Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man.

The Cryptochromes: Blue Light Photoreceptors in Plants and Animals

Plants and animals produce modular developmental units in a periodic fashion. In plants, lateral roots form as repeating units along the root primary axis; however, the developmental mechanism regulating this process is unknown. We found that cyclic expression pulses of a reporter gene mark the position of future lateral roots by establishing prebranch sites and that prebranch site production and root bending are periodic. Microarray and promoter-luciferase studies revealed two sets of genes oscillating in opposite phases at the root tip. Genetic studies show that some oscillating transcriptional regulators are required for periodicity in one or both developmental processes. This molecular mechanism has characteristics that resemble molecular clock–driven activities in animal species.

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Oscillating Gene Expression Determines Competence for Periodic Arabidopsis Root Branching

Filamentous fungi produce a number of small bioactive molecules as part of their secondary metabolism ranging from benign antibiotics such as penicillin to threatening mycotoxins such as aflatoxin. Secondary metabolism can be linked to fungal developmental programs in response to various abiotic or biotic external triggers. The velvet family of regulatory proteins plays a key role in coordinating secondary metabolism and differentiation processes such as asexual or sexual sporulation and sclerotia or fruiting body formation. The velvet family shares a protein domain that is present in most parts of the fungal kingdom from chytrids to basidiomycetes. Most of the current knowledge derives from the model Aspergillus nidulans where VeA, the founding member of the protein family, was discovered almost half a century ago. Different members of the velvet protein family interact with each other and the nonvelvet protein LaeA, primarily in the nucleus. LaeA is a methyltransferase-domain protein that functions as a regulator of secondary metabolism and development. A comprehensive picture of the molecular interplay between the velvet domain protein family, LaeA and other nuclear regulatory proteins in response to various signal transduction pathway starts to emerge from a jigsaw puzzle of several recent studies.

Coordination of secondary metabolism and development in fungi: the velvet family of regulatory proteins.

Ageing leads to a functional deterioration of many brain systems, including the circadian clock--an internal time-keeping system that generates ∼24-hour rhythms in physiology and behaviour. Numerous clinical studies have established a direct correlation between abnormal circadian clock functions and the severity of neurodegenerative and sleep disorders. Latest data from experiments in model organisms, gene expression studies and clinical trials imply that dysfunctions of the circadian clock contribute to ageing and age-associated pathologies, thereby suggesting a functional link between the circadian clock and age-associated decline of brain functions. Potential molecular mechanisms underlying this link include the circadian control of physiological processes such as brain metabolism, reactive oxygen species homeostasis, hormone secretion, autophagy and stem cell proliferation.

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The circadian clock and pathology of the ageing brain

Codon usage biases are found in all eukaryotic and prokaryotic genomes, and preferred codons are more frequently used in highly expressed genes. The effects of codon usage on gene expression were previously thought to be mainly mediated by its impacts on translation. Here, we show that codon usage strongly correlates with both protein and mRNA levels genome-wide in the filamentous fungus Neurospora Gene codon optimization also results in strong up-regulation of protein and RNA levels, suggesting that codon usage is an important determinant of gene expression. Surprisingly, we found that the impact of codon usage on gene expression results mainly from effects on transcription and is largely independent of mRNA translation and mRNA stability. Furthermore, we show that histone H3 lysine 9 trimethylation is one of the mechanisms responsible for the codon usage-mediated transcriptional silencing of some genes with nonoptimal codons. Together, these results uncovered an unexpected important role of codon usage in ORF sequences in determining transcription levels and suggest that codon biases are an adaptation of protein coding sequences to both transcription and translation machineries. Therefore, synonymous codons not only specify protein sequences and translation dynamics, but also help determine gene expression levels.

https://www.pnas.org/content/pnas/113/41/E6117.full.pdf

Codon usage is an important determinant of gene expression levels largely through its effects on transcription

White collar-1 (WC-1) and white collar-2 (WC-2) are essential for light-mediated responses in Neurospora crassa, but the molecular mechanisms underlying gene induction and the roles of other real and putative photoreceptors remain poorly characterized. Unsupervised hierarchical clustering of genome-wide microarrays reveals 5.6% of detectable transcripts, including several novel mediators, that are either early or late light responsive. Evidence is shown for photoreception in the absence of the dominant, and here confirmed, white collar complex (WCC) that regulates both types of light responses. VVD primarily modulates late responses, whereas light-responsive submerged protoperithecia-1 (SUB-1), a GATA family transcription factor, is essential for most late light gene expression. After a 15-min light stimulus, the WCC directly binds the sub-1 promoter. Bioinformatics analysis detects many early light response elements (ELREs), as well as identifying a late light response element (LLRE) required for wild-type activity of late light response promoters. The data provide a global picture of transcriptional response to light, as well as illuminating the cis- and trans-acting elements comprising the regulatory signalling cascade that governs the photobiological response.

/pdf/genome-wide-analysis-of-light-inducible-responses-reveals-37uifcrfos.pdf

Genome-wide analysis of light-inducible responses reveals hierarchical light signalling in Neurospora

band, an allele enabling clear visualization of circadianly regulated spore formation (conidial banding), has remained an integral tool in the study of circadian rhythms for 40 years. bd was mapped using single-nucleotide polymorphisms (SNPs), cloned, and determined to be a T79I point mutation in ras-1. Alterations in light-regulated gene expression in the ras-1bd mutant suggests that the Neurospora photoreceptor WHITE COLLAR-1 is a target of RAS signaling, and increases in transcription of both wc-1 and fluffy show that regulators of conidiation are elevated in ras-1bd. Comparison of ras-1bd with dominant active and dominant-negative ras-1 mutants and biochemical assays of RAS function indicate that RAS-1bd displays a modest enhancement of GDP/GTP exchange and no change in GTPase activity. Because the circadian clock in ras-1bd appears to be normal, ras-1bd apparently acts to amplify a subtle endogenous clock output signal under standard assay conditions. Reactive oxygen species (ROS), which can affect and be affected by RAS signaling, increase conidiation, suggesting a link between generation of ROS and RAS-1 signaling; surprisingly, however, ROS levels are not elevated in ras-1bd. The data suggest that interconnected RAS- and ROS-responsive signaling pathways regulate the amplitude of circadian- and light-regulated gene expression in Neurospora.

/pdf/the-band-mutation-in-neurospora-crassa-is-a-dominant-allele-1e6vj2zc42.pdf

The band mutation in Neurospora crassa is a dominant allele of ras-1 implicating RAS signaling in circadian output

Photoadaptation, the ability to attenuate a light response on prolonged light exposure while remaining sensitive to escalating changes in light intensity, is essential for organisms to decipher time information appropriately, yet the underlying molecular mechanisms are poorly understood. In Neurospora crassa, VIVID (VVD), a small LOV domain containing blue-light photoreceptor protein, affects photoadaptation for most if not all light-responsive genes. We report that there is a physical interaction between VVD and the white collar complex (WCC), the primary blue-light photoreceptor and the transcription factor complex that initiates light-regulated transcriptional responses in Neurospora. Using two previously characterized VVD mutants, we show that the level of interaction is correlated with the level of WCC repression in constant light and that even light-insensitive VVD is sufficient partly to regulate photoadaptation in vivo. We provide evidence that a functional GFP-VVD fusion protein accumulates in the nucleus on light induction but that nuclear localization of VVD does not require light. Constitutively expressed VVD alone is sufficient to change the dynamics of photoadaptation. Thus, our results demonstrate a direct molecular connection between two of the most essential light signaling components in Neurospora, VVD and WCC, illuminating a previously uncharacterized process for light-sensitive eukaryotic cells.

https://www.pnas.org/content/pnas/107/38/16715.full.pdf

Physical interaction between VIVID and white collar complex regulates photoadaptation in Neurospora

Scopoletin was isolated from Sinomonium acutum and studied using four experimental models designed to assess antioxidant properties. The results indicated that scopoletin scavenged superoxide anion in the xanthine/xanthine oxidase reaction system in a concentration-dependent manner, but did not inhibit xanthine oxidase. Scopoletin may therefore be responsible for the superoxide anion scavenging activity seen in Sinomonium acutum extracts and may be of use in preventing superoxide anion-induced damage in vivo.

Antioxidant properties of scopoletin isolated from Sinomonium acutum.

Light, oxygen, or voltage (LOV) protein domains are present in many signaling proteins in bacteria, archaea, protists, plants, and fungi. The LOV protein VIVID (VVD) of the filamentous fungus Neurospora crassa enables the organism to adapt to constant or increasing amounts of light and facilitates proper entrainment of circadian rhythms. Here, we determined the crystal structure of the fully light-adapted VVD dimer and reveal the mechanism by which light-driven conformational change alters the oligomeric state of the protein. Light-induced formation of a cysteinyl-flavin adduct generated a new hydrogen bond network that released the amino (N) terminus from the protein core and restructured an acceptor pocket for binding of the N terminus on the opposite subunit of the dimer. Substitution of residues critical for the switch between the monomeric and the dimeric states of the protein had profound effects on light adaptation in Neurospora. The mechanism of dimerization of VVD provides molecular details that explain how members of a large family of photoreceptors convert light responses to alterations in protein-protein interactions.

Chen-Hui Chen

Papers

Genome-wide analysis of light-inducible responses reveals hierarchical light signalling in Neurospora

The band mutation in Neurospora crassa is a dominant allele of ras-1 implicating RAS signaling in circadian output

Physical interaction between VIVID and white collar complex regulates photoadaptation in Neurospora

Antioxidant properties of scopoletin isolated from Sinomonium acutum.

Structure of a light-activated LOV protein dimer that regulates transcription.