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Casein kinase 1δ (CK1δ) is a simple monomeric enzyme involved in the regulation of a variety of functions, including signal transduction, the circadian clock, and the cell cycle. Although CK1δ is targeted by the ubiquitin ligase APC/CCdh1is not understood how CK1δ expression is regulated to support its multiple functions. Here, we show that kinase activity controls CK1δ homeostasis by coordinating two competing processes: export from the nucleus to ensure distribution of CK1δ between its assembly partners, and proteasomal degradation of unassembled CK1δ in the nucleus to keep the amount of active, potentially deleterious orphan kinase low. During mitosis, CK1δ is released from centrosomes and stabilized by (auto)phosphorylation to preserve it for the subsequent G1 phase.TeaserCompetitive nuclear export and nuclear degradation of active CK1δ ensure efficient partner interaction and keep unassembled kinase levels low.

RNA polymerase II initiates transcription either randomly or in bursts. We examined the light-dependent transcriptional activator White Collar Complex (WCC) ofNeurosporato characterize the transcriptional dynamics of the strongvivid(vvd) promoter and the weakerfrequency(frq) promoter. We show that WCC is not only an activator but also represses transcription by recruiting histone deacetylase 3 (HDA3). Our data suggest that bursts offrqtranscription are governed by a long-lived refractory state established and maintained by WCC and HDA3 at the core promoter, whereas transcription ofvvdis determined by WCC binding dynamics at an upstream activating sequence. Thus, in addition to stochastic binding of transcription factors, transcription factor-mediated repression may also influence transcriptional bursting.TEASERBalanced interaction of transcription factor with coactivator and corepressors determines transcription dynamics and bursting.

The circadian clock of Neurospora crassa is based on a negative transcriptional/translational feedback loops. The frequency (frq) gene controls the morning-specific rhythmic transcription of a sense RNA encoding FRQ, the negative element of the core circadian feedback loop. In addition, a long noncoding antisense RNA, qrf, is rhythmically transcribed in an evening-specific manner. It has been reported that the qrf rhythm relies on transcriptional interference with frq transcription and that complete suppression of qrf transcription impairs the circadian clock. We show here that qrf transcription is dispensable for circadian clock function. Rather, the eveningspecific transcriptional rhythm of qrf is mediated by the morning-specific repressor CSP-1. Since CSP-1 expression is induced by light and glucose, this suggests a rhythmic coordination of qrf transcription with metabolism. However, a possible physiological significance for the circadian clock remains unclear, as suitable assays are not available.

MXDs are transcription repressors that antagonize MYC-mediated gene activation. MYC, when associated with MIZ1, acts also as a repressor of a subset of genes, including p15 and p21. A role for MXDs in regulation of MYC-repressed genes is not known. We report that MXDs activate transcription of p15 and p21 in U2OS cells. This activation required DNA binding by MXDs and their interaction with MIZ1. MXD mutants deficient in MIZ1 binding interacted with the MYC-binding partner MAX and were active as repressors of MYC-activated genes but failed to activate MYC-repressed genes. Mutant MXDs with reduced DNA-binding affinity interacted with MAX and MIZ1 but neither repressed nor activated transcription. Our data show that MXDs and MYC have a reciprocally antagonistic potential to regulate transcription of target genes.

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.

The circadian clock governs rhythmic cellular functions by driving expression of a substantial fraction of the genome and thereby significantly contributes to the adaptation to changing environmental conditions. Using the circadian model organism Neurospora crassa, we show that molecular timekeeping is robust even under severe limitation of carbon sources, however, stoichiometry, phosphorylation and subcellular distribution of the key clock components display drastic alterations. Protein kinase A, protein phosphatase 2A and glycogen synthase kinase are involved in the molecular reorganization of the clock. RNA-seq analysis reveals that the transcriptomic response of metabolism to starvation is highly dependent on the positive clock component WC-1. Moreover, our molecular and phenotypic data indicate that a functional clock facilitates recovery from starvation. We suggest that the molecular clock is a flexible network that allows the organism to maintain a rhythmic physiology and preserve fitness even under long-term nutritional stress.

Circadian clocks are self-sustained oscillators that orchestrate metabolism and physiology in synchrony with the 24-h day–night cycle. They are temperature compensated over a wide range and entrained by daily recurring environmental cues. Eukaryotic circadian clocks are governed by cell-based transcriptional–translational feedback loops (TTFLs). The core components of the TTFLs are largely known and their molecular interactions in many cases well established. Although the core clock components are not or only partly conserved, the molecular wiring of TTFLs is rather similar across kingdoms and phylae. In all known systems, circadian timing relies critically on casein kinase 1 (CK1) and CK1-dependent hyperphosphorylation of core clock proteins, in particular of negative elements of the TTFLs. Yet, we lack concepts as to how phosphorylation by CK1a and other kinases relates to timekeeping on the molecular level. Here we summarize what is known about phosphorylation of core components of the circadian clock of Neurospora crassa and speculate about the molecular basis of circadian timekeeping by hyperphosphorylation of intrinsically disordered regions in clock proteins.

Significance Circadian clocks rely on negative feedback loops. The core circadian inhibitors, FRQ in Neurospora and PERs in animals, are progressively hyperphosphorylated, inactivated, and degraded. CK1 is essential for these clocks. Despite our knowledge of the role of CK1, it is not known how many other kinases are required and how multisite phosphorylation might contribute to circadian timekeeping. We show here that CK1 alone is sufficient to slowly phosphorylate low-affinity sites in FRQ or PER2. The reaction is nearly temperature compensated, and the phosphorylation state of FRQ or PER2 corresponds to the time elapsed since the start of the reaction. Thus, CK1 and FRQ or PER2 form equivalent modules that are in principle capable of measuring time on a circadian scale.

The circadian clock of Neurospora crassa is based on a negative transcriptional/translational feedback loops. The frequency ( frq) gene controls the morning-specific rhythmic transcription of a sense RNA encoding FRQ, the negative element of the core circadian feedback loop. In addition, a long noncoding antisense RNA, qrf, is rhythmically transcribed in an evening-specific manner. It has been reported that the qrf rhythm relies on transcriptional interference with frq transcription and that complete suppression of qrf transcription impairs the circadian clock. We show here that qrf transcription is dispensable for circadian clock function. Rather, the evening-specific transcriptional rhythm of qrf is mediated by the morning-specific repressor CSP-1. Since CSP-1 expression is induced by light and glucose, this suggests a rhythmic coordination of qrf transcription with metabolism. However, a possible physiological significance for the circadian clock remains unclear, as suitable assays are not available.

Checkpoint kinase 2 (CHK-2) is a key component of the DNA damage response (DDR). CHK-2 is activated by the PIP3-kinase-like kinases (PI3KKs) ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR), and in metazoan also by DNA-dependent protein kinase catalytic subunit (DNA-PKcs). These DNA damage-dependent activation pathways are conserved and additional activation pathways of CHK-2 are not known. Here we show that PERIOD-4 (PRD-4), the CHK-2 ortholog of Neurospora crassa, is part of a signaling pathway that is activated when protein translation is compromised. Translation stress induces phosphorylation of PRD-4 by a PI3KK distinct from ATM and ATR. Our data indicate that the activating PI3KK is mechanistic target of rapamycin (mTOR). We provide evidence that translation stress is sensed by unbalancing the expression levels of an unstable protein phosphatase that antagonizes phosphorylation of PRD-4 by mTOR complex 1 (TORC1). Hence, Neurospora mTOR and PRD-4 appear to coordinate metabolic state and cell cycle progression.

Theory predicts that self-sustained oscillations require robust delays and nonlinearities (ultrasensitivity). We study the circadian rhythms in the filamentous fungus Neurospora crassa (wild type period 22.5 hours) to investigate the underlying clock mechanisms. Its transcription translation feedback loop (TTFL) includes the activator White Collar Complex (WCC), the inhibitory FFC complex, and Casein kinase 1a. Moreover, there are multiple phosphorylation sites on FRQ (around 100) and WCC (approximately 95). A phosphoswitch controls the activity status of the FFC complex. We investigate how multiple, slow and random phosphorylations govern delay and nonlinearity in a negative feedback. We model FRQ phosphorylations with turnover of FRQ and CK1a using ordinary differential equations. Our model helps to understand the underlying mechanisms leading to delays and ultrasensitivity. The model shows temporal and steady state switches for the free kinase and phosphorylated FRQ. We show that random phosphorylations and sequestration mechanisms allow high Hill coefficients required for self-sustained oscillations. Our conceptual models help to understand multiple phosphorylations dynamics and to elucidate mechanisms of delayed switch generation.

Summary In many organisms, the circadian clock drives rhythms in the transcription of clock-controlled genes that can be either circadian (∼24-hr period) or ultradian (, Graphical Abstract, Highlights • Coexisting harmonic ∼7-hr and circadian rhythms in fungal clock model organism • Knockout of output regulator CSP1 imposes circadian rhythms over ∼7-hr rhythms • Third harmonic rhythms are a part of key cellular processes and mediated by FF-7 • 7-hr genes are co-regulated in two anti-phasic clusters across genotypes and laboratories, Biological Sciences; Chronobiology; Transcriptomics

MXD proteins are transcription repressors that antagonize the E-box dependent activation of genes by MYC. MYC together with MIZ1 acts also as a repressor of a subset of genes, including cell cycle inhibitor genes such as p15 and p21. A role of MXDs in regulation of MYC-repressed genes is not known. Here we report that MXDs are functionally expressed in U2OS cells and activate transcription of p15 and p21, and other MYC-repressed genes. Activation of transcription was dependent on the interaction of MXDs with MIZ1, and on an intact DNA binding domain. MIZ1-binding deficient MXD mutants interacted with MAX and were active as repressors of MYC-activated genes but failed to activate MYC-repressed genes. Mutant MXDs with reduced DNA binding affinity interacted with MAX and MIZ1 but neither repressed nor activated transcription. Overexpression of MXDs attenuated proliferation of U2OS cells predominantly via MIZ1-dependent induction of p21. Our data show that MXDs and MYC have a reciprocally antagonistic potential to regulate transcription of mutual target genes.

Significance Checkpoint kinase 2 (CHK-2) is a key component of the DNA damage response (DDR) pathway and its activation mechanism is evolutionarily conserved. We show that PERIOD-4 (PRD-4), the CHK-2 ortholog of Neurospora crassa, is part of an additional signaling pathway that is activated when protein translation is compromised. Translation stress induces phosphorylation of PRD-4 by an upstream kinase distinct from those of the DDR pathway. We present evidence that the activating kinase is mTOR. Translation stress is sensed via a decrease in levels of an unstable inhibitor that antagonizes phosphorylation of PRD-4., Checkpoint kinase 2 (CHK-2) is a key component of the DNA damage response (DDR). CHK-2 is activated by the PIP3-kinase-like kinases (PI3KKs) ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR), and in metazoan also by DNA-dependent protein kinase catalytic subunit (DNA-PKcs). These DNA damage-dependent activation pathways are conserved and additional activation pathways of CHK-2 are not known. Here we show that PERIOD-4 (PRD-4), the CHK-2 ortholog of Neurospora crassa, is part of a signaling pathway that is activated when protein translation is compromised. Translation stress induces phosphorylation of PRD-4 by a PI3KK distinct from ATM and ATR. Our data indicate that the activating PI3KK is mechanistic target of rapamycin (mTOR). We provide evidence that translation stress is sensed by unbalancing the expression levels of an unstable protein phosphatase that antagonizes phosphorylation of PRD-4 by mTOR complex 1 (TORC1). Hence, Neurospora mTOR and PRD-4 appear to coordinate metabolic state and cell cycle progression.

The MYC oncogene encodes MYC, a transcription factor that binds the genome through sites termed E-boxes (5'-CACGTG-3'), which are identical to the binding sites of the heterodimeric CLOCK-BMAL1 master circadian transcription factor. Hence, we hypothesized that ectopic MYC expression perturbs the clock by deregulating E-box-driven components of the circadian network in cancer cells. We report here that deregulated expression of MYC or N-MYC disrupts the molecular clock in vitro by directly inducing REV-ERBα to dampen expression and oscillation of BMAL1, and this could be rescued by knockdown of REV-ERB. REV-ERBα expression predicts poor clinical outcome for N-MYC-driven human neuroblastomas that have diminished BMAL1 expression, and re-expression of ectopic BMAL1 in neuroblastoma cell lines suppresses their clonogenicity. Further, ectopic MYC profoundly alters oscillation of glucose metabolism and perturbs glutaminolysis. Our results demonstrate an unsuspected link between oncogenic transformation and circadian and metabolic dysrhythmia, which we surmise to be advantageous for cancer.

Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc-chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a de-ubiquinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome, Associated-molecule-with-the-SH3-Domain-of-STAM (AMSH), which regulates ubiquitin-dependent sorting of cell-surface receptors, and Brcc36, a K63-specific deubiquitnase of BRCC36-containing isopeptidase complex (BRISC) and BRCA1-BRCA2-containing complex (BRCC). We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.

Expression levels and ratios of the long (l) and short (s) isoforms of the Neurospora circadian clock protein FREQUENCY (FRQ) are crucial for temperature compensation of circadian rhythms. We show that the ratio of l-FRQ versus s-FRQ is regulated by thermosensitive splicing of intron 6 of frq, a process removing the translation initiation site of l-FRQ. Thermosensitivity is due to inefficient recognition of nonconsensus splice sites at elevated temperature. The temperature-dependent accumulation of FRQ relative to bulk protein is controlled at the level of translation. The 5′-UTR of frq RNA contains six upstream open reading frames (uORFs) that are in nonconsensus context for translation initiation. Thermosensitive trapping of scanning ribosomes at the uORFs leads to reduced translation of the main ORF and allows adjustment of FRQ levels according to ambient temperature.

As a response to the surrounding environment most organisms regulate their physiology and behavior in rhythmic manner. Such temporal organization is controlled by two global systems, the cell cycle...

Timekeeping by circadian clocks relies upon precise adjustment of expression levels of clock proteins. Here we identify glycogen synthase kinase (GSK) as a novel and critical component of the circadian clock of Neurospora crassa that regulates the abundance of its core transcription factor white collar complex (WCC) on a post-transcriptional level. We show that GSK specifically binds and phosphorylates both subunits of the WCC. Reduced expression of GSK promotes an increased accumulation of WC-1, the limiting factor of the WCC, causing an acceleration of the circadian clock and a shorter free-running period.