Direct transcriptional control of a p38 MAPK pathway by the circadian clock in Neurospora crassa.
ABSTRACT: MAPK signal transduction pathways are important regulators of stress responses, cellular growth, and differentiation. In Neurospora, the circadian clock controls rhythms in phosphorylation of the p38-like MAPK (OS-2); however, the mechanism for this regulation is not known. We show that the WCC, a transcription factor and clock component, binds to the os-4 MAPKKK promoter in response to light and rhythmically in constant darkness, peaking in the subjective morning. Deletion of the WCC binding sites in the os-4 promoter disrupts both os-4 mRNA and OS-2 phosphorylation rhythms. The clock also indirectly regulates rhythmic expression of the histidyl-phosphotransferase gene, hpt-1, which peaks in the evening. Anti-phase expression of positive (OS-4) and negative (HPT-1) MAPK pathway regulators likely coordinate to enhance rhythmic MAPK activation to prepare cells to respond to osmotic stress during the day in the natural environment. Consistent with this idea, we show that wild type cells have a clock-dependent morning kinetic advantage in glycerol accumulation after salt stress as compared to evening treatment. Thus, circadian transcriptional control of MAPK pathway components leads to striking time-of-day-specific effects on the signaling status and physiological response of the pathway.
Project description:Circadian clocks control rhythms in physiology and behavior entrained to 24 h light-dark cycles. Despite of conserved general schemes, molecular circadian clockworks differ between insect species. With RNA interference (RNAi) we examined an ancient circadian clockwork in a basic insect, the hemimetabolous Madeira cockroach Rhyparobia maderae. With injections of double-stranded RNA (dsRNA) of cockroach period (Rm´per), timeless 1 (Rm´tim1), or cryptochrome 2 (Rm´cry2) we searched for essential components of the clock´s core negative feedback loop. Single injections of dsRNA of each clock gene into adult cockroaches successfully and permanently knocked down respective mRNA levels within ~two weeks deleting daytime-dependent mRNA rhythms for Rm´per and Rm´cry2. Rm´perRNAi or Rm´cry2RNAi affected total mRNA levels of both genes, while Rm´tim1 transcription was independent of both, also keeping rhythmic expression. Unexpectedly, circadian locomotor activity of most cockroaches remained rhythmic for each clock gene knockdown employed. It expressed weakened rhythms and unchanged periods for Rm´perRNAi and shorter periods for Rm´tim1RNAi and Rm´cry2RNAi.As a hypothesis of the cockroach´s molecular clockwork, a basic network of switched differential equations was developed to model the oscillatory behavior of clock cells expressing respective clock genes. Data were consistent with two synchronized main groups of coupled oscillator cells, a leading (morning) oscillator, or a lagging (evening) oscillator that couple via mutual inhibition. The morning oscillators express shorter, the evening oscillators longer endogenous periods based on core feedback loops with either PER, TIM1, or CRY2/PER complexes as dominant negative feedback of the clockwork. We hypothesize that dominant morning oscillator cells with shorter periods express PER, but not CRY2, or TIM1 as suppressor of clock gene expression, while two groups of evening oscillator cells with longer periods either comprise TIM1 or CRY2/PER suppressing complexes. Modelling suggests that there is an additional negative feedback next to Rm´PER in cockroach morning oscillator cells.
Project description:The circadian system induces rhythmic variation in a suite of biochemical and physiological processes that serve to optimise plant growth in diel cycles. To be of greatest utility, these rhythmic behaviors are coordinated with regular environmental changes such as the rising and setting of the sun. Photoreceptors, along with metabolites produced during photosynthesis, act to synchronise the internal timing mechanism with lighting cues. We have recently shown that phototropins help maintain robust rhythms of photosynthetic operating efficiency (ϕPSII or Fq'/Fm') under blue light, although rhythmic accumulation of morning-phased circadian transcripts in the nucleus was unaffected. Here we report that evening-phased nuclear clock transcripts were also unaffected. We also observe that rhythms of nuclear clock transcript accumulation are maintained in phototropin mutant plants under a fluctuating lighting regime that induced a loss of Fq'/Fm' rhythms.
Project description:Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest-activity rhythms and relies upon different groups of PERIOD (PER)-expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day-night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons.
Project description:Circadian clocks regulate membrane excitability in master pacemaker neurons to control daily rhythms of sleep and wake. Here, we find that two distinctly timed electrical drives collaborate to impose rhythmicity on Drosophila clock neurons. In the morning, a voltage-independent sodium conductance via the NA/NALCN ion channel depolarizes these neurons. This current is driven by the rhythmic expression of NCA localization factor-1, linking the molecular clock to ion channel function. In the evening, basal potassium currents peak to silence clock neurons. Remarkably, daily antiphase cycles of sodium and potassium currents also drive mouse clock neuron rhythms. Thus, we reveal an evolutionarily ancient strategy for the neural mechanisms that govern daily sleep and wake.
Project description:<h4>Background</h4>Circadian rhythms modulate growth and development in all organisms through interlocking transcriptional-translational feedback loops. The transcriptional loop involves chromatin modifications of central circadian oscillators in mammals and plants. However, the molecular basis for rhythmic epigenetic modifications and circadian regulation is poorly understood.<h4>Results</h4>Here we report a feedback relationship between diurnal regulation of circadian clock genes and histone modifications in Arabidopsis. On one hand, the circadian oscillators CCA1 and LHY regulate diurnal expression of genes coding for the eraser (JMJ14) directly and writer (SDG2) indirectly for H3K4me3 modification, leading to rhythmic H3K4me3 changes in target genes. On the other hand, expression of circadian oscillator genes including CCA1 and LHY is associated with H3K4me3 levels and decreased in the sdg2 mutant but increased in the jmj14 mutant. At the genome-wide level, diurnal rhythms of H3K4me3 and another histone mark H3K9ac are associated with diurnal regulation of 20-30% of the expressed genes. While the majority (86%) of H3K4me3 and H3K9ac target genes overlap, only 13% of morning-phased and 22% of evening-phased genes had both H3K4me3 and H3K9ac peaks, suggesting specific roles of different histone modifications in diurnal gene expression.<h4>Conclusions</h4>Circadian clock genes promote diurnal regulation of SDG2 and JMJ14 expression, which in turn regulate rhythmic histone modification dynamics for the clock and its output genes. This reciprocal regulatory module between chromatin modifiers and circadian clock oscillators orchestrates diurnal gene expression that governs plant growth and development.
Project description:Circadian clocks generate rhythms in cellular functions, including metabolism, to align biological processes with the 24-hour environment. Disruption of this alignment by shift work alters glucose homeostasis. Glucose homeostasis depends on signaling and allosteric control; however, the molecular mechanisms linking the clock to glucose homeostasis remain largely unknown. We investigated the molecular links between the clock and glycogen metabolism, a conserved glucose homeostatic process, in <i>Neurospora crassa</i> We find that glycogen synthase (<i>gsn</i>) mRNA, glycogen phosphorylase (<i>gpn</i>) mRNA, and glycogen levels, accumulate with a daily rhythm controlled by the circadian clock. Because the synthase and phosphorylase are critical to homeostasis, their roles in generating glycogen rhythms were investigated. We demonstrate that while <i>gsn</i> was necessary for glycogen production, constitutive <i>gsn</i> expression resulted in high and arrhythmic glycogen levels, and deletion of <i>gpn</i> abolished <i>gsn</i> mRNA rhythms and rhythmic glycogen accumulation. Furthermore, we show that <i>gsn</i> promoter activity is rhythmic and is directly controlled by core clock component white collar complex (WCC). We also discovered that WCC-regulated transcription factors, VOS-1 and CSP-1, modulate the phase and amplitude of rhythmic <i>gsn</i> mRNA, and these changes are similarly reflected in glycogen oscillations. Together, these data indicate the importance of clock-regulated <i>gsn</i> transcription over signaling or allosteric control of glycogen rhythms, a mechanism that is potentially conserved in mammals and critical to metabolic homeostasis.
Project description:In Drosophila, neuropeptide Pigment Dispersing Factor (PDF) is expressed in small and large ventral Lateral Neurons (sLNv and lLNv), among which sLNv are critical for activity rhythms in constant darkness. Studies show that this is mediated by rhythmic accumulation and likely secretion of PDF from sLNv dorsal projections, which in turn synchronises molecular oscillations in downstream circadian neurons. Using targeted expression of a neurodegenerative protein Huntingtin in LNv, we evoke a selective loss of neuropeptide PDF and clock protein PERIOD from sLNv soma. However, PDF is not lost from sLNv dorsal projections and lLNv. These flies are behaviourally arrhythmic in constant darkness despite persistence of PDF oscillations in sLNv dorsal projections and synchronous PERIOD oscillations in downstream circadian neurons. We find that PDF oscillations in sLNv dorsal projections are not sufficient for sustenance of activity rhythms in constant darkness and this is suggestive of an additional component that is possibly dependent on sLNv molecular clock and PDF in sLNv soma. Additionally, despite loss of PERIOD in sLNv, their activity rhythms entrain to light/dark cycles indicating that sLNv molecular clocks are not necessary for entrainment. Under constant light, these flies lack PDF from both soma and dorsal projections of sLNv, and when subjected to light/dark cycles, show morning and evening anticipation and accurately phased morning and evening peaks. Thus, under light/dark cycles, PDF in sLNv is not necessary for morning anticipation.
Project description:The OS-pathway mitogen-activated protein kinase (MAPK) cascade of Neurospora crassa is responsible for adaptation to osmotic stress. Activation of the MAPK, OS-2, leads to the transcriptional induction of many genes involved in the osmotic stress response. We previously demonstrated that there is a circadian rhythm in the phosphorylation of OS-2 under constant non-stress inducing conditions. Additionally, several osmotic stress-induced genes are known to be regulated by the circadian clock. Therefore, we investigated if rhythms in activation of OS-2 lead to circadian rhythms in other known stress responsive targets. Here we identify three more osmotic stress induced genes as rhythmic: cat-1, gcy-1, and gcy-3. These genes encode a catalase and two predicted glycerol dehydrogenases thought to be involved in the production of glycerol. Rhythms in these genes depend upon the oscillator component FRQ. To investigate how the circadian signal is propagated to these stress induced genes, we examined the role of the OS-responsive transcription factor, ASL-1, in mediating circadian gene expression. We find that while the asl-1 transcript is induced by several stresses including an osmotic shock, asl-1 mRNA accumulation is not rhythmic. However, we show that ASL-1 is required for generating normal circadian rhythms of some OS-pathway responsive transcripts (bli-3, ccg-1, cat-1, gcy-1 and gcy-3) in the absence of an osmotic stress. These data are consistent with the possibility that post-transcriptional regulation of ASL-1 by the rhythmically activated OS-2 MAPK could play a role in generating rhythms in downstream targets.
Project description:Regulation of circadian behavior and physiology by the <i>Drosophila</i> brain clock requires communication from central clock neurons to downstream output regions, but the mechanism by which clock cells regulate downstream targets is not known. We show here that the pars intercerebralis (PI), previously identified as a target of the morning cells in the clock network, also receives input from evening cells. We determined that morning and evening clock neurons have time-of-day-dependent connectivity to the PI, which is regulated by specific peptides as well as by fast neurotransmitters. Interestingly, PI cells that secrete the peptide DH44, and control rest:activity rhythms, are inhibited by clock inputs while insulin-producing cells (IPCs) are activated, indicating that the same clock cells can use different mechanisms to drive cycling in output neurons. Inputs of morning cells to IPCs are relevant for the circadian rhythm of feeding, reinforcing the role of the PI as a circadian relay that controls multiple behavioral outputs. Our findings provide mechanisms by which clock neurons signal to nonclock cells to drive rhythms of behavior.
Project description:BACKGROUND: Circadian clocks control rhythmic expression of a large number of genes in coordination with the 24 hour day-night cycle. The mechanisms generating circadian rhythms, their amplitude and circadian phase are dependent on a transcriptional network of immense complexity. Moreover, the contribution of post-transcriptional mechanisms in generating rhythms in RNA abundance is not known. RESULTS: Here, we analyzed the clock-controlled transcriptome of Neurospora crassa together with temporal profiles of elongating RNA polymerase II. Our data indicate that transcription contributes to the rhythmic expression of the vast majority of clock-controlled genes (ccgs) in Neurospora. The ccgs accumulate in two main clusters with peak transcription and expression levels either at dawn or dusk. Dawn-phased genes are predominantly involved in catabolic and dusk-phased genes in anabolic processes, indicating a clock-controlled temporal separation of the physiology of Neurospora. Genes whose expression is strongly dependent on the core circadian activator WCC fall mainly into the dawn-phased cluster while rhythmic genes regulated by the glucose-dependent repressor CSP1 fall predominantly into the dusk-phased cluster. Surprisingly, the number of rhythmic transcripts increases about twofold in the absence of CSP1, indicating that rhythmic expression of many genes is attenuated by the activity of CSP1. CONCLUSIONS: The data indicate that the vast majority of transcript rhythms in Neurospora are generated by dawn and dusk specific transcription. Our observations suggest a substantial plasticity of the circadian transcriptome with respect to the number of rhythmic genes as well as amplitude and phase of the expression rhythms and emphasize a major role of the circadian clock in the temporal organization of metabolism and physiology.