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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.

m6A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m6A-modified base. The recruitment of the mRNA m6A methyltransferase writer complex to phase-separated nuclear speckles is likely to be crucial in its regulation; however, control over the activity of the complex remains unclear. Supported by our observation that a core catalytic subunit of the methyltransferase complex, METTL3, is endogenously colocalized within nuclear speckles as well as in noncolocalized puncta, we tracked the components of the complex with a Cry2-METTL3 fusion construct to disentangle key domains and interactions necessary for the phase separation of METTL3. METTL3 is capable of self-interaction and likely provides the multivalency to drive condensation. Condensates in cells necessarily contain myriad components, each with partition coefficients that establish an entropic barrier that can regulate entry into the condensate. In this regard, we found that, in contrast to the constitutive binding of METTL14 to METTL3 in both the diffuse and the dense phase, WTAP only interacts with METTL3 in dense phase and thereby distinguishes METTL3/METTL14 single complexes in the dilute phase from METTL3/METTL14 multicomponent condensates. Finally, control over METTL3/METTL14 condensation is determined by its small molecule cofactor, S-adenosylmethionine (SAM), which regulates conformations of two gate loops, and some cancer-associated mutations near gate loops can impair METTL3 condensation. Therefore, the link between SAM binding and the control of writer complex phase state suggests that the regulation of its phase state is a potentially critical facet of its functional regulation.

The ∼20,000 cells of the suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinate subordinate cellular clocks across the organism, driving adaptive daily rhythms of physiology and behavior. The canonical model for SCN timekeeping pivots around transcriptional/translational feedback loops (TTFL) whereby PERIOD (PER) and CRYPTOCHROME (CRY) clock proteins associate and translocate to the nucleus to inhibit their own expression. The fundamental individual and interactive behaviors of PER and CRY in the SCN cellular environment and the mechanisms that regulate them are poorly understood. We therefore used confocal imaging to explore the behavior of endogenous PER2 in the SCN of PER2::Venus reporter mice, transduced with viral vectors expressing various forms of CRY1 and CRY2. In contrast to nuclear localization in wild-type SCN, in the absence of CRY proteins, PER2 was predominantly cytoplasmic and more mobile, as measured by fluorescence recovery after photobleaching. Virally expressed CRY1 or CRY2 relocalized PER2 to the nucleus, initiated SCN circadian rhythms, and determined their period. We used translational switching to control CRY1 cellular abundance and found that low levels of CRY1 resulted in minimal relocalization of PER2, but yet, remarkably, were sufficient to initiate and maintain circadian rhythmicity. Importantly, the C-terminal tail was necessary for CRY1 to localize PER2 to the nucleus and to initiate SCN rhythms. In CRY1-null SCN, CRY1Δtail opposed PER2 nuclear localization and correspondingly shortened SCN period. Through manipulation of CRY proteins, we have obtained insights into the spatiotemporal behaviors of PER and CRY sitting at the heart of the TTFL molecular mechanism.

Disruption of circadian rhythms increases the risk of several types of cancer. Mammalian cryptochromes (CRY1 and CRY2) are circadian transcriptional repressors that are related to DNA-repair enzymes. While CRYs lack DNA-repair activity, they modulate the transcriptional response to DNA damage, and CRY2 can promote SKP1 cullin 1-F-box (SCF)FBXL3-mediated ubiquitination of c-MYC and other targets. Here, we characterize five mutations in CRY2 observed in human cancers in The Cancer Genome Atlas. We demonstrate that two orthologous mutations of mouse CRY2 (D325H and S510L) accelerate the growth of primary mouse fibroblasts expressing high levels of c-MYC. Neither mutant affects steady-state levels of overexpressed c-MYC, and they have divergent impacts on circadian rhythms and on the ability of CRY2 to interact with SCFFBXL3 Unexpectedly, stable expression of either CRY2 D325H or of CRY2 S510L robustly suppresses P53 target-gene expression, suggesting that this may be a primary mechanism by which they influence cell growth.

Sexual reproduction constrains progeny to inherit allelic genes from both parents. Selective acquisition of target genes from only one parent in the F1 generation of plants has many potential applications including the elimination of undesired alleles and acceleration of trait stacking. CRISPR/Cas9-based gene drives can generate biased transmission of a preferred allele and convert heterozygotes to homozygotes in insects and mice, but similar strategies have not been implementable in plants because of a lack of efficient homology-directed repair (HDR). Here, we place a gene drive, which consists of cassettes that produce Cas9, guide RNAs (gRNA), and fluorescent markers, into the CRYPTOCHROME 1 (CRY1) gene through CRISPR/Cas9-mediated HDR, resulting in cry1drive lines. After crossing the cry1drive/cry1drive lines to wild type, we observe F1 plants which have DNA at the CRY1 locus from only the cry1drive/cry1drive parent. Moreover, a non-autonomous trans-acting gene drive, in which the gene drive unit and the target gene are located on different chromosomes, converts a heterozygous mutation in the target gene to homozygous. Our results demonstrate that homozygous F1 plants can be obtained through zygotic conversion using a CRISPR/Cas9-based gene drive.

We developed a method for single-cell resolution longitudinal bioluminescence imaging of PERIOD (PER) protein and TIMELESS (TIM) oscillations in cultured male adult Drosophila brains that captures circadian circuit-wide cycling under simulated day/night cycles. Light input analysis confirms that CRYPTOCHROME (CRY) is the primary circadian photoreceptor and mediates clock disruption by constant light (LL), and that eye light input is redundant to CRY; 3-h light phase delays (Friday) followed by 3-h light phase advances (Monday morning) simulate the common practice of staying up later at night on weekends, sleeping in later on weekend days then returning to standard schedule Monday morning [weekend light shift (WLS)]. PER and TIM oscillations are highly synchronous across all major circadian neuronal subgroups in unshifted light schedules for 11 d. In contrast, WLS significantly dampens PER oscillator synchrony and rhythmicity in most circadian neurons during and after exposure. Lateral ventral neuron (LNv) oscillations are the first to desynchronize in WLS and the last to resynchronize in WLS. Surprisingly, the dorsal neuron group-3 (DN3s) increase their within-group synchrony in response to WLS. In vivo, WLS induces transient defects in sleep stability, learning, and memory that temporally coincide with circuit desynchrony. Our findings suggest that WLS schedules disrupt circuit-wide circadian neuronal oscillator synchrony for much of the week, thus leading to observed behavioral defects in sleep, learning, and memory.

Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4COP1/SPAs, has been reported for plant CRYs so far. Here we show that Cul3LRBs is the second E3 ligase of CRY2 in Arabidopsis. We demonstrate the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2, at the CCE domain of CRY2, to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4COP1/SPAs and Cul3LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature.

Circadian rhythms are generated by interlocked transcription-translation feedback loops that establish cell-autonomous biological timing of ∼24 h. Mutations in core clock genes that alter their stability or affinity for one another lead to changes in circadian period. The human CRY1Δ11 mutant lengthens circadian period to cause delayed sleep phase disorder (DSPD), characterized by a very late onset of sleep. CRY1 is a repressor that binds to the transcription factor CLOCK:BMAL1 to inhibit its activity and close the core feedback loop. We previously showed how the PHR (photolyase homology region) domain of CRY1 interacts with distinct sites on CLOCK and BMAL1 to sequester the transactivation domain from coactivators. However, the Δ11 variant alters an intrinsically disordered tail in CRY1 downstream of the PHR. We show here that the CRY1 tail, and in particular the region encoded by exon 11, modulates the affinity of the PHR domain for CLOCK:BMAL1. The PHR-binding epitope in exon 11 is necessary and sufficient to disrupt the interaction between CRY1 and the subunit CLOCK. Moreover, PHR-tail interactions are conserved in the paralog CRY2 and reduced when either CRY is bound to the circadian corepressor PERIOD2. Discovery of this autoregulatory role for the mammalian CRY1 tail and conservation of PHR-tail interactions in both mammalian cryptochromes highlights functional conservation with plant and insect cryptochromes, which also utilize PHR-tail interactions to reversibly control their activity.

Sexual reproduction constrains progeny to inherit allelic genes from both parents. Selective acquisition of target genes from only one parent in the F1 generation of plants has many potential applications including the elimination of undesired alleles and acceleration of trait stacking. CRISPR/Cas9-based gene drives can generate biased transmission of a preferred allele and convert heterozygotes to homozygotes in insects and mice, but similar strategies have not been implementable in plants because of a lack of efficient homology-directed repair (HDR). Here, we place a gene drive, which consists of cassettes that produce Cas9, guide RNAs (gRNA), and fluorescent markers, into the CRYPTOCHROME 1 (CRY1) gene through CRISPR/Cas9-mediated HDR, resulting in cry1drive lines. After crossing the cry1drive/cry1drive lines to wild type, we observe F1 plants which have DNA at the CRY1 locus from only the cry1drive/cry1drive parent. Moreover, a non-autonomous trans-acting gene drive, in which the gene drive unit and the target gene are located on different chromosomes, converts a heterozygous mutation in the target gene to homozygous. Our results demonstrate that homozygous F1 plants can be obtained through zygotic conversion using a CRISPR/Cas9-based gene drive.

Cryptochromes (CRYs) are evolutionarily conserved photoreceptors that mediate various light-induced responses in bacteria, plants, and animals. Plant cryptochromes govern a variety of critical growth and developmental processes including seed germination, flowering time and entrainment of the circadian clock. CRY's photocycle involves reduction of their flavin adenine dinucleotide (FAD)-bound chromophore, which is completely oxidized in the dark and semi to fully reduced in the light signaling-active state. Despite the progress in characterizing cryptochromes, important aspects of their photochemistry, regulation, and light-induced structural changes remain to be addressed. In this study, we determine the crystal structure of the photosensory domain of Arabidopsis CRY2 in a tetrameric active state. Systematic structure-based analyses of photo-activated and inactive plant CRYs elucidate distinct structural elements and critical residues that dynamically partake in photo-induced oligomerization. Our study offers an updated model of CRYs photoactivation mechanism as well as the mode of its regulation by interacting proteins.

Cryptochrome (CRY) proteins play an essential role in regulating mammalian circadian rhythms. CRY is composed of a structured N-terminal domain known as the photolyase homology region (PHR), which is tethered to an intrinsically disordered C-terminal tail. The PHR domain is a critical hub for binding other circadian clock components such as CLOCK, BMAL1, PERIOD, or the ubiquitin ligases FBXL3 and FBXL21. While the isolated PHR domain is necessary and sufficient to generate circadian rhythms, removing or modifying the cryptochrome tails modulates the amplitude and/or periodicity of circadian rhythms, suggesting that they play important regulatory roles in the molecular circadian clock. In this commentary, we will discuss how recent studies of these intrinsically disordered tails are helping to establish a general and evolutionarily conserved model for CRY function, where the function of PHR domains is modulated by reversible interactions with their intrinsically disordered tails. Video abstract.

The nucleus accumbens (NAc), a central component of the midbrain dopamine reward circuit, exhibits disturbed circadian rhythms in the postmortem brains of depressed patients. We hypothesized that normal mood regulation requires proper circadian timing in the NAc, and that mood disorders are associated with dysfunctions of the NAc cellular circadian clock. In mice exhibiting stress-induced depression-like behavior (helplessness), we found altered circadian clock function and high nighttime expression of the core circadian clock component CRYPTOCHROME (CRY) in the NAc. In the NAc of helpless mice, we found that higher expression of CRY is associated with decreased activation of dopamine 1 receptor-expressing medium spiny neurons (D1R-MSNs). Furthermore, D1R-MSN-specific CRY-knockdown in the NAc reduced susceptibility to stress-induced helplessness and increased NAc neuronal activation at night. Finally, we show that CRY inhibits D1R-induced G protein activation, likely by interacting with the Gs protein. Altered circadian rhythms and CRY expression were also observed in human fibroblasts from major depressive disorder patients. Our data reveal a causal role for CRY in regulating the midbrain dopamine reward system, and provide a mechanistic link between the NAc circadian clock and vulnerability to depression.

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

Cryptochromes are ubiquitous flavin-binding light sensors closely related to DNA-repairing photolyases. The animal-like cryptochrome CraCRY from the green alga Chlamydomonas reinhardtii challenges the paradigm of cryptochromes as pure blue-light receptors by acting as a (6-4) photolyase, using 8-hydroxy-5-deazaflavin (8-HDF) as a light-harvesting antenna with a 17.4 Å distance to flavin and showing spectral sensitivity up to 680 nm. The expanded action spectrum is attributed to the presence of the flavin neutral radical (FADH•) in the dark, despite a rapid FADH• decay observed in vitro in samples exclusively carrying flavin. Herein, the red-light response of CraCRY carrying flavin and 8-HDF was studied, revealing a 3-fold prolongation of the FADH• lifetime in the presence of 8-HDF. Millisecond time-resolved ultraviolet-visible spectroscopy showed the red-light-induced formation and decay of an absorbance band at 458 nm concomitant with flavin reduction. Time-resolved Fourier transform infrared (FTIR) spectroscopy and density functional theory attributed these changes to the deprotonation of 8-HDF, challenging the paradigm of 8-HDF being permanently deprotonated in photolyases. FTIR spectra showed changes in the hydrogen bonding network of asparagine 395, a residue suggested to indirectly control flavin protonation, indicating the involvement of N395 in the stabilization of FADH•. Fluorescence spectroscopy revealed a decrease in the energy transfer efficiency of 8-HDF upon flavin reduction, possibly linked to 8-HDF deprotonation. The discovery of the interdependence of flavin and 8-HDF beyond energy transfer processes highlights the essential role of the antenna, introducing a new concept enabling CraCRY and possibly other bifunctional cryptochromes to fulfill their dual function.

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

Drosophila CRYPTOCHROME (dCRY) mediates electrophysiological depolarization and circadian clock resetting in response to blue or ultraviolet (UV) light. These light-evoked biological responses operate at different timescales and possibly through different mechanisms. Whether electron transfer down a conserved chain of tryptophan residues underlies biological responses following dCRY light activation has been controversial. To examine these issues in in vivo and in ex vivo whole-brain preparations, we generated transgenic flies expressing tryptophan mutant dCRYs in the conserved electron transfer chain and then measured neuronal electrophysiological phototransduction and behavioral responses to light. Electrophysiological-evoked potential analysis shows that dCRY mediates UV and blue-light-evoked depolarizations that are long lasting, persisting for nearly a minute. Surprisingly, dCRY appears to mediate red-light-evoked depolarization in wild-type flies, absent in both cry-null flies, and following acute treatment with the flavin-specific inhibitor diphenyleneiodonium in wild-type flies. This suggests a previously unsuspected functional signaling role for a neutral semiquinone flavin state (FADH•) for dCRY. The W420 tryptophan residue located closest to the FAD-dCRY interaction site is critical for blue- and UV-light-evoked electrophysiological responses, while other tryptophan residues within electron transfer distance to W420 do not appear to be required for light-evoked electrophysiological responses. Mutation of the dCRY tryptophan residue W342, more distant from the FAD interaction site, mimics the cry-null behavioral light response to constant light exposure. These data indicate that light-evoked dCRY electrical depolarization and clock resetting are mediated by distinct mechanisms.

Ambient temperature sensing by phytochrome B (PHYB) in Arabidopsis is thought to operate mainly at night. Here we show that PHYB plays an equally critical role in temperature sensing during the daytime. In daytime thermosensing, PHYB signals primarily through the temperature-responsive transcriptional regulator PIF4, which requires the transcriptional activator HEMERA (HMR). HMR does not regulate PIF4 transcription, instead, it interacts directly with PIF4, to activate the thermoresponsive growth-relevant genes and promote warm-temperature-dependent PIF4 accumulation. A missense allele hmr-22, which carries a loss-of-function D516N mutation in HMR's transcriptional activation domain, fails to induce the thermoresponsive genes and PIF4 accumulation. Both defects of hmr-22 could be rescued by expressing a HMR22 mutant protein fused with the transcriptional activation domain of VP16, suggesting a causal relationship between HMR-mediated activation of PIF4 target-genes and PIF4 accumulation. Together, this study reveals a daytime PHYB-mediated thermosensing mechanism, in which HMR acts as a necessary activator for PIF4-dependent induction of temperature-responsive genes and PIF4 accumulation.

Cryptochromes (CRYs) are blue light receptors that mediate light regulation of plant growth and development. Land plants possess various numbers of cryptochromes, CRY1 and CRY2, which serve overlapping and partially redundant functions in different plant species. Cryptochromes exist as physiologically inactive monomers in darkness; photoexcited cryptochromes undergo homodimerization to increase their affinity to the CRY-signaling proteins, such as CIBs (CRY2-interacting bHLH), PIFs (Phytochrome-Interacting Factors), AUX/IAA (Auxin/INDOLE-3-ACETIC ACID), and the COP1-SPAs (Constitutive Photomorphogenesis 1-Suppressors of Phytochrome A) complexes. These light-dependent protein-protein interactions alter the activity of the CRY-signaling proteins to change gene expression and developmental programs in response to light. In the meantime, photoexcitation also changes the affinity of cryptochromes to the CRY-regulatory proteins, such as BICs (Blue-light Inhibitors of CRYs) and PPKs (Photoregulatory Protein Kinases), to modulate the activity, modification, or abundance of cryptochromes and photosensitivity of plants in response to the changing light environment.

In response to blue light, cryptochromes photoexcite and interact with signal partners to transduce signal almost synchronously in plants. The detailed mechanism of CRY-mediated light signaling remains unclear: the photobiochemical reactions of cryptochrome are transient and synchronous, thus making the monitoring and analysis of each step difficult in plant cells. In this study, we reconstituted the Arabidopsis CRY2 signaling pathway in mammalian cells and investigated the biological role of Arabidopsis CRY2 in this heterologous system, eliminating the interferences of other plant proteins. Our results demonstrated that, besides being the light receptor, Arabidopsis CRY2 binds to DNA directly and acts as a transcriptional activator in a blue-light-enhanced manner. Similar to classic transcription factors, we found that the transcriptional activity of CRY2 is regulated by its dimerization and phosphorylation. In addition, CRY2 cooperates with CIB1 to regulate transcription by enhancing the DNA affinity and transcriptional activity of CIB1 under blue light.

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1-/- , and Cry2-/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle.

Polyploidy is a major speciation process in vascular plants, and is postulated to be particularly important in shaping the diversity of extant ferns. However, limitations in the availability of bi-parental markers for ferns have greatly limited phylogenetic investigation of polyploidy in this group. With a large number of allopolyploid species, the genus Botrychium is a classic example in ferns where recurrent polyploidy is postulated to have driven frequent speciation events. Here, we use PacBio sequencing and the PURC bioinformatics pipeline to capture all homeologous or allelic copies of four long (∼1 kb) low-copy nuclear regions from a sample of 45 specimens (25 diploids and 20 polyploids) representing 37 Botrychium taxa, and three outgroups. This sample includes most currently recognized Botrychium species in Europe and North America, and the majority of our specimens were genotyped with co-dominant nuclear allozymes to ensure species identification. We analyzed the sequence data using maximum likelihood (ML) and Bayesian inference (BI) concatenated-data ("gene tree") approaches to explore the relationships among Botrychium species. Finally, we estimated divergence times among Botrychium lineages and inferred the multi-labeled polyploid species tree showing the origins of the polyploid taxa, and their relationships to each other and to their diploid progenitors. We found strong support for the monophyly of the major lineages within Botrychium and identified most of the parental donors of the polyploids; these results largely corroborate earlier morphological and allozyme-based investigations. Each polyploid had at least two distinct homeologs, indicating that all sampled polyploids are likely allopolyploids (rather than autopolyploids). Our divergence-time analyses revealed that these allopolyploid lineages originated recently-within the last two million years-and thus that the genus has undergone a recent radiation, correlated with multiple independent allopolyploidizations across the phylogeny. Also, we found strong parental biases in the formation of allopolyploids, with individual diploid species participating multiple times as either the maternal or paternal donor (but not both). Finally, we discuss the role of polyploidy in the evolutionary history of Botrychium and the interspecific reproductive barriers possibly involved in these parental biases.

Contents Summary 547 I. Introduction 547 II. Phytochromes mediate light-induced transcription of BICs to inactivate cryptochromes 548 III. PPKs phosphorylate light-signaling proteins and histones to affect plant development 548 IV. Prospect 550 Acknowledgements 550 References 550 SUMMARY: Plants perceive and respond to light signals by multiple sensory photoreceptors, including phytochromes and cryptochromes, which absorb different wavelengths of light to regulate genome expression and plant development. Photophysiological analyses have long revealed the coordinated actions of different photoreceptors, a phenomenon referred to as the photoreceptor coaction. The mechanistic explanations of photoreceptor coactions are not fully understood. The function of direct protein-protein interaction of phytochromes and cryptochromes and common signaling molecules of these photoreceptors, such as SPA1/COP1 E3 ubiquitin ligase complex and bHLH transcription factors PIFs, would partially explain phytochrome-cryptochrome coactions. In addition, newly discovered proteins that block cryptochrome photodimerization or catalyze cryptochrome phosphorylation may also participate in the phytochrome and cryptochrome coaction. This Tansley insight, which is not intended to make a comprehensive review of the studies of photoreceptor coactions, attempts to highlight those recent findings and their possible roles in the photoreceptor coaction.

The circadian system regulates numerous physiological processes including immune responses. Here, we show that mice deficient of the circadian clock genes Cry1 and Cry2 [Cry double knockout (DKO)] develop an autoimmune phenotype including high serum IgG concentrations, serum antinuclear antibodies, and precipitation of IgG, IgM, and complement 3 in glomeruli and massive infiltration of leukocytes into the lungs and kidneys. Flow cytometry of lymphoid organs revealed decreased pre-B cell numbers and a higher percentage of mature recirculating B cells in the bone marrow, as well as increased numbers of B2 B cells in the peritoneal cavity of Cry DKO mice. The B cell receptor (BCR) proximal signaling pathway plays a critical role in autoimmunity regulation. Activation of Cry DKO splenic B cells elicited markedly enhanced tyrosine phosphorylation of cellular proteins compared with cells from control mice, suggesting that overactivation of the BCR-signaling pathway may contribute to the autoimmunity phenotype in the Cry DKO mice. In addition, the expression of C1q, the deficiency of which contributes to the pathogenesis of systemic lupus erythematosus, was significantly down-regulated in Cry DKO B cells. Our results suggest that B cell development, the BCR-signaling pathway, and C1q expression are regulated by circadian clock CRY proteins and that their dysregulation through loss of CRY contributes to autoimmunity.

Cryptochromes are blue light receptors that regulate various light responses in plants. Arabidopsis cryptochrome 1 (CRY1) and cryptochrome 2 (CRY2) mediate blue light inhibition of hypocotyl elongation and long-day (LD) promotion of floral initiation. It has been reported recently that two negative regulators of Arabidopsis cryptochromes, Blue light Inhibitors of Cryptochromes 1 and 2 (BIC1 and BIC2), inhibit cryptochrome function by blocking blue light-dependent cryptochrome dimerization. However, it remained unclear how cryptochromes regulate the BIC gene activity. Here we show that cryptochromes mediate light activation of transcription of the BIC genes, by suppressing the activity of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), resulting in activation of the transcription activator ELONGATED HYPOCOTYL 5 (HY5) that is associated with chromatins of the BIC promoters. These results demonstrate a CRY-BIC negative-feedback circuitry that regulates the activity of each other. Surprisingly, phytochromes also mediate light activation of BIC transcription, suggesting a novel photoreceptor co-action mechanism to sustain blue light sensitivity of plants under the broad spectra of solar radiation in nature.

Plant cryptochromes undergo blue light-dependent phosphorylation to regulate their activity and abundance, but the protein kinases that phosphorylate plant cryptochromes have remained unclear. Here we show that photoexcited Arabidopsis cryptochrome 2 (CRY2) is phosphorylated in vivo on as many as 24 different residues, including 7 major phosphoserines. We demonstrate that four closely related Photoregulatory Protein Kinases (previously referred to as MUT9-like kinases) interact with and phosphorylate photoexcited CRY2. Analyses of the ppk123 and ppk124 triple mutants and amiR4k artificial microRNA-expressing lines demonstrate that PPKs catalyse blue light-dependent CRY2 phosphorylation to both activate and destabilize the photoreceptor. Phenotypic analyses of these mutant lines indicate that PPKs may have additional substrates, including those involved in the phytochrome signal transduction pathway. These results reveal a mechanism underlying the co-action of cryptochromes and phytochromes to coordinate plant growth and development in response to different wavelengths of solar radiation in nature.

The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD) binding pocket of CRYPTOCHROME2 (CRY2), a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin kinase (Rfk), an FAD biosynthetic enzyme, enhanced CRY degradation. RFK protein levels and FAD concentrations oscillate in the nucleus, suggesting that they are subject to circadian control. Knockdown of Rfk combined with a riboflavin-deficient diet altered the CRY levels in mouse liver and the expression profiles of clock and clock-controlled genes (especially those related to metabolism including glucose homeostasis). We conclude that light-independent mechanisms of FAD regulate CRY and contribute to proper circadian oscillation of metabolic genes in mammals.

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ∼24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here we show that CRY1 binds directly to the PAS domain core of CLOCK:BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solution X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.

Blue light affects many aspects of plant growth and development throughout the plant lifecycle. Plant cryptochromes (CRYs) are UV-A/blue light photoreceptors that play pivotal roles in regulating blue light-mediated physiological responses via the regulated expression of more than one thousand genes. Photoactivated CRYs regulate transcription via two distinct mechanisms: indirect promotion of the activity of transcription factors by inactivation of the COP1/SPA E3 ligase complex or direct activation or inactivation of at least two sets of basic helix-loop-helix transcription factor families by physical interaction. Hence, CRYs govern intricate mechanisms that modulate activities of transcription factors to regulate multiple aspects of blue light-responsive photomorphogenesis. Here, we review recent progress in dissecting the pathways of CRY signaling and discuss accumulating evidence that shows how CRYs regulate broad physiological responses to blue light.

Drosophila melanogaster CRYPTOCHROME (CRY) mediates behavioral and electrophysiological responses to blue light coded by circadian and arousal neurons. However, spectroscopic and biochemical assays of heterologously expressed CRY suggest that CRY may mediate functional responses to UV-A (ultraviolet A) light as well. To determine the relative contributions of distinct phototransduction systems, we tested mutants lacking CRY and mutants with disrupted opsin-based phototransduction for behavioral and electrophysiological responses to UV light. CRY and opsin-based external photoreceptor systems cooperate for UV light-evoked acute responses. CRY mediates behavioral avoidance responses related to executive choice, consistent with its expression in central brain neurons.

Cryptochromes are evolutionarily related to the light-dependent DNA repair enzyme photolyase, serving as major regulators of circadian rhythms in insects and vertebrate animals. There are two types of cryptochromes in the animal kingdom: Drosophila-like CRYs that act as nonvisual photopigments linking circadian rhythms to the environmental light/dark cycle, and vertebrate-like CRYs that do not appear to sense light directly, but control the generation of circadian rhythms by acting as transcriptional repressors. Some animals have both types of CRYs, while others possess only one. Cryptochromes have two domains, the photolyase homology region (PHR) and an extended, intrinsically disordered C-terminus. While all animal CRYs share a high degree of sequence and structural homology in their PHR domains, the C-termini are divergent in both length and sequence identity. Recently, cryptochrome function has been shown to extend beyond its pivotal role in circadian clocks, participating in regulation of the DNA damage response, cancer progression and glucocorticoid signaling, as well as being implicated as possible magnetoreceptors. In this review, we provide a historical perspective on the discovery of animal cryptochromes, examine similarities and differences of the two types of animal cryptochromes and explore some of the divergent roles for this class of proteins.

Cryptochromes are blue-light receptors that regulate development and the circadian clock in plants and animals. We found that Arabidopsis cryptochrome 2 (CRY2) undergoes blue light-dependent homodimerization to become physiologically active. We identified BIC1 (blue-light inhibitor of cryptochromes 1) as an inhibitor of plant cryptochromes that binds to CRY2 to suppress the blue light-dependent dimerization, photobody formation, phosphorylation, degradation, and physiological activities of CRY2. We hypothesize that regulated dimerization governs homeostasis of the active cryptochromes in plants and other evolutionary lineages.

In many legumes, roots that are exposed to light do not form nodules. Here, we report that blue light inhibits nodulation in Lotus japonicus roots inoculated with Mesorhizobium loti. Using RNA interference, we suppressed the expression of the phototropin and cryptochrome genes in L. japonicus hairy roots. Under blue light, plants transformed with an empty vector did not develop nodules, whereas plants exhibiting suppressed expression of cry1 and cry2 genes formed nodules. We also measured rhizobial growth to investigate whether the inhibition of nodulation could be caused by a reduced population of rhizobia in response to light. Although red light had no effect on rhizobial growth, blue light had a strong inhibitory effect. Rhizobial growth under blue light was partially restored in signature-tagged mutagenesis (STM) strains in which LOV-HK/PAS- and photolyase-related genes were disrupted. Moreover, when Ljcry1A and Ljcry2B-silenced plants were inoculated with the STM strains, nodulation was additively increased. Our data show that blue light receptors in both the host plant and the symbiont have a profound effect on nodule development. The exact mechanism by which these photomorphogenetic responses function in the symbiosis needs further study, but they are clearly involved in optimizing legume nodulation.

Cryptochromes are blue light receptors regulated by light-dependent ubiquitination and degradation in both plant and animal lineages. The Arabidopsis genome encodes two cryptochromes, CRY1 and CRY2, of which CRY2 undergoes blue light-dependent ubiquitination and 26S proteasome-dependent degradation. The molecular mechanism regulating blue light-dependent proteolysis of CRY2 is still not fully understood. We found that the F-box proteins ZEITLUPE (ZTL) and Lov Kelch Protein2 (LKP2), which mediate blue light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1-SPA1 protein complex in blue light-dependent CRY2 degradation is more likely to be attributable to its cullin 4 (CUL4)-based E3 ubiquitin ligase activity than its activity as the cryptochrome signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4 mutant, the cop1-5 null mutant and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele (axr6-3), especially under the non-permissive temperature. Based on these and other results presented, we propose that photoexcited CRY2 undergoes Lys48-linked polyubiquitination catalyzed by the CUL4- and CUL1-based E3 ubiquitin ligases.

Familial Advanced Sleep Phase (FASP) is a heritable human sleep phenotype characterized by very early sleep and wake times. We identified a missense mutation in the human Cryptochrome 2 (CRY2) gene that co-segregates with FASP in one family. The mutation leads to replacement of an alanine residue at position 260 with a threonine (A260T). In mice, the CRY2 mutation causes a shortened circadian period and reduced phase-shift to early-night light pulse associated with phase-advanced behavioral rhythms in the light-dark cycle. The A260T mutation is located in the phosphate loop of the flavin adenine dinucleotide (FAD) binding domain of CRY2. The mutation alters the conformation of CRY2, increasing its accessibility and affinity for FBXL3 (an E3 ubiquitin ligase), thus promoting its degradation. These results demonstrate that CRY2 stability controlled by FBXL3 plays a key role in the regulation of human sleep wake behavior.

Light is the primary signal that calibrates circadian neural circuits and thus coordinates daily physiological and behavioral rhythms with solar entrainment cues. Drosophila and mammalian circadian circuits consist of diverse populations of cellular oscillators that exhibit a wide range of dynamic light responses, periods, phases, and degrees of synchrony. How heterogeneous circadian circuits can generate robust physiological rhythms while remaining flexible enough to respond to synchronizing stimuli has long remained enigmatic. Cryptochrome is a short-wavelength photoreceptor that is endogenously expressed in approximately half of Drosophila circadian neurons. In a previous study, physiological light response was measured using real-time bioluminescence recordings in Drosophila whole-brain explants, which remain intrinsically light-sensitive. Here we apply analysis of real-time bioluminescence experimental data to show detailed dynamic ensemble representations of whole circadian circuit light entrainment at single neuron resolution. Organotypic whole-brain explants were either maintained in constant darkness (DD) for 6 days or exposed to a phase-advancing light pulse on the second day. We find that stronger circadian oscillators support robust overall circuit rhythmicity in DD, whereas weaker oscillators can be pushed toward transient desynchrony and damped amplitude to facilitate a new state of phase-shifted network synchrony. Additionally, we use mathematical modeling to examine how a network composed of distinct oscillator types can give rise to complex dynamic signatures in DD conditions and in response to simulated light pulses. Simulations suggest that complementary coupling mechanisms and a combination of strong and weak oscillators may enable a robust yet flexible circadian network that promotes both synchrony and entrainment. A more complete understanding of how the properties of oscillators and their signaling mechanisms facilitate their distinct roles in light entrainment may allow us to direct and augment the circadian system to speed recovery from jet lag, shift work, and seasonal affective disorder.

Lithium is widely used as a treatment of bipolar disorder, a neuropsychiatric disorder associated with disrupted circadian rhythms. Lithium is known to lengthen period and increase amplitude of circadian rhythms. One possible pathway for these effects involves inhibition of glycogen synthase kinase-3β (GSK-3β), which regulates degradation of CRY2, a canonical clock protein determining circadian period. CRY1 is also known to play important roles in regulating circadian period and phase, although there is no evidence that it is similarly phosphorylated by GSK-3β. In this paper, we tested the hypothesis that lithium affects circadian rhythms through CRYs. We cultured fibroblasts and slices of the suprachiasmatic nucleus (SCN), the master circadian pacemaker of the brain, from Cry1-/-, Cry2-/-, or wild-type (WT) mice bearing the PER2:LUC circadian reporter. Lithium was applied in the culture medium, and circadian rhythms of PER2 expression were measured. In WT and Cry2-/- fibroblasts, 10mM lithium increased PER2 expression and rhythm amplitude but not period, and 1mM lithium did not affect either period or amplitude. In non-rhythmic Cry1-/- fibroblasts, 10mM lithium increased PER2 expression. In SCN slices, 1mM lithium lengthened period ∼1h in all genotypes, but did not affect amplitude except in Cry2-/- SCN. Thus, the amplitude-enhancing effect of lithium in WT fibroblasts was unaffected by Cry2 knockout and occurred in the absence of period-lengthening, whereas the period-lengthening effect of lithium in WT SCN was unaffected by Cry1 or Cry2 knockout and occurred in the absence of rhythm amplification, suggesting that these two effects of lithium on circadian rhythms are independent of CRYs and of each other.

Genes involved in circadian regulation, such as circadian locomotor output cycles kaput [CLOCK], cryptochrome [CRY1] and period [PER], have been associated with sleep outcomes in prior animal and human research. However, it is unclear whether polymorphisms in these genes are associated with the sleep disturbances commonly experienced by adults living with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Thus, the purpose of this study was to describe polymorphisms in selected circadian genes that are associated with sleep duration or disruption as well as the sleep-wake rhythm strength and phase timing among adults living with HIV/AIDS. A convenience sample of 289 adults with HIV/AIDS was recruited from HIV clinics and community sites in the San Francisco Bay Area. A wrist actigraph was worn for 72 h on weekdays to estimate sleep duration or total sleep time (TST), sleep disruption or percentage of wake after sleep onset (WASO) and several circadian rhythm parameters: mesor, amplitude, the ratio of mesor to amplitude (circadian quotient), and 24-h autocorrelation. Circadian phase measures included clock time for peak activity (acrophase) from actigraphy movement data, and bed time and final wake time from actigraphy and self-report. Genotyping was conducted for polymorphisms in five candidate genes involved in circadian regulation: CLOCK, CRY1, PER1, PER2 and PER3. Demographic and clinical variables were evaluated as potential covariates. Interactions between genotype and HIV variables (i.e. viral load, years since HIV diagnosis) were also evaluated. Controlling for potentially confounding variables (e.g. race, gender, CD4+ T-cell count, waist circumference, medication use, smoking and depressive symptoms), CLOCK was associated with WASO, 24-h autocorrelation and objectively-measured bed time; CRY1 was associated with circadian quotient; PER1 was associated with mesor and self-reported habitual wake time; PER2 was associated with TST, mesor, circadian quotient, 24-h autocorrelation and bed and wake times; PER3 was associated with amplitude, 24-h autocorrelation, acrophase and bed and wake times. Most of the observed associations involved a significant interaction between genotype and HIV. In this chronic illness population, polymorphisms in several circadian genes were associated with measures of sleep disruption and timing. These findings extend the evidence for an association between genetic variability in circadian regulation and sleep outcomes to include the sleep-wake patterns experienced by adults living with HIV/AIDS. These results provide direction for future intervention research related to circadian sleep-wake behavior patterns.

Cryptochromes are blue-light absorbing flavoproteins with multiple signaling roles. In plants, cryptochrome (cry1, cry2) biological activity has been linked to flavin photoreduction via an electron transport chain to the protein surface comprising 3 evolutionarily conserved tryptophan residues known as the 'Trp triad.' Mutation of any of the Trp triad residues abolishes photoreduction in isolated cryptochrome protein in vitro and therefore had been suggested as essential for electron transfer to the flavin. However, photoreduction of the flavin in Arabidopsis cry2 proteins occurs in vivo even with mutations in the Trp triad, indicating the existence of alternative electron transfer pathways to the flavin. These pathways are potentiated by metabolites in the intracellular environment including ATP, ADP, AMP, and NADH. In the present work we extend these observations to Arabidopsis cryptochrome 1 and demonstrate that Trp triad substitution mutants at W400F and W324F positions which are not photoreduced in vitro can be photoreduced in whole cell extracts, albeit with reduced efficiency. We further show that the flavin signaling state (FADH°) is stabilized in an in vivo context. These data illustrate that in vivo modulation by metabolites in the cellular environment may play an important role in cryptochrome signaling, and are discussed with respect to possible effects on the conformation of the C-terminal domain to generate the biologically active conformational state.

Female reproductive function changes during aging with the estrous cycle becoming more irregular during the transition to menopause. We found that intermittent shifts of the light-dark cycle disrupted regularity of estrous cycles in middle-aged female mice, whose estrous cycles were regular under unperturbed 24-hr light-dark cycles. Although female mice deficient in Cry1 or Cry2, the core components of the molecular circadian clock, exhibited regular estrous cycles during youth, they showed accelerated senescence characterized by irregular and unstable estrous cycles and resultant infertility in middle age. Notably, tuning the period length of the environmental light-dark cycles closely to the endogenous one inherent in the Cry-deficient females restored the regularity of the estrous cycles and, consequently, improved fertility in middle age. These results suggest that reproductive potential can be strongly influenced by age-related changes in the circadian system and normal reproductive functioning can be rescued by the manipulation of environmental timing signals.

Cryptochromes in different evolutionary lineages act as either photoreceptors or light-independent transcription repressors. The flavin cofactor of both types of cryptochromes can be photoreduced in vitro by electron transportation via three evolutionarily conserved tryptophan residues known as the "Trp triad." It was hypothesized that Trp triad-dependent photoreduction leads directly to photoexcitation of cryptochrome photoreceptors. We tested this hypothesis by analyzing mutations of Arabidopsis cryptochrome 1 (CRY1) altered in each of the three Trp-triad tryptophan residues (W324, W377, and W400). Surprisingly, in contrast to a previous report all photoreduction-deficient Trp-triad mutations of CRY1 remained physiologically and biochemically active in Arabidopsis plants. ATP did not enhance rapid photoreduction of the wild-type CRY1, nor did it rescue the defective photoreduction of the CRY1(W324A) and CRY1(W400F) mutants that are photophysiologically active in vivo. The lack of correlation between rapid flavin photoreduction or the effect of ATP on the rapid flavin photoreduction and the in vivo photophysiological activities of plant cryptochromes argues that the Trp triad-dependent photoreduction is not required for the function of cryptochromes and that further efforts are needed to elucidate the photoexcitation mechanism of cryptochrome photoreceptors.

The molecular circadian clock in mammals is generated from transcriptional activation by the bHLH-PAS transcription factor CLOCK-BMAL1 and subsequent repression by PERIOD and CRYPTOCHROME (CRY). The mechanism by which CRYs repress CLOCK-BMAL1 to close the negative feedback loop and generate 24-h timing is not known. Here we show that, in mouse fibroblasts, CRY1 competes for binding with coactivators to the intrinsically unstructured C-terminal transactivation domain (TAD) of BMAL1 to establish a functional switch between activation and repression of CLOCK-BMAL1. TAD mutations that alter affinities for co-regulators affect the balance of repression and activation to consequently change the intrinsic circadian period or eliminate cycling altogether. Our results suggest that CRY1 fulfills its role as an essential circadian repressor by sequestering the TAD from coactivators, and they highlight regulation of the BMAL1 TAD as a critical mechanism for establishing circadian timing.

Transmembrane receptors are the predominant conduit through which cells sense and transduce extracellular information into intracellular biochemical signals. Current methods to control and study receptor function, however, suffer from poor resolution in space and time and often employ receptor overexpression, which can introduce experimental artefacts. We report a genetically encoded approach, termed Clustering Indirectly using Cryptochrome 2 (CLICR), for spatiotemporal control over endogenous transmembrane receptor activation, enabled through the optical regulation of target receptor clustering and downstream signalling using noncovalent interactions with engineered Arabidopsis Cryptochrome 2 (Cry2). CLICR offers a modular platform to enable photocontrol of the clustering of diverse transmembrane receptors including fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR) and integrins in multiple cell types including neural stem cells. Furthermore, light-inducible manipulation of endogenous receptor tyrosine kinase (RTK) activity can modulate cell polarity and establish phototaxis in fibroblasts. The resulting spatiotemporal control over cellular signalling represents a powerful new optogenetic framework for investigating and controlling cell function and fate.

Arabidopsis cryptochrome 2 (CRY2) is a blue light receptor that mediates light inhibition of hypocotyl elongation and long-day promotion of floral initiation. CRY2 is known to undergo blue light-dependent phosphorylation, which is believed to serve regulatory roles in the function of CRY2. We report here on a biochemical and genetics study of CRY2 phosphorylation. Using mass spectrometry analysis, we identified three serine residues in the CCE domain of CRY2 (S598, S599, and S605) that undergo blue light-dependent phosphorylation in Arabidopsis seedlings. A study of serine-substitution mutations in the CCE domain of CRY2 demonstrates that CRY2 contains two types of phosphorylation in the CCE domain, one in the serine cluster that causes electrophoretic mobility upshift and the other outside the serine cluster that does not seem to cause mobility upshift. We showed that mutations in the serine residues within and outside the serine cluster diminished blue light-dependent CRY2 phosphorylation, degradation, and physiological activities. These results support the hypothesis that blue light-dependent phosphorylation of the CCE domain determines the photosensitivity of Arabidopsis CRY2.

Blue light activation of the photoreceptor CRYPTOCHROME (CRY) evokes rapid depolarization and increased action potential firing in a subset of circadian and arousal neurons in Drosophila melanogaster. Here we show that acute arousal behavioral responses to blue light significantly differ in mutants lacking CRY, as well as mutants with disrupted opsin-based phototransduction. Light-activated CRY couples to membrane depolarization via a well conserved redox sensor of the voltage-gated potassium (K(+)) channel β-subunit (Kvβ) Hyperkinetic (Hk). The neuronal light response is almost completely absent in hk(-/-) mutants, but is functionally rescued by genetically targeted neuronal expression of WT Hk, but not by Hk point mutations that disable Hk redox sensor function. Multiple K(+) channel α-subunits that coassemble with Hk, including Shaker, Ether-a-go-go, and Ether-a-go-go-related gene, are ion conducting channels for CRY/Hk-coupled light response. Light activation of CRY is transduced to membrane depolarization, increased firing rate, and acute behavioral responses by the Kvβ subunit redox sensor.

ObjectiveThe diagnostic boundaries of sleep disorders are under considerable debate. The main sleep disorders are partly heritable; therefore, defining heritable pathophysiologic mechanisms could delineate diagnoses and suggest treatment. We collected clinical data and DNA from consenting patients scheduled to undergo clinical polysomnograms, to expand our understanding of the polymorphisms associated with the phenotypes of particular sleep disorders.MethodsPatients at least 21 years of age were recruited to contribute research questionnaires, and to provide access to their medical records, saliva for deoxyribonucleic acid (DNA), and polysomnographic data. From these complex data, 38 partly overlapping phenotypes were derived indicating complaints, subjective and objective sleep timing, and polysomnographic disturbances. A custom chip was used to genotype 768 single-nucleotide polymorphisms (SNPs). Additional assays derived ancestry-informative markers (eg, 751 participants of European ancestry). Linear regressions controlling for age, gender, and ancestry were used to assess the associations of each phenotype with each of the SNPs, highlighting those with Bonferroni-corrected significance.ResultsIn peroxisome proliferator-activated receptor gamma, coactivator 1 beta (PPARGC1B), rs6888451 was associated with several markers of obstructive sleep apnea. In aryl hydrocarbon receptor nuclear translocator-like (ARNTL), rs10766071 was associated with decreased polysomnographic sleep duration. The association of rs3923809 in BTBD9 with periodic limb movements in sleep was confirmed. SNPs in casein kinase 1 delta (CSNK1D rs11552085), cryptochrome 1 (CRY1 rs4964515), and retinoic acid receptor-related orphan receptor A (RORA rs11071547) were less persuasively associated with sleep latency and time of falling asleep.ConclusionsSNPs associated with several sleep phenotypes were suggested, but due to risks of false discovery, independent replications are needed before the importance of these associations can be assessed, followed by investigation of molecular mechanisms.

Intracellular circadian clocks, composed of clock genes that act in transcription-translation feedback loops, drive global rhythmic expression of the mammalian transcriptome and allow an organism to anticipate to the momentum of the day. Using a novel clock-perturbing peptide, we established a pivotal role for casein kinase (CK)-2-mediated circadian BMAL1-Ser90 phosphorylation (BMAL1-P) in regulating central and peripheral core clocks. Subsequent analysis of the underlying mechanism showed a novel role of CRY as a repressor for protein kinase. Co-immunoprecipitation experiments and real-time monitoring of protein-protein interactions revealed that CRY-mediated periodic binding of CK2β to BMAL1 inhibits BMAL1-Ser90 phosphorylation by CK2α. The FAD binding domain of CRY1, two C-terminal BMAL1 domains, and particularly BMAL1-Lys537 acetylation/deacetylation by CLOCK/SIRT1, were shown to be critical for CRY-mediated BMAL1-CK2β binding. Reciprocally, BMAL1-Ser90 phosphorylation is prerequisite for BMAL1-Lys537 acetylation. We propose a dual negative-feedback model in which a CRY-dependent CK2-driven posttranslational BMAL1-P-BMAL1 loop is an integral part of the core clock oscillator.

The light-based control of ion channels has been transformative for the neurosciences, but the optogenetic toolkit does not stop there. An expanding number of proteins and cellular functions have been shown to be controlled by light, and the practical considerations in deciding between reversible optogenetic systems (such as systems that use light-oxygen-voltage domains, phytochrome proteins, cryptochrome proteins and the fluorescent protein Dronpa) are well defined. The field is moving beyond proof of concept to answering real biological questions, such as how cell signalling is regulated in space and time, that were difficult or impossible to address with previous tools.

Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH(·) radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·(-), from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396(-). Its negative charge could trigger conformational changes necessary for signal transduction.

Cryptochromes are blue light receptors that regulate light responses in plants, including various crops. The molecular mechanism of plant cryptochromes has been extensively investigated in Arabidopsis thaliana, but it has not been reported in any crop species. Here, we report a study of the mechanism of soybean (Glycine max) cryptochrome2 (CRY2a). We found that CRY2a regulates leaf senescence, which is a life history trait regulated by light and photoperiods via previously unknown mechanisms. We show that CRY2a undergoes blue light-dependent interaction with the soybean basic helix-loop-helix transcription activator CIB1 (for cryptochrome-interacting bHLH1) that specifically interacts with the E-box (CANNTG) DNA sequences. Analyses of transgenic soybean plants expressing an elevated or reduced level of the CRY2a or CIB1 demonstrate that CIB1 promotes leaf senescence, whereas CRY2a suppresses leaf senescence. Results of the gene expression and molecular interaction analyses support the hypothesis that CIB1 activates transcription of senescence-associated genes, such as WRKY DNA BINDING PROTEIN53b (WRKY53b), and leaf senescence. CIB1 interacts with the E-box-containing promoter sequences of the WRKY53b chromatin, whereas photoexcited CRY2a interacts with CIB1 to inhibit its DNA binding activity. These findings argue that CIB-dependent transcriptional regulation is an evolutionarily conserved CRY-signaling mechanism in plants, and this mechanism is opted in evolution to mediate light regulation of different aspects of plant development in different plant species.

Prader-Willi syndrome (PWS), a genetic disorder of obesity, intellectual disability and sleep abnormalities, is caused by loss of non-coding RNAs on paternal chromosome 15q11-q13. The imprinted minimal PWS locus encompasses a long non-coding RNA (lncRNA) transcript processed into multiple SNORD116 small nucleolar RNAs and the spliced exons of the host gene, 116HG. However, both the molecular function and the disease relevance of the spliced lncRNA 116HG are unknown. Here, we show that 116HG forms a subnuclear RNA cloud that co-purifies with the transcriptional activator RBBP5 and active metabolic genes, remains tethered to the site of its transcription and increases in size in post-natal neurons and during sleep. Snord116del mice lacking 116HG exhibited increased energy expenditure corresponding to the dysregulation of diurnally expressed Mtor and circadian genes Clock, Cry1 and Per2. These combined genomic and metabolic analyses demonstrate that 116HG regulates the diurnal energy expenditure of the brain. These novel molecular insights into the energy imbalance in PWS should lead to improved therapies and understanding of lncRNA roles in complex neurodevelopmental and metabolic disorders.