiation for 17 d or for 6 h, to study the effects of blue, UV-A, and UV-B radiation. Both CRYs and UVR8 independently enabled growth and survival of plants under solar levels of UV, while their joint absence was lethal under UV-B. CRYs mediated gene expression under blue light. UVR8 mediated gene expression under UV-B radiation, and in the absence of CRYs, also under UV-A. This negative regulation of UVR8-mediated gene expression by CRYs was also observed for UV-B. The accumulation of flavonoids was also consistent with this interaction between CRYs and UVR8. In conclusion, we provide evidence for an antagonistic interaction between CRYs and UVR8 and a role of UVR8 in UV-A perception.

Cryptochrome (CRY) is a short-wavelength light-sensitive photoreceptor expressed in a subset of circadian neurons and eyes in Drosophila that regulates light-evoked circadian clock resetting. Acutely, light evokes rapid electrical excitation of the ventral lateral subset of circadian neurons and confers circadian-modulated avoidance behavioral responses to short-wavelength light. Recent work shows dramatically different avoidance versus attraction behavioral responses to short-wavelength light in day-active versus night-active mosquitoes and that these behavioral responses are attenuated by CRY protein degradation by constant light exposure in mosquitoes. To determine whether CRY1s mediate species-specific coding for behavioral and electrophysiological light responses, we used an "empty neuron" approach and transgenically expressed diurnal Aedes aegypti (AeCRY1) versus nocturnal Anopheles gambiae (AgCRY1) in a cry-null Drosophila background. AeCRY1 is much less light sensitive than either AgCRY1 or DmCRY as shown by partial behavioral rhythmicity following constant light exposure. Remarkably, expression of nocturnal AgCRY1 confers low survival to constant white light as does expression of AeCRY1 to a lesser extent. AgCRY1 mediates significantly stronger electrophysiological cell-autonomous responses to 365 nm ultraviolet (UV) light relative to AeCRY1. AgCRY1 expression mediates electrophysiological sensitivity to 635 nm red light, whereas AeCRY1 does not, consistent with species-specific mosquito red light responses. AgCRY1 and DmCRY mediate intensity-dependent avoidance behavior to UV light at different light intensity thresholds, whereas AeCRY1 does not, thus mimicking mosquito and fly behaviors. These findings highlight CRY as a key non-image-forming visual photoreceptor that mediates physiological and behavioral light responses in a species-specific fashion.

Cryptochromes and photolyases are structurally related but have different biological functions in signalling and DNA repair. Proteobacteria and cyanobacteria harbour a new class of cryptochromes, called CryPro. We have solved the 2.7 Å structure of one of its members, cryptochrome B from Rhodobacter sphaeroides, which is a regulator of photosynthesis gene expression. The structure reveals that, in addition to the photolyase-like fold, CryB contains two cofactors only conserved in the CryPro subfamily: 6,7-dimethyl-8-ribityl-lumazine in the antenna-binding domain and a [4Fe-4S] cluster within the catalytic domain. The latter closely resembles the iron-sulphur cluster harbouring the large primase subunit PriL, indicating that PriL is evolutionarily related to the CryPro class of cryptochromes.

Irises isolated from the eyes of diverse species constrict when exposed to light. Depending on species this intrinsic photomechanical transduction response (PMTR) requires either melanopsin or cryptochrome (CRY) photopigment proteins, generated by their respective association with retinoid or flavin adenine dinucleotide (FAD) chromophores. Although developmentally relevant circadian rhythms are also synchronized and reset by these same proteins, the cell type, mechanism, and specificity of photomechanical transduction (PMT) and its relationship to circadian processes remain poorly understood. Here we show that PMTRs consistent with CRY activation by 430 nm blue light occur in developing chicken iris striated muscle, identify relevant mechanisms, and demonstrate that similar PMTRs occur in striated iris and pectoral muscle fibers, prevented in both cases by knocking down CRY gene transcript levels. Supporting CRY activation, iris PMTRs were reduced by inhibiting flavin reductase, but unaffected by melanopsin antagonism. The largest iris PMTRs paralleled the developmental predominance of striated over smooth muscle fibers, and shared their requirement for extracellular Ca2+ influx and release of intracellular Ca2+. Photo-stimulation of identified striated myotubes maintained in dissociated culture revealed the cellular and molecular bases of PMT. Myotubes in iris cell cultures responded to 435 nm light with increased intracellular Ca2+ and contractions, mimicking iris PMTRs and their spectral sensitivity. Interestingly PMTRs featuring contractions and requiring extracellular Ca2+ influx and release of intracellular Ca2+ were also displayed by striated myotubes derived from pectoral muscle. Consistent with these findings, cytosolic CRY1 and CRY2 proteins were detected in both iris and pectoral myotubes, and knocking down myotube CRY1/CRY2 gene transcript levels specifically blocked PMTRs in both cases. Thus CRY-mediated PMT is not unique to iris, but instead reflects a more general feature of developing striated muscle fibers. Because CRYs are core timing components of circadian clocks and CRY2 is critical for circadian regulation of myogenic differentiation CRY-mediated PMT may interact with cell autonomous clocks to influence the progression of striated muscle development.

Cryptochromes (CRYs) are evolutionarily conserved blue-light receptors that evolved from bacterial photolyases that repair damaged DNA. Today, CRYs have lost their ability to repair damaged DNA; however, prior reports suggest that human CRYs can respond to DNA damage. Currently, the role of CRYs in the DNA damage response (DDR) is lacking, especially in plants. Therefore, we evaluated the role of plant CRYs in DDR along with UBP12/13 deubiquitinases, which interact with and regulate the CRY2 protein. We found that cry1cry2 was hypersensitive, while ubp12ubp13 was hyposensitive to UVC-induced DNA damage. Elevated UV-induced cyclobutane pyrimidine dimers (CPDs) and the lack of DNA repair protein RAD51 accumulation in cry1cry2 plants indicate that CRYs are required for DNA repair. On the contrary, CPD levels diminished and RAD51 protein levels elevated in plants lacking UBP12 and UBP13, indicating their role in DDR repression. Temporal transcriptomic analysis revealed that DDR-induced transcriptional responses were subdued in cry1cry2 , but elevated in ubp12ubp13 compared to WT. Through transcriptional modeling of the timecourse transcriptome, we found that genes quickly induced by UVC (15 min) are targets of CAMTA 1-3 transcription factors, which we found are required for DDR. This transcriptional regulation seems, however, diminished in the cry1cry2 mutant, indicating that CAMTAs are required for CRY2-mediated DDR. Furthermore, we observed enhanced CRY2-UBP13 interaction and formation of CRY2 nuclear speckles under UVC, suggesting that UVC activates CRY2 similarly to blue light. Together, our data reveal the temporal dynamics of the transcriptional events underlying UVC-induced genotoxicity and expand our knowledge of the role of CRY and UBP12/13 in DDR.

Light is one of the most important environmental factors affecting growth and geographic distribution of forestry plants. Moso bamboo is the largest temperate bamboo on earth and an important non-woody forestry species that serves not only important functions in the economy of rural areas but also carbon sequestration in the world. Due to its decades-long reproductive timing, the germplasm of moso bamboo cannot be readily improved by conventional breeding methods, arguing for a greater need to study the gene function and regulatory mechanisms of this species. We systematically studied the photoregulatory mechanisms of the moso bamboo (Phyllostachys edulis) cryptochrome 1, PheCRY1. Our results show that, similar to its Arabidopsis counterpart, the bamboo PheCRY1s are functionally restricted to the blue light inhibition of cell elongation without an apparent activity in promoting floral initiation. We demonstrate that PheCRY1s undergo light-dependent oligomerization that is inhibited by PheBIC1s, and light-dependent phosphorylation that is catalyzed by PhePPKs. We hypothesize that light-induced phosphorylation of PheCRY1s facilitate their degradation, which control availability of the PheCRY1 proteins and photosensitivity of bamboo plants. Our results demonstrate the evolutionary conservation of not only the function but also photoregulatory mechanism of PheCRY1 in this monocot forestry species.