Domains required for the interaction of the central negative element FRQ with its transcriptional activator WCC within the core circadian clock of Neurospora.

In the negative feedback loop composing the Neurospora circadian clock, the core element, FREQUENCY (FRQ), binds with FRQ-interacting RNA helicase (FRH) and casein kinase 1 to form the FRQ-FRH complex (FFC) which represses its own expression by interacting with and promoting phosphorylation of its transcriptional activators White Collar-1 (WC-1) and WC-2 (together forming the White Collar complex, WCC). Physical interaction between FFC and WCC is a prerequisite for the repressive phosphorylations, and although the motif on WCC needed for this interaction is known, the reciprocal recognition motif(s) on FRQ remains poorly defined. To address this, we assessed FFC-WCC in a series of frq segmental-deletion mutants, confirming that multiple dispersed regions on FRQ are necessary for its interaction with WCC. Biochemical analysis shows that interaction between FFC and WCC but not within FFC or WCC can be disrupted by high salt, suggesting that electrostatic forces drive the association of the two complexes. As a basic sequence on WC-1 was previously identified as a key motif for WCC-FFC assembly, our mutagenetic analysis targeted negatively charged residues of FRQ, leading to identification of three Asp/Glu clusters in FRQ that are indispensable for FFC-WCC formation. Surprisingly, in several frq Asp/Glu-to-Ala mutants that vastly diminish FFC-WCC interaction, the core clock still oscillates robustly with an essentially wildtype period, indicating that the interaction between the positive and negative elements in the feedback loop is required for the operation of the circadian clock but is not a determinant of the period length.

Differential regulation of phosphorylation, structure, and stability of circadian clock protein FRQ isoforms.

Neurospora crassa is an important model organism for circadian clock research. The Neurospora core circadian component FRQ protein has two isoforms, large FRQ (l-FRQ) and small FRQ (s-FRQ), of which l-FRQ bears an additional N-terminal 99-amino acid fragment. However, how the FRQ isoforms operate differentially in regulating the circadian clock remains elusive. Here, we show l-FRQ and s-FRQ play different roles in regulating the circadian negative feedback loop. Compared to s-FRQ, l-FRQ is less stable and undergoes hypophosphorylation and faster degradation. The phosphorylation of the C-terminal l-FRQ 794-aa fragment was markedly higher than that of s-FRQ, suggesting the l-FRQ N-terminal 99-aa region may regulate the phosphorylation of the entire FRQ protein. Quantitative label-free LC/MS analysis identified several peptides that were differentially phosphorylated between l-FRQ and s-FRQ, which were distributed in FRQ in an interlaced fashion. Furthermore, we identified two novel phosphorylation sites, S765 and T781; mutations S765A and T781A showed no significant effects on conidiation rhythmicity, although T781 conferred FRQ stability. These findings demonstrate that FRQ isoforms play differential roles in the circadian negative feedback loop and undergo different regulations of phosphorylation, structure, and stability. The l-FRQ N-terminal 99-aa region plays an important role in regulating the phosphorylation, stability, conformation, and function of the FRQ protein. As the FRQ circadian clock counterparts in other species also have isoforms or paralogues, these findings will also further our understanding of the underlying regulatory mechanisms of the circadian clock in other organisms based on the high conservation of circadian clocks in eukaryotes.

Functional analysis of 110 phosphorylation sites on the circadian clock protein FRQ identifies clusters determining period length and temperature compensation.

In the negative feedback loop driving the Neurospora circadian oscillator, the negative element, FREQUENCY (FRQ), inhibits its own expression by promoting phosphorylation of its heterodimeric transcriptional activators, White Collar-1 (WC-1) and WC-2. FRQ itself also undergoes extensive time-of-day-specific phosphorylation with over 100 phosphosites previously documented. Although disrupting individual or certain clusters of phosphorylation sites has been shown to alter circadian period lengths to some extent, it is still elusive how all the phosphorylations on FRQ control its activity. In this study, we systematically investigated the role in period determination of all 110 reported FRQ phosphorylation sites, using mutagenesis and luciferase reporter assays. Surprisingly, robust FRQ phosphorylation is still detected even when 84 phosphosites were eliminated altogether; further mutating another 26 phosphoresidues completely abolished FRQ phosphorylation. To identify phosphoresidue(s) on FRQ impacting circadian period length, a series of clustered frq phosphomutants covering all the 110 phosphosites were generated and examined for period changes. When phosphosites in the N-terminal and middle regions of FRQ were eliminated, longer periods were typically seen while removal of phosphorylation in the C-terminal tail resulted in extremely short periods, among the shortest reported. Interestingly, abolishing the 11 phosphosites in the C-terminal tail of FRQ not only results in an extremely short period, but also impacts temperature compensation (TC), yielding an overcompensated circadian oscillator. In addition, the few phosphosites in the middle of FRQ are also found to be crucial for TC. When different groups of FRQ phosphomutations were combined intramolecularly, expected additive effects were generally observed except for one novel case of intramolecular epistasis, where arrhythmicity resulting from one cluster of phosphorylation site mutants was restored by eliminating phosphorylation at another group of sites.

Neurospora casein kinase 1a recruits the circadian clock protein FRQ via the C-terminal lobe of its kinase domain.

Timing by the circadian clock of Neurospora is associated with hyperphosphorylation of frequency (FRQ), which depends on anchoring casein kinase 1a (CK1a) to FRQ. It is not known how CK1a is anchored so that approximately 100 sites in FRQ can be targeted. Here, we identified two regions in CK1a, p1 and p2, that are required for anchoring to FRQ. Mutation of p1 or p2 impairs progressive hyperphosphorylation of FRQ. A p1-mutated strain is viable but its circadian clock is non-functional, whereas a p2-mutated strain is non-viable. Our data suggest that p1 and potentially also p2 in CK1a provide an interface for interaction with FRQ. Anchoring via p1-p2 leaves the active site of CK1a accessible for phosphorylation of FRQ at multiple sites.

Casein kinase 1 and disordered clock proteins form functionally equivalent, phospho-based circadian modules in fungi and mammals.

Circadian clocks are timing systems that rhythmically adjust physiology and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks are based on transcriptional-translational feedback loops (TTFLs). Yet TTFL-core components such as Frequency (FRQ) in Neurospora and Periods (PERs) in animals are not conserved, leaving unclear how a 24-h period is measured on the molecular level. Here, we show that CK1 is sufficient to promote FRQ and mouse PER2 (mPER2) hyperphosphorylation on a circadian timescale by targeting a large number of low-affinity phosphorylation sites. Slow phosphorylation kinetics rely on site-specific recruitment of Casein Kinase 1 (CK1) and access of intrinsically disordered segments of FRQ or mPER2 to bound CK1 and on CK1 autoinhibition. Compromising CK1 activity and substrate binding affects the circadian clock in Neurospora and mammalian cells, respectively. We propose that CK1 and the clock proteins FRQ and PERs form functionally equivalent, phospho-based timing modules in the core of the circadian clocks of fungi and animals.

Cellular Calcium Levels Influenced by NCA-2 Impact Circadian Period Determination in Neurospora.

Intracellular calcium signaling has been implicated in the control of a variety of circadian processes in animals and plants, but its role in microbial clocks has remained largely cryptic. To examine the role of intracellular Ca2+ in the Neurospora clock, we screened mutants with knockouts of calcium transporter genes and identified a gene encoding a calcium exporter, nca-2, uniquely as having significant period effects. The loss of NCA-2 results in an increase in the cytosolic calcium level, and this leads to hyper-phosphorylation of core clock components, FRQ and WC-1, and a short period, as measured by both the core oscillator and the overt clock. Genetic analyses showed that mutations in certain frq phospho-sites and in Ca2+-calmodulin-dependent kinase 2 (camk-2) are epistatic to nca-2 in controlling the pace of the oscillator. These data are consistent with a model in which elevated intracellular Ca2+ leads to the increased activity of CAMK-2, leading to enhanced FRQ phosphorylation, accelerated closure of the circadian feedback loop, and a shortened circadian period length. At a mechanistic level, some CAMKs undergo more auto-phosphorylations in the Δnca-2 mutant, consistent with high calcium levels in the Δnca-2 mutant influencing the enzymatic activities of CAMKs. NCA-2 interacts with multiple proteins, including CSP-6, a protein known to be required for circadian output. Most importantly, the expression of nca-2 is circadian clock-controlled at both the transcriptional and translational levels, and this in combination with the period effects seen in strains lacking NCA-2 firmly places calcium signaling within the larger circadian system, where it acts as both an input to and an output from the core clock. IMPORTANCE Circadian rhythms are based on cell-autonomous, auto-regulatory feedback loops formed by interlocked positive and negative arms, and they regulate myriad molecular and cellular processes in most eukaryotes, including fungi. Intracellular calcium signaling is also a process that impacts a broad range of biological events in most eukaryotes. Clues have suggested that calcium signaling can influence circadian oscillators through multiple pathways; however, mechanistic details have been lacking in microorganisms. When we built on prior work describing calcium transporters in the fungus Neurospora, one such transporter, NCA-2, was identified as a regulator of circadian period length. Increased intracellular calcium levels caused by the loss of NCA-2 resulted in overactivation of calcium-responsive protein kinases, in turn leading to a shortened circadian period length. Importantly, the expression of NCA-2 is itself controlled by the molecular clock. In this way, calcium signaling can be seen as providing both input to and output from the circadian system.

Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora.

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of "clock genes." Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes (frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation (vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δgtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δgtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δgtr2 is defective in entrainment. In all of these assays, Δgtr2 is similar to Δvta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.

The Resonance and Adaptation of Neurospora crassa Circadian and Conidiation Rhyth ms to Short Light-Dark Cycles.

Circadian clocks control the physiological and behavioral rhythms to adapt to the environment with a period of ~24 h. However, the influences and mechanisms of the extreme light/dark cycles on the circadian clock remain unclear. We showed that, in Neurospora crassa, both the growth and the microconidia production contribute to adaptation in LD12:12 (12 h light/12 h dark, periodically). Mathematical modeling and experiments demonstrate that in short LD cycles, the expression of the core clock protein FREQUENCY was entrained to the LD cycles when LD > 3:3 while it free ran when T ≤ LD3:3. The conidial rhythmicity can resonate with a series of different LD conditions. Moreover, we demonstrate that the existence of unknown blue light photoreceptor(s) and the circadian clock might promote the conidiation rhythms that resonate with the environment. The ubiquitin E3 ligase FWD-1 and the previously described CRY-dependent oscillator system were implicated in regulating conidiation under short LD conditions. These findings shed new light on the resonance of Neurospora circadian clock and conidiation rhythms to short LD cycles, which may benefit the understandings of both the basic regulatory aspects of circadian clock and the adaptation of physiological rhythms to the extreme conditions.

Corrigendum: The Forensic Restrictiveness Questionnaire: Development, Validation, and Revision.

[This corrects the article DOI: 10.3389/fpsyt.2019.00805.].

Perceptions of Restrictiveness in Forensic Mental Health: Do Demographic, Clinical, and Legal Characteristics Matter?

Where safe, forensic mental health systems should provide care in the least restrictive environment possible. Doing so can maximize patient autonomy and empowerment while minimizing unnecessary social disconnection and stigmatization. This study investigated whether patients' perceptions of restrictiveness were associated with demographic, clinical, and legal characteristics. The Forensic Restrictiveness Questionnaire (FRQ) was used to measure perceptions of restrictiveness in 235 patients in low-, medium-, and high-secure settings in England. The results showed that restrictiveness scores were significantly higher for patients who experienced an adverse event in the past week or were diagnosed with a personality disorder compared to those with a mental illness. A regression analysis suggested that only diagnosis was predictive of FRQ scores when controlling for perceptions of ward atmosphere and quality of life. Age, length of stay, ethnicity, level of security, legal section, and offence type were not associated with FRQ scores. Future research should investigate the roles that individual symptoms, insight into illness, mood, personality, and expectations of care have in influencing perceptions of restrictiveness.

A Comparison of the Neurospora and Drosophila Clocks.

In Neurospora and other fungi, the protein frequency (FRQ) is an integral part and a negative element in the fungal circadian oscillator. In Drosophila and many other higher organisms, the protein period (PER) is an integral part and a negative element of their circadian oscillator. Employing bioinformatic techniques, such as BLAST, CLUSTAL, and MEME (Multiple Em for Motif Elicitation), 11 regions (sequences) of potential similarity were found between the fungal FRQ and the Drosophila PER. Many of these FRQ regions are conserved in many fungal FRQ(s). Many of these PER regions are conserved in many insects. In addition, these regions are also of biological significance since mutations in these regions lead to changes in the circadian clock of Neurospora and Drosophila. Many of these regions of similarity between FRQ and PER are also conserved between the Drosophila PER and the mouse PER (mPER2). This suggests conserved and important regions for all 3 proteins and a common ancestor, possibly in those amoeba, such as Capsaspora, that sits at the base of the phylogenetic tree where fungi and animals diverged. Two additional examples of a possible common ancestor between Neurospora and Drosophila were found. One, the white collar (WC-1) protein of Neurospora and the Drosophila PER, shows significant similarity in its Per/Arnt/Sim (PAS) motifs to the PAS motif of an ARNT-like protein found in the amoeba, Capsaspora. Two, both of the positive elements in each system (i.e., WC-1 in Neurospora and cycle [CYC] in Drosophila), show significant similarity to this Capsaspora ARNT protein. A discussion of these findings centers on the long-time debate about the origins of the many different clock systems (i.e., independent evolution or common ancestor as well as to the question of how new genes are formed).

Principles of the animal molecular clock learned from Neurospora.

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.

The Forensic Restrictiveness Questionnaire: Development, Validation, and Revision.

Introduction: Forensic psychiatric care is often practiced in closed institutions. These highly regulated, secure, and prescriptive environments arguably reduce patient autonomy, self-expression, and personhood. Taken together these settings are restrictive as patients' active participation in clinical, organizational, community, and personal life-worlds are curtailed. The consequences of patients' experiences of restrictiveness have not been explored empirically. This study aimed to develop a psychometrically-valid measure of experiences of restrictiveness. This paper presents the development, validation, and revision of the Forensic Restrictiveness Questionnaire (FRQ). Methods: In total, 235 patients recruited from low, medium, and high secure hospitals across England completed the FRQ. The dimensionality of the 56-item FRQ was tested using Principle Axis Factor Analysis and parallel analysis. Internal consistency was explored with Cronbach's α. Ward climate (EssenCES) and quality of life (FQL-SV) questionnaires were completed by participants as indicators of convergent validity. Exploratory Factor Analysis (EFA) and Cronbach's α guided the removal of items that did not scale adequately. Results: The analysis indicated good psychometric properties. EFA revealed a unidimensional structure, suggesting a single latent factor. Convergent validity was confirmed as the FRQ was significantly negatively correlated with quality of life (Spearman's ρ = -0.72) and ward climate (Spearman's ρ = -0.61). Internal consistency was strong (α = 0.93). Forty-one items were removed from the pilot FRQ. The data indicate that a final 15-item FRQ is a valid and internally reliable measure. Conclusion: The FRQ offers a novel and helpful method for clinicians and researchers to measure and explore forensic patients' experiences of restrictiveness within secure hospitals.

Circadian Rhythms in Fungi: Structure/Function/Evolution of Some Clock Components.

The fungal clock, especially that in Neurospora crassa, is composed of several proteins, notably FRQ, WC-1, and WC-2, which interact at the protein level and at the level of transcription. It is shown here that regions of the FRQ that are highly conserved in many fungal species show significant similarity to regions of proteins found in the amoebae Capsaspora and Acanthamoebae. These 2 amoebae were specifically explored because they have been suggested, based on extensive evidence, to be related to precursors of the modern fungi. Those proteins in Capsaspora/Acanthamoebae with some similarity to FRQ are LARP (an RNA-binding protein), ARNT (which has a PAS motif), and heat shock factor (HSF). These regions of LARP and HSF that show similarity to FRQ are highly conserved between plants, animals, and amoeba. This suggests that these regions were present at the time of the divergence of plants, fungi, insects, and animals, and therefore, they could be plausible precursors to regions of the fungal FRQ. These particular regions of FRQ that show similarity to LARP and HSF are also of functional significance since mutations in these regions of the Neurospora FRQ led to changes in the rhythm. The FRQ proteins from 13 different species of fungi were analyzed via motif analysis (MEME), and 11 different motifs were found. This provides some understanding as to the minimum requirements for an FRQ protein. Many of these FRQ motifs can be matched up with known domains in FRQ. In addition, these 13 different species of fungi were screened for the presence/absence of 7 additional genes/proteins that play some role in fungal clocks.

The Phospho-Code Determining Circadian Feedback Loop Closure and Output in Neurospora.

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.

Expression of putative circadian clock components in the arbuscular mycorrhizal fungus Rhizoglomus irregulare.

Arbuscular mycorrhizal fungi (AMF) are obligatory plant symbionts that live underground, so few studies have examined their response to light. Responses to blue light by other fungi can be mediated by White Collar-1 (WC-1) and WC-2 proteins. These wc genes, together with the frequency gene (frq), also form part of the endogenous circadian clock. The clock mechanism has never been studied in AMF, although circadian growth of their hyphae in the field has been reported. Using both genomic and transcriptomic data, we have found homologs of wc-1, wc-2, and frq and related circadian clock genes in the arbuscular mycorrhizal fungus Rhizoglomus irregulare (synonym Rhizophagus irregularis). Gene expression of wc-1, wc-2, and frq was analyzed using RT-qPCR on RNA extracted from germinating spores and from fungal material cultivated in vitro with transformed carrot roots. We found that all three core clock genes were expressed in both pre- and post-mycorrhizal stages of R. irregulare growth. Similar to the model fungus Neurospora crassa, the core circadian oscillator gene frq was induced by brief light stimulation. The presence of circadian clock and output genes in R. irregulare opens the door to the study of circadian clocks in the fungal partner of plant-AMF symbiosis. Our finding also provides new insight into the evolution of the circadian frq gene in fungi.

Up-Frameshift Protein UPF1 Regulates Neurospora crassa Circadian and Diurnal Growth Rhythms.

Nonsense-mediated RNA decay (NMD) is a crucial post-transcriptional regulatory mechanism that recognizes and eliminates aberrantly processed transcripts, and mediates the expression of normal gene transcripts. In this study, we report that in the filamentous fungus Neurospora crassa, the NMD factors play a conserved role in regulating the surveillance of NMD targets including premature termination codon (PTC)-containing transcripts and normal transcripts. The circadian rhythms in all of the knockout strains of upf1-3 genes, which encode the Up-frameshift proteins, were aberrant. The upf1 knockout strain displays a shortened circadian period, which can be restored by constantly expressing exogenous Up-frameshift protein 1 (UPF1). UPF1 regulates the circadian clock by modulating the splicing of the core clock gene frequency (frq) through spliceosome and spliceosome-related arginine/serine-rich splicing factors, which partly account for the short periods in the upf1 knockout strain. We also demonstrated that the clock genes including White Collar (WC)-1, WC-2, and FRQ are involved in controlling the diurnal growth rhythm, and UPF1 may affect the growth rhythms by mediating the FRQ protein levels in the daytime. These findings suggest that the NMD factors play important roles in regulating the circadian clock and diurnal growth rhythms in Neurospora.

PRD-1, a Component of the Circadian System of Neurospora crassa, Is a Member of the DEAD-box RNA Helicase Family.

The circadian rhythms found in almost all organisms are driven by molecular oscillators, including transcription/translation feedback loops (TTFLs). However, TTFL-independent oscillators can drive rhythms in both eukaryotes and prokaryotes. The fungus Neurospora crassa is a model organism for studying the molecular mechanism of the circadian clock. Although a circadian TTFL involving the proteins FRQ, WC-1, and WC-2 is well-characterized in N. crassa, rhythms can still be observed in the absence of this feedback loop. These rhythms are said to be driven by 1 or more FRQ-less oscillator(s) (FLOs). The prd-1 mutation lengthens the period in frq wild type and was previously shown to severely disrupt FRQ-less rhythms in frq null mutants under several different conditions; therefore, the prd-1 gene product is a candidate for a component of a FLO. We report here that prd-1 also disrupts free-running rhythms in wc-1 null mutants, confirming its effects on FRQ-less rhythms. We have now mapped and identified the prd-1 gene as NCU07839, a DEAD-box RNA helicase dbp-2 Complementation with the wild-type gene corrects the rhythm defects of the prd-1 mutant in the complete circadian system (when the FRQ-based TTFL is intact) and also the free-running FRQ-less rhythm on low choline. A PRD-1-GFP fusion protein localizes to the nucleus. The prd-1 mutant has a single base pair change in the first base of an intron that results in abnormally spliced transcripts. FRQ-less rhythms on low choline, or entrained to heat pulses, were only marginally affected in strains carrying deletions of 2 other RNA helicases (prd-6 and msp-8). We conclude that PRD-1 is a member of an RNA helicase family that may be specifically involved in regulating rhythmicity in N. crassa in both the complete circadian system and FLO(s).

Suppression of WHITE COLLAR-independent frequency Transcription by Histone H3 Lysine 36 Methyltransferase SET-2 Is Necessary for Clock Function in Neurospora.

The circadian system in Neurospora is based on the transcriptional/translational feedback loops and rhythmic frequency (frq) transcription requires the WHITE COLLAR (WC) complex. Our previous paper has shown that frq could be transcribed in a WC-independent pathway in a strain lacking the histone H3K36 methyltransferase, SET-2 (su(var)3-9-enhancer-of-zeste-trithorax-2) (1), but the mechanism was unclear. Here we disclose that loss of histone H3K36 methylation, due to either deletion of SET-2 or H3K36R mutation, results in arrhythmic frq transcription and loss of overt rhythmicity. Histone acetylation at frq locus increases in set-2(KO) mutant. Consistent with these results, loss of H3K36 methylation readers, histone deacetylase RPD-3 (reduced potassium dependence 3) or EAF-3 (essential SAS-related acetyltransferase-associated factor 3), also leads to hyperacetylation of histone at frq locus and WC-independent frq expression, suggesting that proper chromatin modification at frq locus is required for circadian clock operation. Furthermore, a mutant strain with three amino acid substitutions (histone H3 lysine 9, 14, and 18 to glutamine) was generated to mimic the strain with hyperacetylation state of histone H3. H3K9QK14QK18Q mutant exhibits the same defective clock phenotype as rpd-3(KO) mutant. Our results support a scenario in which H3K36 methylation is required to establish a permissive chromatin state for circadian frq transcription by maintaining proper acetylation status at frq locus.