Nutritional compensation of the circadian clock is a conserved process influenced by gene expression regulation and mRNA stability.

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.

Histone deacetylation and cytosine methylation compartmentalize heterochromatic regions in the genome organization of Neurospora crassa.

Chromosomes must correctly fold in eukaryotic nuclei for proper genome function. Eukaryotic organisms hierarchically organize their genomes, including in the fungus Neurospora crassa, where chromatin fiber loops compact into Topologically Associated Domain-like structures formed by heterochromatic region aggregation. However, insufficient data exist on how histone posttranslational modifications (PTMs), including acetylation, affect genome organization. In Neurospora, the HCHC complex [composed of the proteins HDA-1, CDP-2 (Chromodomain Protein-2), Heterochromatin Protein-1, and CHAP (CDP-2 and HDA-1 Associated Protein)] deacetylates heterochromatic nucleosomes, as loss of individual HCHC members increases centromeric acetylation, and alters the methylation of cytosines in DNA. Here, we assess whether the HCHC complex affects genome organization by performing Hi-C in strains deleted of the cdp-2 or chap genes. CDP-2 loss increases intra- and interchromosomal heterochromatic region interactions, while loss of CHAP decreases heterochromatic region compaction. Individual HCHC mutants exhibit different patterns of histone PTMs genome-wide, as CDP-2 deletion increases heterochromatic H4K16 acetylation, yet smaller heterochromatic regions lose H3K9 trimethylation and gain interheterochromatic region interactions; CHAP loss produces minimal acetylation changes but increases heterochromatic H3K9me3 enrichment. Loss of both CDP-2 and the DIM-2 DNA methyltransferase causes extensive genome disorder as heterochromatic-euchromatic contacts increase despite additional H3K9me3 enrichment. Our results highlight how the increased cytosine methylation in HCHC mutants ensures genome compartmentalization when heterochromatic regions become hyperacetylated without HDAC activity.

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.

PRD-2 mediates clock-regulated perinuclear localization of clock gene RNAs within the circadian cycle of Neurospora.

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.

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.

The "life-span" of lytic polysaccharide monooxygenases (LPMOs) correlates to the number of turnovers in the reductant peroxidase reaction.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that degrade the insoluble crystalline polysaccharides cellulose and chitin. Besides the H2O2 cosubstrate, the cleavage of glycosidic bonds by LPMOs depends on the presence of a reductant needed to bring the enzyme into its reduced, catalytically active Cu(I) state. Reduced LPMOs that are not bound to substrate catalyze reductant peroxidase reactions, which may lead to oxidative damage and irreversible inactivation of the enzyme. However, the kinetics of this reaction remain largely unknown, as do possible variations between LPMOs belonging to different families. Here, we describe the kinetic characterization of two fungal family AA9 LPMOs, TrAA9A of Trichoderma reesei and NcAA9C of Neurospora crassa, and two bacterial AA10 LPMOs, ScAA10C of Streptomyces coelicolor and SmAA10A of Serratia marcescens. We found peroxidation of ascorbic acid and methyl-hydroquinone resulted in the same probability of LPMO inactivation (pi), suggesting that inactivation is independent of the nature of the reductant. We showed the fungal enzymes were clearly more resistant toward inactivation, having pi values of less than 0.01, whereas the pi for SmAA10A was an order of magnitude higher. However, the fungal enzymes also showed higher catalytic efficiencies (kcat/KM(H2O2)) for the reductant peroxidase reaction. This inverse linear correlation between the kcat/KM(H2O2) and pi suggests that, although having different life spans in terms of the number of turnovers in the reductant peroxidase reaction, LPMOs that are not bound to substrates have similar half-lives. These findings have not only potential biological but also industrial implications.

Cloning and expression of Neurospora crassa cellobiohydrolase II in Pichia pastoris.

A major cellobiohydrolase of Neurospora crassa CBH2 was successfully expressed in Pichia pastoris. The maximum Avicelase activity in shake flask among seven transformants which selected on 4.0 g/L G418 plates was 0.61 U/mL. The optimal pH and temperature for Avicelase activity of the recombinant CBH2 were determined to be 4.8 and 60 °C, respectively. The new CBH2 maintained 63.5 % Avicelase activity in the range of pH 4.0-10.4, and 60.2 % Avicelase activity in the range of 30-90 °C. After incubation at 70-90 °C for 1 h, the Avicelase activity retained 60.5 % of its initial activity. The presence of Zn2+, Ca2+ or Cd2+ enhanced the Avicelase activity of the CBH2, of which Cd2+ at 10 mM causing the highest increase. The recombinant CBH2 was used to enhance the Avicel hydrolysis by improving the exo-exo-synergism between CBH2 and CBH1 in N.crassa cellulase. The enzymatic hydrolysis yield was increased by 38.1 % by adding recombinant CBH2 and CBH1, and the yield was increased by 215.4 % when the temperature is raised to 70 °C. This work provided a CBH2 with broader pH range and better heat resistance, which is a potential enzyme candidate in food, textile, pulp and paper industries, and other industrial fields.

H3K56 deacetylation and H2A.Z deposition are required for aberrant heterochromatin spreading.

Crucial mechanisms are required to restrict self-propagating heterochromatin spreading within defined boundaries and prevent euchromatic gene silencing. In the filamentous fungus Neurospora crassa, the JmjC domain protein DNA METHYLATION MODULATOR-1 (DMM-1) prevents aberrant spreading of heterochromatin, but the molecular details remain unknown. Here, we revealed that DMM-1 is highly enriched in a well-defined 5-kb heterochromatin domain upstream of the cat-3 gene, hereby called 5H-cat-3 domain, to constrain aberrant heterochromatin spreading. Interestingly, aberrant spreading of the 5H-cat-3 domain observed in the dmm-1KO strain is accompanied by robust deposition of histone variant H2A.Z, and deletion of H2A.Z abolishes aberrant spreading of the 5H-cat-3 domain into adjacent euchromatin. Furthermore, lysine 56 of histone H3 is deacetylated at the expanded heterochromatin regions, and mimicking H3K56 acetylation with an H3K56Q mutation effectively blocks H2A.Z-mediated aberrant spreading of the 5H-cat-3 domain. Importantly, genome-wide analyses demonstrated the general roles of H3K56 deacetylation and H2A.Z deposition in aberrant spreading of heterochromatin. Together, our results illustrate a previously unappreciated regulatory process that mediates aberrant heterochromatin spreading.

Structural insights into the dual activities of the two-barrel RNA polymerase QDE-1.

Neurospora crassa protein QDE-1, a member of the two-barrel polymerase superfamily, possesses both DNA- and RNA-dependent RNA polymerase (DdRP and RdRP) activities. The dual activities are essential for the production of double-stranded RNAs (dsRNAs), the precursors of small interfering RNAs (siRNAs) in N. crassa. Here, we report five complex structures of N-terminal truncated QDE-1 (QDE-1ΔN), representing four different reaction states: DNA/RNA-templated elongation, the de novo initiation of RNA synthesis, the first step of nucleotide condensation during de novo initiation and initial NTP loading. The template strand is aligned by a bridge-helix and double-psi beta-barrels 2 (DPBB2), the RNA product is held by DPBB1 and the slab domain. The DNA template unpairs with the RNA product at position -7, but the RNA template remains paired. The NTP analog coordinates with cations and is precisely positioned at the addition site by a rigid trigger loop and a proline-containing loop in the active center. The unique C-terminal tail from the QDE-1 dimer partner inserts into the substrate-binding cleft and plays regulatory roles in RNA synthesis. Collectively, this work elucidates the conserved mechanisms for DNA/RNA-dependent dual activities by QDE-1 and other two-barrel polymerase superfamily members.

Structure of the translating Neurospora ribosome arrested by cycloheximide.

Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX's mechanism of action based on the cryo-electron microscopy structure of actively translating Neurospora crassa ribosomes bound with CHX at 2.7-Å resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from Neurospora, yeast, and humans all contain a highly conserved CHX binding site. We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating N. crassa ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.

The transcriptional factor Clr-5 is involved in cellulose degradation through regulation of amino acid metabolism in Neurospora crassa.

BACKGROUND: Filamentous fungi are efficient degraders of plant biomass and the primary producers of commercial cellulolytic enzymes. While the transcriptional regulation mechanisms of cellulases have been continuously explored in lignocellulolytic fungi, the induction of cellulase production remains a complex multifactorial system, with several aspects still largely elusive. RESULTS: In this study, we identified a Zn2Cys6 transcription factor, designated as Clr-5, which regulates the expression of cellulase genes by influencing amino acid metabolism in Neurospora crassa during growth on cellulose. The deletion of clr-5 caused a significant decrease in secreted protein and cellulolytic enzyme activity of N. crassa, which was partially alleviated by supplementing with yeast extract. Transcriptomic profiling revealed downregulation of not only the genes encoding main cellulases but also those related to nitrogen metabolism after disruption of Clr-5 under Avicel condition. Clr-5 played a crucial role in the utilization of multiple amino acids, especially leucine and histidine. When using leucine or histidine as the sole nitrogen source, the Δclr-5 mutant showed significant growth defects on both glucose and Avicel media. Comparative transcriptomic analysis revealed that the transcript levels of most genes encoding carbohydrate-active enzymes and those involved in the catabolism and uptake of histidine, branched-chain amino acids, and aromatic amino acids, were remarkably reduced in strain Δclr-5, compared with the wild-type N. crassa when grown in Avicel medium with leucine or histidine as the sole nitrogen source. These findings underscore the important role of amino acid metabolism in the regulation of cellulase production in N. crassa. Furthermore, the function of Clr-5 in regulating cellulose degradation is conserved among ascomycete fungi. CONCLUSIONS: These findings regarding the novel transcription factor Clr-5 enhance our comprehension of the regulatory connections between amino acid metabolism and cellulase production, offering fresh prospects for the development of fungal cell factories dedicated to cellulolytic enzyme production in bio-refineries.

Optimized fluorescent proteins for 4-color and photoconvertible live-cell imaging in Neurospora crassa.

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.

DNA interstrand crosslink repair by XPF-ERCC1 homologue confers ultraviolet resistance in Neurospora crassa.

Ultraviolet (UV) light is a mutagen that causes DNA damage. Some UV-sensitive Neurospora crassa strains have been reported to exhibit a partial photoreactivation defect (PPD) phenotype, and the possible cause of this has been unknown for more than half a century. In this study, in the process of elucidating the possible causes of a PPD phenotype, we discovered that the XPF homologue MUS-38 is involved in repairing the UV-induced DNA interstrand crosslink (ICL) in N. crassa. Furthermore, the sensitivity of the Δmus-38 and Δmus-44 strains to ICL agents was significantly higher than that of other nucleotide excision repair (NER)-related gene knockout (KO) strains, indicating that the MUS-38/MUS-44 complex is involved in an NER-independent ICL repair mechanism. Based on reports concerning the mammalian homologues XPF and ERCC1 we obtained separation-of-function mutants defective only in NER in mus-38 and mus-44. Additionally, the photoreactivation ability of these mutants was significantly higher than that of the KO strains. These results indicate that the PPD phenotype is caused by a defect in the repair-ability of ICL induced by UV and that an NER-independent ICL repair by MUS-38 and MUS-44 confers resistance to UV in N. crassa.

Effect of itaconic acid production on Neurospora crassa in consolidated bioprocessing of cellulose.

A system for itaconic acid synthesis from cellulose by Neurospora crassa was established, resulting in the highest yield of itaconic acid was 354.08 + 35.99 mg/L. Meanwhile, cellulase activity increased significantly, without any strain modifications for improved cellulase production. Multi-omics analyses showed that itaconic acid synthesis reduced energy production, leading to decreases in trehalose, cell wall, fatty acids synthesis and downregulations in MAPK signaling pathway, cell cycle and meiosis. More importantly, the low-energy environment enhanced the energy-efficient cellobionic acid/gluconic acid pathway, and the cellulase composition also changed significantly, manifested as the up-regulation of LPMOs and the down-regulation of ß-glucosidases. Enhancing LPMOs-cellobionic acid/gluconic acid system has the potential to reduce energy consumption of the consolidated bioprocessing. These findings offer an overview of resource allocations by N. crassa in response to itaconic acid synthesis and highlight a series of intriguing connections between itaconic acid synthesis and cellulase synthesis in consolidated bioprocessing.

Codon Usage Influences the Local Rate of Translation Elongation to Regulate Co-translational Protein Folding.

Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and has been proposed to regulate translation efficiency, accuracy, and protein folding based on the assumption that codon usage affects translation dynamics. The roles of codon usage in translation, however, are not clear and have been challenged by recent ribosome profiling studies. Here we used a Neurospora cell-free translation system to directly monitor the velocity of mRNA translation. We demonstrated that the preferred codons enhance the rate of translation elongation, whereas non-optimal codons slow elongation. Codon usage also controls ribosome traffic on mRNA. These conclusions were supported by ribosome profiling results in vitro and in vivo with template mRNAs designed to increase the signal-to-noise ratio. Finally, we demonstrate that codon usage regulates protein function by affecting co-translational protein folding. These results resolve a long-standing fundamental question and suggest the existence of a codon usage code for protein folding.

Molecular characterization of a fungal gasdermin-like protein.

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulatorof cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassarcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1-1 and rcd-1-2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1-dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1-1/rcd-1-2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1 This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.

The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus.

Filamentous fungi, such as Neurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.

Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation.

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.

Evolutionarily ancient BAH-PHD protein mediates Polycomb silencing.

Methylation of histone H3 lysine 27 (H3K27) is widely recognized as a transcriptionally repressive chromatin modification but the mechanism of repression remains unclear. We devised and implemented a forward genetic scheme to identify factors required for H3K27 methylation-mediated silencing in the filamentous fungus Neurospora crassa and identified a bromo-adjacent homology (BAH)-plant homeodomain (PHD)-containing protein, EPR-1 (effector of polycomb repression 1; NCU07505). EPR-1 associates with H3K27-methylated chromatin, and loss of EPR-1 de-represses H3K27-methylated genes without loss of H3K27 methylation. EPR-1 is not fungal-specific; orthologs of EPR-1 are present in a diverse array of eukaryotic lineages, suggesting an ancestral EPR-1 was a component of a primitive Polycomb repression pathway.