Biomedical subjects

Jennifer J Loros

Publications and source records attributed to Jennifer J Loros.

At least 19 recordsLinked to original sources

PubMed2006

How fungi keep time: circadian system in Neurospora and other fungi.

The circadian system in Neurospora remains a premier model system for understanding circadian rhythms, and evidence has now begun to accumulate suggesting broad conservation of rhythmicity amongst the filamentous fungi. A well-described transcription-translation-based negative feedback loop involving the FREQUENCY, WHITE COLLAR-1 and WHITE COLLAR-2 proteins is integral to the Neurospora system. Recent advances include descriptions of the surprisingly complex frequency transcription unit, an enhanced appreciation of the roles of kinases and their regulation in the generation of the circadian rhythm and their links to the cell cycle, and strong evidence for an additional WHITE COLLAR-associated feedback loop. Documentation of sequence homologs of integral circadian and photoresponsive proteins amongst the 42 available sequenced fungal genomes suggests unexpected roles for circadian timing among both pathogens and saprophytes.

Biological Clocks↗

PubMed2006

CLOCK leaves its mark on histones.

Transcriptional regulation of circadian rhythms in mammals involves a negative-feedback loop whereby a CLOCK-BMAL1 complex activates transcription of the genes encoding the proteins PER1 (Period 1), PER2, CRY1 (Cryptochrome 1) and CRY2, which feed back to inhibit the activity of CLOCK-BMAL1. It is becoming evident that chromatin remodeling has a crucial role in the transcriptional regulation of genes governing circadian rhythms. This is highlighted in a recent report that shows that CLOCK has histone acetyltransferase activity, thus, demonstrating that molecular components of the biological clock can themselves catalyze chromatin modifications.

ARNTL Transcription Factors↗

PubMed2006

The Neurospora checkpoint kinase 2: a regulatory link between the circadian and cell cycles.

The clock gene period-4 (prd-4) in Neurospora was identified by a single allele displaying shortened circadian period and altered temperature compensation. Positional cloning followed by functional tests show that PRD-4 is an ortholog of mammalian checkpoint kinase 2 (Chk2). Expression of prd-4 is regulated by the circadian clock and, reciprocally, PRD-4 physically interacts with the clock component FRQ, promoting its phosphorylation. DNA-damaging agents can reset the clock in a manner that depends on time of day, and this resetting is dependent on PRD-4. Thus, prd-4, the Neurospora Chk2, identifies a molecular link that feeds back conditionally from circadian output to input and the cell cycle.

Amino Acid Sequence↗

PubMed2006

Circadian rhythmicity by autocatalysis.

The temperature compensated in vitro oscillation of cyanobacterial KaiC phosphorylation, the first example of a thermodynamically closed system showing circadian rhythmicity, only involves the three Kai proteins (KaiA, KaiB, and KaiC) and ATP. In this paper, we describe a model in which the KaiA- and KaiB-assisted autocatalytic phosphorylation and dephosphorylation of KaiC are the source for circadian rhythmicity. This model, based upon autocatalysis instead of transcription-translation negative feedback, shows temperature-compensated circadian limit-cycle oscillations with KaiC phosphorylation profiles and has period lengths and rate constant values that are consistent with experimental observations.

Bacterial Proteins↗

PubMed2005

The relationship between FRQ-protein stability and temperature compensation in the Neurospora circadian clock.

Temperature compensation is an important property of all biological clocks. In Neurospora crassa, negative-feedback regulation on the frequency (frq) gene's transcription by the FRQ protein plays a central role in the organism's circadian pacemaker. Earlier model calculations predicted that the stability of FRQ should determine the period length of Neurospora's circadian rhythm as well as the rhythm's temperature compensation. Here, we report experimental FRQ protein stabilities in frq mutants at 20 degrees C and 25 degrees C, and estimates of overall activation energies for mutant FRQ protein degradation. The results are consistent with earlier model predictions, i.e., temperature compensation of Neurospora's circadian rhythm is a highly regulated process where the stability of FRQ is an important factor in determining Neurospora's circadian period as well as the clock's temperature compensation. The partial loss of temperature compensation in frq7 and frq(S513I) mutants can be described by a simple negative-feedback model (the Goodwin oscillator) when the experimentally obtained activation energies of FRQ degradation for theses mutants are incorporated into the model.

Circadian Rhythm↗

PubMed2005

The PAS/LOV protein VIVID supports a rapidly dampened daytime oscillator that facilitates entrainment of the Neurospora circadian clock.

A light-entrainable circadian clock controls development and physiology in Neurospora crassa. Existing simple models for resetting based on light pulses (so-called nonparametric entrainment) predict that constant light should quickly send the clock to an arrhythmic state; however, such a clock would be of little use to an organism in changing photoperiods in the wild, and we confirm that true, albeit dampened, rhythmicity can be observed in extended light. This rhythmicity requires the PAS/LOV protein VIVID (VVD) that acts, in the light, to facilitate expression of an oscillator that is related to, but distinguishable from, the classic FREQUENCY/WHITE-COLLAR complex (FRQ/WCC)-based oscillator that runs in darkness. VVD prevents light resetting of the clock at dawn but, by influencing frq RNA turnover, promotes resetting at dusk, thereby allowing the clock to run through the dawn transition and take its phase cues from dusk. Consistent with this, loss of VVD yields a clock whose performance follows the simple predictions of earlier models, and overexpression of VVD restores rhythmicity in the light and sensitivity of phase to the duration of the photoperiod.

Circadian Rhythm↗

PubMed2005

Temperature-modulated alternative splicing and promoter use in the Circadian clock gene frequency.

The expression of FREQUENCY, a central component of the circadian clock in Neurospora crassa, shows daily cycles that are exquisitely sensitive to the environment. Two forms of FRQ that differ in length by 99 amino acids, LFRQ and SFRQ, are synthesized from alternative initiation codons and the change in their ratio as a function of temperature contributes to robust rhythmicity across a range of temperatures. We have found frq expression to be surprisingly complex, despite our earlier prediction of a simple transcription unit based on limited cDNA sequencing. Two distinct environmentally regulated major promoters drive primary transcripts whose environmentally influenced alternative splicing gives rise to six different major mRNA species as well as minor forms. Temperature-sensitive alternative splicing determines AUG choice and, as a consequence, the ratio of LFRQ to SFRQ. Four of the six upstream ORFs are spliced out of the vast majority of frq mRNA species. Alternative splice site choice in the 5' UTR and relative use of two major promoters are also influenced by temperature, and the two promoters are differentially regulated by light. Evolutionary comparisons with the Sordariaceae reveal conservation of 5' UTR sequences, as well as significant conservation of the alternative splicing events, supporting their relevance to proper regulation of clock function.

Alternative Splicing↗

PubMed2005

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.

Blotting, Northern↗

PubMed2005

Assignment of an essential role for the Neurospora frequency gene in circadian entrainment to temperature cycles.

Circadian systems include slave oscillators and central pacemakers, and the cores of eukaryotic circadian clocks described to date are composed of transcription and translation feedback loops (TTFLs). In the model system Neurospora, normal circadian rhythmicity requires a TTFL in which a White Collar complex (WCC) activates expression of the frequency (frq) gene, and the FRQ protein feeds back to attenuate that activation. To further test the centrality of this TTFL to the circadian mechanism in Neurospora, we used low-amplitude temperature cycles to compare WT and frq-null strains under conditions in which a banding rhythm was elicited. WT cultures were entrained to these temperature cycles. Unlike those normal strains, however, frq-null mutants did not truly entrain to the same cycles. Their peaks and troughs always occurred in the cold and warm periods, respectively, strongly suggesting that the rhythm in Neurospora lacking frq function simply is driven by the temperature cycles. Previous reports suggested that a FRQ-less oscillator (FLO) could be entrained to temperature cycles, rather than being driven, and speculated that the FLO was the underlying circadian-rhythm generator. These inferences appear to derive from the use of a phase reference point affected by both the changing waveform and the phase of the oscillation. Examination of several other phase markers as well as results of additional experimental tests indicate that the FLO is, at best, a slave oscillator to the TTFL, which underlies circadian rhythm generation in Neurospora.

Animals↗

PubMed2005

Analysis of circadian rhythms in Neurospora: overview of assays and genetic and molecular biological manipulation.

The eukaryotic filamentous fungus Neurospora crassa is a tractable model system that has provided numerous insights into the molecular basis of circadian rhythms. In the core circadian clock feedback loop, WC-1 and WC-2 interact via PAS domains to heterodimerize, and this complex acts both as the circadian photoreceptor and, in the dark, as a transcription factor that promotes the expression of the frq gene. In the negative step of the loop, dimers of FRQ feed back to block the activity of the WC-1/WC-2 complex (WCC) and, in a positive step, to promote the synthesis of WC-1. Several kinases phosphorylate FRQ, leading to its ubiquitination and turnover, releasing the WC-1/WC-2 dimer to reactivate frq expression and restart the circadian cycle. Light and temperature entrainment of the clock arise from rapid light induction of frq expression and from the effect of elevated temperatures in driving higher levels of FRQ. Noncircadian candidate slave oscillators, termed FRQ-less oscillators (FLOs), have been described, each of which appears to regulate aspects of Neurospora growth or development. Overall, the core FRQ/WCC feedback loop coordinates the circadian system by regulating downstream clock-controlled genes either directly or via regulation of driven FLOs. This article provides a brief synopsis of the system and describes current assays for the Neurospora clock. Methods for genetic and molecular manipulation of the core clock are summarized, and accompanying chapters address more specifically aspects of photobiology and output.

Circadian Rhythm↗

PubMed2005

Analysis of circadian output rhythms of gene expression in Neurospora and mammalian cells in culture.

The true biology of chronobiology lies in the spectrum of processes that are controlled by the circadian clock. Although this biology plays out at the level of the whole organism, it derives, finally, from clock-driven changes in physiology, and frequently in gene expression, that occur at the level of individual cells. For this reason, analysis of gene expression rhythms measured in cell culture or in organisms that elaborate only a few cell types provides insights not possible in multicellular organisms. In this context we have used mammalian fibroblasts in culture as well as the eukaryotic filamentous fungus Neurospora crassa to study circadian output, in particular the output rhythms in gene expression that underlie so much of circadian biology. Each cell type has its own advantages: Data from mammalian cells are obviously immediately pertinent to animal cell rhythms, but the system allows little genetics and only limited amounts of material can be collected. Alternatively, Neurospora allows genetic and molecular analyses and is useful for developing concepts and models of output that can be examined in other contexts. This methods article focuses on these two systems for analysis, providing an overview of how control is presently viewed followed by current methods for its analysis.

Animals↗

PubMed2004

Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism.

We present an analysis of over 1,100 of the approximately 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.

Animals↗

PubMed2004

A nitrate-induced frq-less oscillator in Neurospora crassa.

When nitrate is the only nitrogen source, Neurospora crassa's nitrate reductase (NR) shows endogenous oscillations in its nitrate reductase activity (NRA) on a circadian time scale. These NRA oscillations can be observed in darkness or continuous light conditions and also in a frq(9) mutant in which no functional FRQ protein is formed. Even in a white-collar-1 knockout mutant, NRA oscillations have been observed, although with a highly reduced amplitude. This indicates that the NRA oscillations are not a simple output rhythm of the white-collar-driven frq oscillator but may be generated by another oscillator that contains the nit-3 autoregulatory negative feedback loop as a part. In this negative feedback loop, a product in the reaction chain catalyzed by nitrate reductase, probably glutamine, induces repression of the nitrate reductase gene and thus downregulates its own production. This is the first example of an endogenous, nutritionally induced daily rhythm with known molecular components that is observed in the absence of an intact FRQ protein.

Darkness↗

PubMed2004

The neurospora circadian system.

The eukaryotic filamentous fungus Neurospora crassa has proven to be a durable and dependable model system for the analysis of the cellular and molecular bases of circadian rhythms. Pioneering genetic analyses identified clock genes, and beginning with the cloning of frequency (frq), work over the past 2 decades has revealed the molecular basis of a core circadian clock feedback loop that has illuminated our understanding of circadian oscillators in microbes, plants, and animals. In this transcription/translation-based feedback loop, a heterodimer of the White Collar-1 (WC-1) and WC-2 proteins acts both as the circadian photoreceptor and, in the dark, as a transcription factor that promotes the expression of the frq gene. FRQ dimerizes and feeds back to block the activity of its activators (making a negative feedback loop), as well as feeding forward to promote the synthesis of its activator, WC-1. Phosphorylation of FRQ by several kinases leads to its ubiquitination and turnover, releasing the WC-1/WC-2 dimer to reactivate frq expression and restart the circadian cycle. Light resetting of the clock can be understood through the rapid light induction of frq expression and temperature resetting through the influence of elevated temperatures in driving higher levels of FRQ. Several FRQ- and WC-independent, noncircadian FRQ-less oscillators (FLOs) have been described, each of which appears to regulate aspects of Neurospora growth or development. Overall, the FRQ/white collar complex feedback loop appears to coordinate the circadian system through its activity to regulate downstream-target clock-controlled genes, either directly or via regulation of driven FLOs.

Animals↗

PubMed2003

Rhythmic binding of a WHITE COLLAR-containing complex to the frequency promoter is inhibited by FREQUENCY.

The biological clock of Neurospora crassa includes interconnected transcriptional and translational feedback loops that cause both the transcript and protein encoded by the frequency gene (frq) to undergo the robust daily oscillations in abundance, which are essential for clock function. To understand better the mechanism generating rhythmic frq transcript, reporter constructs were used to show that the oscillation in frq message is transcriptionally regulated, and a single cis-acting element in the frq promoter, the Clock Box (C box), is both necessary and sufficient for this rhythmic transcription. Nuclear protein extracts used in binding assays revealed that a White Collar (WC)-1- and WC-2-containing complex (WCC) binds to the C box in a time-of-day-specific manner. Overexpression of an ectopic copy of FRQ or addition of in vitro-generated FRQ resulted in reduced WCC binding to the C box. These data suggest that oscillations in frq transcript result from WCC binding to the frq promoter and activating transcription with subsequent changes in FRQ levels having an inverse effect on WCC binding. In this way rhythmic expression and turnover of FRQ drives the rhythm in its own transcription.

Amino Acid Sequence↗

PubMed2003

Role for antisense RNA in regulating circadian clock function in Neurospora crassa.

The prevalence of antisense RNA in eukaryotes is not known and only a few naturally occurring antisense transcripts have been assigned a function. However, the recent identification of a large number of putative antisense transcripts strengthens the view that antisense RNAs might affect a wider variety of processes than previously thought. Here we show that in the model organism Neurospora crassa entrainment of the circadian clock, which is critical for the correct temporal expression of genes and their products, is controlled partly by an antisense RNA arising from a clock component locus. In a wild-type strain, levels of antisense frequency (frq) transcripts cycle in antiphase to sense frq transcripts in the dark, and are inducible by light. In mutant strains in which the induction of antisense frq RNA by light is abolished, the time of the internal clock is delayed relative to the wild-type strain, and resetting of the clock by light is altered. These data provide an unexpected link between antisense RNA and circadian timing and provide a new example of a eukaryotic cellular process regulated by naturally occurring antisense RNA.

Circadian Rhythm↗

PubMed2003

Roles for WHITE COLLAR-1 in circadian and general photoperception in Neurospora crassa.

The transcription factors WHITE COLLAR-1 (WC-1) and WHITE COLLAR-2 (WC-2) interact to form a heterodimeric complex (WCC) that is essential for most of the light-mediated processes in Neurospora crassa. WCC also plays a distinct non-light-related role as the transcriptional activator in the FREQUENCY (FRQ)/WCC feedback loop that is central to the N. crassa circadian system. Although an activator role was expected for WC-1, unanticipated phenotypes resulting from some wc-1 alleles prompted a closer examination of an allelic series for WC-1 that has uncovered roles for this central regulator in constant darkness and in response to light. We analyzed the phenotypes of five different wc-1 mutants for expression of FRQ and WC-1 in constant darkness and following light induction. While confirming the absolute requirement of WC-1 for light responses, the data suggest multiple levels of control for light-regulated genes.

Circadian Rhythm↗

PubMed2003

The frequency gene is required for temperature-dependent regulation of many clock-controlled genes in Neurospora crassa.

The circadian clock of Neurospora broadly regulates gene expression and is synchronized with the environment through molecular responses to changes in ambient light and temperature. It is generally understood that light entrainment of the clock depends on a functional circadian oscillator comprising the products of the wc-1 and wc-2 genes as well as those of the frq gene (the FRQ/WCC oscillator). However, various models have been advanced to explain temperature regulation. In nature, light and temperature cues reinforce one another such that transitions from dark to light and/or cold to warm set the clock to subjective morning. In some models, the FRQ/WCC circadian oscillator is seen as essential for temperature-entrained clock-controlled output; alternatively, this oscillator is seen exclusively as part of the light pathway mediating entrainment of a cryptic "driving oscillator" that mediates all temperature-entrained rhythmicity, in addition to providing the impetus for circadian oscillations in general. To identify novel clock-controlled genes and to examine these models, we have analyzed gene expression on a broad scale using cDNA microarrays. Between 2.7 and 5.9% of genes were rhythmically expressed with peak expression in the subjective morning. A total of 1.4-1.8% of genes responded consistently to temperature entrainment; all are clock controlled and all required the frq gene for this clock-regulated expression even under temperature-entrainment conditions. These data are consistent with a role for frq in the control of temperature-regulated gene expression in N. crassa and suggest that the circadian feedback loop may also serve as a sensor for small changes in ambient temperature.

Biological Clocks↗