RESUMO
In the negative feedback loop driving fungal and animal circadian oscillators, negative elements (FREQUENCY [FRQ], PERIODS [PERs], and CRYPTOCHROMES [CRYs]) are understood to inhibit their own expression, in part by promoting the phosphorylation of their heterodimeric transcriptional activators (e.g., White Collar-1 [WC-1]-WC-2 [White Collar complex; WCC] and BMAL1/Circadian Locomotor Output Cycles Kaput [CLOCK]). However, correlations between heterodimer activity and phosphorylation are weak, contradictions exist, and mechanistic details are almost wholly lacking. We report mapping of 80 phosphosites on WC-1 and 15 on WC-2 and elucidation of the time-of-day-specific code, requiring both a group of phosphoevents on WC-1 and two distinct clusters on WC-2, that governs circadian repression, leading to feedback loop closure. Combinatorial control via phosphorylation also governs rhythmic WCC binding to the promoters of clock-controlled genes mediating the essential first step in circadian output, a group encoding both transcription factors and signaling proteins. These data provide a basic mechanistic understanding for fundamental events underlying circadian negative feedback and output, key aspects of circadian biology.
Assuntos
Ritmo Circadiano/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Neurospora crassa/genética , Fatores de Transcrição/genética , Fatores de Transcrição ARNTL/genética , Retroalimentação Fisiológica , Regulação Fúngica da Expressão Gênica , Neurospora crassa/fisiologia , Fosforilação , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genéticaRESUMO
Compensation is a defining principle of a true circadian clock, where its approximately 24-hour period length is relatively unchanged across environmental conditions. Known compensation effectors directly regulate core clock factors to buffer the oscillator's period length from variables in the environment. Temperature Compensation mechanisms have been experimentally addressed across circadian model systems, but much less is known about the related process of Nutritional Compensation, where circadian period length is maintained across physiologically relevant nutrient levels. Using the filamentous fungus Neurospora crassa, we performed a genetic screen under glucose and amino acid starvation conditions to identify new regulators of Nutritional Compensation. Our screen uncovered 16 novel mutants, and together with 4 mutants characterized in prior work, a model emerges where Nutritional Compensation of the fungal clock is achieved at the levels of transcription, chromatin regulation, and mRNA stability. However, eukaryotic circadian Nutritional Compensation is completely unstudied outside of Neurospora. To test for conservation in cultured human cells, we selected top hits from our fungal genetic screen, performed siRNA knockdown experiments of the mammalian orthologs, and characterized the cell lines with respect to compensation. We find that the wild-type mammalian clock is also compensated across a large range of external glucose concentrations, as observed in Neurospora, and that knocking down the mammalian orthologs of the Neurospora compensation-associated genes CPSF6 or SETD2 in human cells also results in nutrient-dependent period length changes. We conclude that, like Temperature Compensation, Nutritional Compensation is a conserved circadian process in fungal and mammalian clocks and that it may share common molecular determinants.
Assuntos
Relógios Circadianos , Neurospora crassa , Nutrientes , Estabilidade de RNA , Humanos , Relógios Circadianos/genética , Ritmo Circadiano/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Estabilidade de RNA/genética , Nutrientes/metabolismoRESUMO
Some longstanding dogmas in the circadian field warrant reexamination in light of recent studies focused on the role of post-translational modifications and intrinsic disorder in core circadian clock proteins of mice and fungi. Such dogmas include the role of turnover in circadian feedback loops and the origin myths describing evolutionary relatedness among circadian clocks. In this Essay, the authors recapitulate recent findings on circadian clock protein regulation by taking an unconventional approach in the form of a dialog between Wizard and Apprentice.
Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Animais , Evolução Biológica , Proteínas CLOCK , Retroalimentação Fisiológica , Fungos , Humanos , Camundongos , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
The transcription-translation negative feedback loops underlying animal and fungal circadian clocks are remarkably similar in their molecular regulatory architecture and, although much is understood about their central mechanism, little is known about the spatiotemporal dynamics of the gene products involved. A common feature of these circadian oscillators is a significant temporal delay between rhythmic accumulation of clock messenger RNAs (mRNAs) encoding negative arm proteins, for example, frq in Neurospora and Per1-3 in mammals, and the appearance of the clock protein complexes assembled from the proteins they encode. Here, we report use of single-molecule RNA fluorescence in situ hybridization (smFISH) to show that the fraction of nuclei actively transcribing the clock gene frq changes in a circadian manner, and that these mRNAs cycle in abundance with fewer than five transcripts per nucleus at any time. Spatial point patterning statistics reveal that frq is spatially clustered near nuclei in a time of day-dependent manner and that clustering requires an RNA-binding protein, PRD-2 (PERIOD-2), recently shown also to bind to mRNA encoding another core clock component, casein kinase 1. An intrinsically disordered protein, PRD-2 displays behavior in vivo and in vitro consistent with participation in biomolecular condensates. These data are consistent with a role for phase-separating RNA-binding proteins in spatiotemporally organizing clock mRNAs to facilitate local translation and assembly of clock protein complexes.
Assuntos
Proteínas CLOCK , Relógios Circadianos , Ritmo Circadiano , Proteínas Fúngicas , Neurospora crassa , Proteínas Circadianas Period , RNA Mensageiro , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hibridização in Situ Fluorescente , Neurospora crassa/genética , Neurospora crassa/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Temperature compensation of circadian clocks is an unsolved problem with relevance to the general phenomenon of biological compensation. We identify casein kinase 2 (CK2) as a key regulator of temperature compensation of the Neurospora clock by determining that two long-standing clock mutants, chrono and period-3, displaying distinctive alterations in compensation encode the beta1 and alpha subunits of CK2, respectively. Reducing the dose of these subunits, particularly beta1, significantly alters temperature compensation without altering the enzyme's Q(10). By contrast, other kinases and phosphatases implicated in clock function do not play appreciable roles in temperature compensation. CK2 exerts its effects on the clock by directly phosphorylating FREQUENCY (FRQ), and this phosphorylation is compromised in CK2 hypomorphs. Finally, mutation of certain putative CK2 phosphosites on FRQ, shown to be phosphorylated in vivo, predictably alters temperature compensation profiles effectively phenocopying CK2 mutants.
Assuntos
Caseína Quinase II/fisiologia , Ritmo Circadiano , Neurospora crassa/enzimologia , Neurospora crassa/fisiologia , Caseína Quinase II/química , Caseína Quinase II/genética , Dosagem de Genes , Mutação , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , TemperaturaRESUMO
Fungal cells are quite unique among life in their organization and structure, and yet implementation of many tools recently developed for fluorescence imaging in animal systems and yeast has been slow in filamentous fungi. Here we present analysis of properties of fluorescent proteins in Neurospora crassa as well as describing genetic tools for the expression of these proteins that may be useful beyond cell biology applications. The brightness and photostability of ten different fluorescent protein tags were compared in a well-controlled system; six different promoters are described for the assessment of the fluorescent proteins and varying levels of expression, as well as a customizable bidirectional promoter system. We present an array of fluorescent proteins suitable for use across the visible light spectrum to allow for 4-color imaging, in addition to a photoconvertible fluorescent protein that enables a change in the color of a small subset of proteins in the cell. These tools build on the rich history of cell biology research in filamentous fungi and provide new tools to help expand research capabilities.
Assuntos
Neurospora crassa , Animais , Neurospora crassa/genética , Neurospora crassa/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Corantes/metabolismoRESUMO
Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols.
Assuntos
Neurospora crassa , Parede Celular , Digestão , Neurospora crassa/genética , RNA , Ribonucleases/genéticaRESUMO
Protein conformation dictates a great deal of protein function. A class of naturally unstructured proteins, termed intrinsically disordered proteins (IDPs), demonstrates that flexibility in structure can be as important mechanistically as rigid structure. At the core of the circadian transcription/translation feedback loop in Neurospora crassa is the protein FREQUENCY (FRQ), shown here shown to share many characteristics of IDPs. FRQ in turn binds to FREQUENCY-Interacting RNA Helicase (FRH), whose clock function has been assumed to relate to its predicted helicase function. However, mutational analyses reveal that the helicase function of FRH is not essential for the clock, and a region of FRH distinct from the helicase region is essential for stabilizing FRQ against rapid degradation via a pathway distinct from its typical ubiquitin-mediated turnover. These data lead to the hypothesis that FRQ is an IDP and that FRH acts nonenzymatically, stabilizing FRQ to enable proper clock circuitry/function.
Assuntos
Proteínas CLOCK/metabolismo , Ritmo Circadiano , Proteínas Fúngicas/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Neurospora crassa/enzimologia , RNA Helicases/metabolismo , Proteínas CLOCK/genética , Ritmo Circadiano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Genótipo , Proteínas Intrinsicamente Desordenadas/genética , Mutação , Neurospora crassa/genética , Neurospora crassa/crescimento & desenvolvimento , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteólise , RNA Helicases/genética , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
Circadian clocks are ubiquitous in eukaryotic organisms where they are used to anticipate regularly occurring diurnal and seasonal environmental changes. Nevertheless, little is known regarding pathways connecting the core clock to its output pathways. Here, we report that the HAD family phosphatase CSP-6 is required for overt circadian clock output but not for the core oscillation. The loss of function Δcsp-6 deletion mutant is overtly arrhythmic on race tubes under free running conditions; however, reporter assays confirm that the FREQUENCY-WHITE COLLAR COMPLEX core circadian oscillator is functional, indicating a discrete block between oscillator and output. CSP-6 physically interacts with WHI-2, Δwhi-2 mutant phenotypes resemble Δcsp-6, and the CSP-6/WHI-2 complex physically interacts with WC-1, all suggesting that WC-1 is a direct target for CSP-6/WHI-2-mediated dephosphorylation and consistent with observed WC-1 hyperphosphorylation in Δcsp-6. To identify the source of the block to output, known clock-controlled transcription factors were screened for rhythmicity in Δcsp-6, identifying loss of circadian control of ADV-1, a direct target of WC-1, as responsible for the loss of overt rhythmicity. The CSP-6/WHI-2 complex thus participates in the clock output pathway by regulating WC-1 phosphorylation to promote proper transcriptional/translational activation of adv-1/ADV-1; these data establish an unexpected essential role for post-translational modification parallel to circadian transcriptional regulation in the early steps of circadian output.
Assuntos
Ritmo Circadiano/genética , Proteínas Fúngicas/fisiologia , Hidrolases/fisiologia , Neurospora crassa/genética , Monoéster Fosfórico Hidrolases/fisiologia , Relógios Circadianos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Hidrolases/genética , Neurospora crassa/enzimologia , Organismos Geneticamente Modificados , Fosforilação , Ligação Proteica , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
From cyanobacteria to mammals, organisms have evolved timing mechanisms to adapt to environmental changes in order to optimize survival and improve fitness. To anticipate these regular daily cycles, many organisms manifest â¼24h cell-autonomous oscillations that are sustained by transcription-translation-based or post-transcriptional negative-feedback loops that control a wide range of biological processes. With an eye to identifying emerging common themes among cyanobacterial, fungal, and animal clocks, some major recent developments in the understanding of the mechanisms that regulate these oscillators and their output are discussed. These include roles for antisense transcription, intrinsically disordered proteins, codon bias in clock genes, and a more focused discussion of post-transcriptional and translational regulation as a part of both the oscillator and output.
Assuntos
Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Ritmo Circadiano/genética , Retroalimentação Fisiológica , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Animais , Ritmo Circadiano/efeitos da radiação , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Códon , Sequência Conservada , Cianobactérias/genética , Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Fungos/genética , Fungos/metabolismo , Fungos/efeitos da radiação , Interação Gene-Ambiente , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Luz , Transdução de Sinal LuminosoRESUMO
Study of Neurospora, a model system evolutionarily related to animals and sharing a circadian system having nearly identical regulatory architecture to that of animals, has advanced our understanding of all circadian rhythms. Work on the molecular bases of the Oscillator began in Neurospora before any clock genes were cloned and provided the second example of a clock gene, frq, as well as the first direct experimental proof that the core of the Oscillator was built around a transcriptional translational negative feedback loop (TTFL). Proof that FRQ was a clock component provided the basis for understanding how light resets the clock, and this in turn provided the generally accepted understanding for how light resets all animal and fungal clocks. Experiments probing the mechanism of light resetting led to the first identification of a heterodimeric transcriptional activator as the positive element in a circadian feedback loop, and to the general description of the fungal/animal clock as a single step TTFL. The common means through which DNA damage impacts the Oscillator in fungi and animals was first described in Neurospora. Lastly, the systematic study of Output was pioneered in Neurospora, providing the vocabulary and conceptual framework for understanding how Output works in all cells. This model system has contributed to the current appreciation of the role of Intrinsic Disorder in clock proteins and to the documentation of the essential roles of protein post-translational modification, as distinct from turnover, in building a circadian clock.
Assuntos
Relógios Circadianos , Neurospora crassa , Animais , Relógios Circadianos/genética , Ritmo Circadiano , Proteínas Fúngicas/genética , Neurospora crassa/genética , Fatores de TranscriçãoRESUMO
In the Neurospora crassa circadian clock, a protein complex of frequency (FRQ), casein kinase 1a (CK1a), and the FRQ-interacting RNA Helicase (FRH) rhythmically represses gene expression by the white-collar complex (WCC). FRH crystal structures in several conformations and bound to ADP/RNA reveal differences between FRH and the yeast homolog Mtr4 that clarify the distinct role of FRH in the clock. The FRQ-interacting region at the FRH N-terminus has variable structure in the absence of FRQ A known mutation that disrupts circadian rhythms (R806H) resides in a positively charged surface of the KOW domain, far removed from the helicase core. We show that changes to other similarly located residues modulate interactions with the WCC and FRQ A V142G substitution near the N-terminus also alters FRQ and WCC binding to FRH, but produces an unusual short clock period. These data support the assertion that FRH helicase activity does not play an essential role in the clock, but rather FRH acts to mediate contacts among FRQ, CK1a and the WCC through interactions involving its N-terminus and KOW module.
Assuntos
Relógios Circadianos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Neurospora crassa/enzimologia , RNA Helicases/química , RNA Helicases/metabolismo , Cristalografia por Raios X , Proteínas Fúngicas/genética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , RNA Helicases/genéticaRESUMO
MOTIVATION: Decreasing costs are making it feasible to perform time series proteomics and genomics experiments with more replicates and higher resolution than ever before. With more replicates and time points, proteome and genome-wide patterns of expression are more readily discernible. These larger experiments require more batches exacerbating batch effects and increasing the number of bias trends. In the case of proteomics, where methods frequently result in missing data this increasing scale is also decreasing the number of peptides observed in all samples. The sources of batch effects and missing data are incompletely understood necessitating novel techniques. RESULTS: Here we show that by exploiting the structure of time series experiments, it is possible to accurately and reproducibly model and remove batch effects. We implement Learning and Imputation for Mass-spec Bias Reduction (LIMBR) software, which builds on previous block-based models of batch effects and includes features specific to time series and circadian studies. To aid in the analysis of time series proteomics experiments, which are often plagued with missing data points, we also integrate an imputation system. By building LIMBR for imputation and time series tailored bias modeling into one straightforward software package, we expect that the quality and ease of large-scale proteomics and genomics time series experiments will be significantly increased. AVAILABILITY AND IMPLEMENTATION: Python code and documentation is available for download at https://github.com/aleccrowell/LIMBR and LIMBR can be downloaded and installed with dependencies using 'pip install limbr'. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Software , Genoma , Genômica , Espectrometria de Massas , ProteômicaRESUMO
Regulatable promoters are important genetic tools, particularly for assigning function to essential and redundant genes. They can also be used to control the expression of enzymes that influence metabolic flux or protein secretion, thereby optimizing product yield in bioindustry. This review will focus on regulatable systems for use in filamentous fungi, an important group of organisms whose members include key research models, devastating pathogens of plants and animals, and exploitable cell factories. Though we will begin by cataloging those promoters that are controlled by nutritional or chemical means, our primary focus will rest on those who can be controlled by a literal flip-of-the-switch: promoters of light-regulated genes. The vvd promoter of Neurospora will first serve as a paradigm for how light-driven systems can provide tight, robust, tunable, and temporal control of either autologous or heterologous fungal proteins. We will then discuss a theoretical approach to, and practical considerations for, the development of such promoters in other species. To this end, we have compiled genes from six previously published light-regulated transcriptomic studies to guide the search for suitable photoregulatable promoters in your fungus of interest.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Genes Fúngicos/genética , Luz , Neurospora crassa/genética , Neurospora crassa/efeitos da radiação , Regiões Promotoras Genéticas/genéticaRESUMO
Most organisms on earth sense light through the use of chromophore-bearing photoreceptive proteins with distinct and characteristic photocycle lengths, yet the biological significance of this adduct decay length is neither understood nor has been tested. In the filamentous fungus Neurospora crassa VIVID (VVD) is a critical player in the process of photoadaptation, the attenuation of light-induced responses and the ability to maintain photosensitivity in response to changing light intensities. Detailed in vitro analysis of the photochemistry of the blue light sensing, FAD binding, LOV domain of VVD has revealed residues around the site of photo-adduct formation that influence the stability of the adduct state (light state), that is, altering the photocycle length. We have examined the biological significance of VVD photocycle length to photoadaptation and report that a double substitution mutant (vvdI74VI85V), previously shown to have a very fast light to dark state reversion in vitro, shows significantly reduced interaction with the White Collar Complex (WCC) resulting in a substantial photoadaptation defect. This reduced interaction impacts photoreceptor transcription factor WHITE COLLAR-1 (WC-1) protein stability when N. crassa is exposed to light: The fast-reverting mutant VVD is unable to form a dynamic VVD-WCC pool of the size required for photoadaptation as assayed both by attenuation of gene expression and the ability to respond to increasing light intensity. Additionally, transcription of the clock gene frequency (frq) is sensitive to changing light intensity in a wild-type strain but not in the fast photo-reversion mutant indicating that the establishment of this dynamic VVD-WCC pool is essential in general photobiology and circadian biology. Thus, VVD photocycle length appears sculpted to establish a VVD-WCC reservoir of sufficient size to sustain photoadaptation while maintaining sensitivity to changing light intensity. The great diversity in photocycle kinetics among photoreceptors may be viewed as reflecting adaptive responses to specific and salient tasks required by organisms to respond to different photic environments.
Assuntos
Adaptação Fisiológica/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Luz , Neurospora crassa/genética , Fotorreceptores Microbianos/genética , Fatores de Transcrição/metabolismo , Relógios Circadianos , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Loci Gênicos , Genótipo , Neurospora crassa/metabolismo , Fotorreceptores Microbianos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
Mutants in the period-1 (prd-1) gene, characterized by a recessive allele, display a reduced growth rate and period lengthening of the developmental cycle controlled by the circadian clock. We refined the genetic location of prd-1 and used whole genome sequencing to find the mutation defining it, confirming the identity of prd-1 by rescuing the mutant circadian phenotype via transformation. PRD-1 is an RNA helicase whose orthologs, DDX5 [DEAD (Asp-Glu-Ala-Asp) Box Helicase 5] and DDX17 in humans and DBP2 (Dead Box Protein 2) in yeast, are implicated in various processes, including transcriptional regulation, elongation, and termination, ribosome biogenesis, and mRNA decay. Although prd-1 mutants display a long period (â¼25 h) circadian developmental cycle, they interestingly display a WT period when the core circadian oscillator is tracked using a frq-luciferase transcriptional fusion under conditions of limiting nutritional carbon; the core oscillator in the prd-1 mutant strain runs with a long period under glucose-sufficient conditions. Thus, PRD-1 clearly impacts the circadian oscillator and is not only part of a metabolic oscillator ancillary to the core clock. PRD-1 is an essential protein, and its expression is neither light-regulated nor clock-regulated. However, it is transiently induced by glucose; in the presence of sufficient glucose, PRD-1 is in the nucleus until glucose runs out, which elicits its disappearance from the nucleus. Because circadian period length is carbon concentration-dependent, prd-1 may be formally viewed as a clock mutant with defective nutritional compensation of circadian period length.
Assuntos
Relógios Circadianos/fisiologia , Neurospora crassa/fisiologia , Proteínas Circadianas Period/genética , RNA Helicases/fisiologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Carbono/metabolismo , Glucose/metabolismo , Dados de Sequência Molecular , Proteínas Circadianas Period/fisiologiaRESUMO
Circadian systems are comprised of multiple proteins functioning together to produce feedback loops driving robust, approximately 24 hr rhythms. In all circadian systems, proteins in these loops are regulated through myriad physically and temporally distinct posttranslational modifications (PTMs). To better understand how PTMs impact a circadian oscillator, we implemented a proteomics-based approach by combining purification of endogenous FREQUENCY (FRQ) and its interacting partners with quantitative mass spectrometry (MS). We identify and quantify time-of-day-specific protein-protein interactions in the clock and show how these provide a platform for temporal and physical separation between the dual roles of FRQ. Additionally, by unambiguously identifying over 75 phosphorylated residues, following their quantitative change over a circadian cycle, and examining the phenotypes of strains that have lost these sites, we demonstrate how spatially and temporally regulated phosphorylation has opposing effects directly on overt circadian rhythms and FRQ stability.
Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Proteínas Fúngicas/metabolismo , Neurospora crassa/metabolismo , Proteômica , Mapeamento Cromossômico , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Neurospora crassa/genética , Fosforilação , Mapeamento de Interação de ProteínasRESUMO
In the negative feedback loop comprising the Neurospora circadian oscillator, the White Collar Complex (WCC) formed from White Collar-1 (WC-1) and White Collar-2 (WC-2) drives transcription of the circadian pacemaker gene frequency (frq). Although FRQ-dependent repression of WCC has been extensively studied, the mechanism by which the WCC initiates a circadian cycle remains elusive. Structure/function analysis of WC-1 eliminated domains previously thought to transactivate frq expression but instead identified amino acids 100-200 as essential for frq circadian expression. A proteomics-based search for coactivators with WCC uncovered the SWI/SNF (SWItch/Sucrose NonFermentable) complex: SWI/SNF interacts with WCC in vivo and in vitro, binds to the Clock box in the frq promoter, and is required both for circadian remodeling of nucleosomes at frq and for rhythmic frq expression; interestingly, SWI/SNF is not required for light-induced frq expression. These data suggest a model in which WC-1 recruits SWI/SNF to remodel and loop chromatin at frq, thereby activating frq expression to initiate the circadian cycle.
Assuntos
Ritmo Circadiano , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Modelos Biológicos , Mutação , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/química , Transcrição GênicaRESUMO
Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation-based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from â¼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter-luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level.
Assuntos
Relógios Circadianos , Regulação Fúngica da Expressão Gênica , Neurospora crassa/genética , Transcriptoma/genética , Metabolismo Energético/genética , Retroalimentação Fisiológica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Neurospora crassa/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Transdução de Sinais/genéticaRESUMO
Light plays an important role for most organisms on this planet, serving either as a source of energy or information for the adaptation of biological processes to specific times of day. The fungal kingdom is estimated to contain well over a million species, possibly 10-fold more, and it is estimated that a majority of the fungi respond to light, eliciting changes in several physiological characteristics including pathogenesis, development and secondary metabolism. Two model organisms for photobiological studies have taken centre-stage over the last few decades--Neurospora crassa and Aspergillus nidulans. In this review, we will first discuss our understanding of the light response in N. crassa, about which the most is known, and will then juxtapose N. crassa with A. nidulans, which, as will be described below, provides an excellent template for understanding photosensory cross-talk. Finally, we will end with a commentary on the variability of the light response among other relevant fungi, and how our molecular understanding in the aforementioned model organisms still provides a strong base for dissecting light responses in such species.