Jay C. Dunlap

Molecular Bases for Circadian Clocks

been PERFRQT, reflecting the Drosophila period gene Dartmouth Medical School and the Neurospora frequency gene (the fruits of the Hanover, New Hampshire 03755 first decades of genetic and molecular genetic analysis of clocks) and the fact that the Drosophila timeless gene, tim, was still in the process of arriving. This era was Life is a cyclical chemical process that is regulated in spent convincing ourselves that such genes really were four dimensions. We distinguish parts of the cycle: de-the key to understanding how clocks work. Flies and velopment describes the changes from single cell to fungi were PERFRQT systems for working out basic adult, and aging the changes from adult to death. Birth tools, paradigms, and approaches—gene products whose to death, a cycle, and there are cycles within cycles— expression levels themselves oscillate, the importance circannual rhythms, menstrual cycles, semilunar cycles, of negative feedback, criteria to begin to distinguish and daily 24 hr or circadian cycles. which oscillatory gene products might contribute to the Twice a year we get a reminder of the importance of action of an internal timer as distinct from being output our internal circadian biological clocks. Daylight sav-(reviewed in Dunlap, 1996), and a universal appreciation ings: in October we fall back just an hour, and yet we of the importance of genetics. If overall this left us with wake up an hour early on Monday anyway and think a less than PERFRQT understanding of timing in gen-meals are late—but only for a day, until our clocks are eral, at least many found optimism in the sense that we reset. The reminder is about the way we process envi-were, finally, asking the right questions. This naturally ronmental information and time, namely that we use segued into an interlude where light resetting was ex-external time cues (light and temperature changes that plained by two different mechanisms, through transcrip-track the day without) to set an internal clock that guides tional induction of oscillator components in Neurospora the day within. This internal clock is the lens through (Crosthwaite et al., 1995) or protein turnover in Drosoph-which we survey acute external factors; it takes the lead ila (reviewed in Young, 1998). But by mid 1997 the word in determining what we perceive as time. was PASWCCLK (the first clock components with known It used to be that research in chronobiology moved biochemical functions [transcriptional activators], the along at a gentlemanly pace. …

Cross-species microarray hybridization to identify developmentally regulated genes in the filamentous fungus Sordaria macrospora

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies that protect the developing ascospores and ensure their proper discharge. Several regulatory genes essential for fruiting body development were previously isolated by complementation of the sterile mutants pro1, pro11 and pro22. To establish the genetic relationships between these genes and to identify downstream targets, we have conducted cross-species microarray hybridizations using cDNA arrays derived from the closely related fungus Neurospora crassa and RNA probes prepared from wild-type S. macrospora and the three developmental mutants. Of the 1,420 genes which gave a signal with the probes from all the strains used, 172 (12%) were regulated differently in at least one of the three mutants compared to the wild type, and 17 (1.2%) were regulated differently in all three mutant strains. Microarray data were verified by Northern analysis or quantitative real time PCR. Among the genes that are up- or down-regulated in the mutant strains are genes encoding the pheromone precursors, enzymes involved in melanin biosynthesis and a lectin-like protein. Analysis of gene expression in double mutants revealed a complex network of interaction between the pro gene products.

A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors

The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.

Light-Induced Resetting of a Mammalian Circadian Clock Is Associated with Rapid Induction of the mPer1 Transcript

To understand how light might entrain a mammalian circadian clock, we examined the effects of light on mPer1, a sequence homolog of Drosophila per, that exhibits robust rhythmic expression in the SCN. mPer1 is rapidly induced by short duration exposure to light at levels sufficient to reset the clock, and dose-response curves reveal that mPer1 induction shows both reciprocity and a strong correlation with phase shifting of the overt rhythm. Thus, in both the phasing of dark expression and the response to light mPer1 is most similar to the Neurospora clock gene frq. Within the SCN there appears to be localization of the induction phenomenon, consistent with the localization of both light-sensitive and light-insensitive oscillators in this circadian center.

Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism

SUMMARY We present an analysis of over 1,100 of the ∼10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

Light-induced resetting of a circadian clock is mediated by a rapid increase in frequency transcript.

To understand how light entrains circadian clocks, we examined the effects of light on a gene known to encode a state variable of a circadian oscillator, the frequency (frq) gene. frq is rapidly induced by short pulses of visible light; clock resetting is correlated with frq induction and is blocked by drugs that block the synthesis of protein or translatable RNA. The speed and magnitude of frq induction suggest that this may be the initial clock-specific event in light resetting. Light induction overcomes frq negative autoregulation so that frq expression can remain high in constant light. These data explain how a simple unidirectional signal (light and the induction of frq) may be turned into a bidirectional clock response (time of day-specific advances and delays). This light entrainment model is easily generalized and may be the common mechanism by which the intracellular feedback cycles that comprise circadian clocks are brought into synchrony with external cycles in the real world.

Alternative Initiation of Translation and Time-Specific Phosphorylation Yield Multiple Forms of the Essential Clock Protein FREQUENCY

The frequency (frq) gene encodes central components of the transcription/translation-based negative-feedback loop comprising the core of the Neurospora circadian oscillator; posttranscriptional regulation associated with FRQ is surprisingly complex. Alternative use of translation initiation sites gives rise to two forms of FRQ whose levels peak 4-6 hr following the peak of frq transcript. Each form of FRQ is progressively phosphorylated over the course of the day, thus providing a number of temporally distinct FRQ products. The kinetics of these regulatory processes suggest a view of the clock where relatively rapid events involving translational regulation in the synthesis of FRQ and negative feedback of FRQ on frq transcript levels are followed by slower posttranslational regulation, ultimately driving the turnover of FRQ and reactivation of the frq gene.

Circadian programs of transcriptional activation, signaling, and protein turnover revealed by microarray analysis of mammalian cells.

Many aspects of physiology and behavior are temporally organized into daily 24 hr rhythms, driven by an endogenous circadian clock. Studies in eukaryotes have identified a network of interacting genes forming interlocked autoregulatory feedback loops which underlie overt circadian organization in single cells. While in mammals the master oscillator resides in the suprachiasmatic nuclei of the hypothalamus, semiautonomous circadian oscillators also exist in peripheral tissues and in immortalized fibroblasts, where rhythmicity is induced following a serum shock. We used this model system in combination with high-density cDNA microarrays to examine the magnitude and quality of clock control of gene expression in mammalian cells. Supported by application of novel bioinformatics tools, we find approximately 2% of genes, including expected canonical clock genes, to show consistent rhythmic circadian expression across five independent experiments. Rhythmicity in most of these genes is novel, and they fall into diverse functional groups, highlighted by a predominance of transcription factors, ubiquitin-associated factors, proteasome components, and Ras/MAPK signaling pathway components. When grouped according to phase, 68% of the genes were found to peak during estimated subjective day, 32% during estimated subjective night, with a tendency to peak at a phase corresponding to anticipation of dawn or dusk.

The PAS protein VIVID defines a clock-associated feedback loop that represses light input, modulates gating, and regulates clock resetting.

vvd, a gene regulating light responses in Neurospora, encodes a novel member of the PAS/LOV protein superfamily. VVD defines a circadian clock-associated autoregulatory feedback loop that influences light resetting, modulates circadian gating of input by connecting output and input, and regulates light adaptation. Rapidly light induced, vvd is an early repressor of light-regulated processes. Further, vvd is clock controlled; the clock gates light induction of vvd and the clock gene frq so identical signals yield greater induction in the morning. Mutation of vvd severely dampens gating, especially of frq, consistent with VVD modulating gating and phasing light-resetting responses. vvd null strains display distinct alterations in the phase-response curve to light. Thus VVD, although not part of the clock, contributes significantly to regulation within the Neurospora circadian system.

Conformational Switching in the Fungal Light Sensor Vivid

The Neurospora crassa photoreceptor Vivid tunes blue-light responses and modulates gating of the circadian clock. Crystal structures of dark-state and light-state Vivid reveal a light, oxygen, or voltage Per-Arnt-Sim domain with an unusual N-terminal cap region and a loop insertion that accommodates the flavin cofactor. Photoinduced formation of a cystein-flavin adduct drives flavin protonation to induce an N-terminal conformational change. A cysteine-to-serine substitution remote from the flavin adenine dinucleotide binding site decouples conformational switching from the flavin photocycle and prevents Vivid from sending signals in Neurospora. Key elements of this activation mechanism are conserved by other photosensors such as White Collar-1, ZEITLUPE, ENVOY, and flavin-binding, kelch repeat, F-BOX 1 (FKF1).