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.

MXD/MIZ1 transcription regulatory complexes activate the expression of MYC-repressed genes.

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.

The circadian cryptochrome, CRY1, is a pro-tumorigenic factor that rhythmically modulates DNA repair.

Mechanisms regulating DNA repair processes remain incompletely defined. Here, the circadian factor CRY1, an evolutionally conserved transcriptional coregulator, is identified as a tumor specific regulator of DNA repair. Key findings demonstrate that CRY1 expression is androgen-responsive and associates with poor outcome in prostate cancer. Functional studies and first-in-field mapping of the CRY1 cistrome and transcriptome reveal that CRY1 regulates DNA repair and the G2/M transition. DNA damage stabilizes CRY1 in cancer (in vitro, in vivo, and human tumors ex vivo), which proves critical for efficient DNA repair. Further mechanistic investigation shows that stabilized CRY1 temporally regulates expression of genes required for homologous recombination. Collectively, these findings reveal that CRY1 is hormone-induced in tumors, is further stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation. These studies identify the circadian factor CRY1 as pro-tumorigenic and nominate CRY1 as a new therapeutic target.

A pathway linking translation stress to checkpoint kinase 2 signaling in Neurospora crassa.

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.

Help from my friends-cooperation of BMAL1 with noncircadian transcription factors.

The circadian clock in the suprachiasmatic nucleus (SCN) of mammals drives 24-h rhythms of sleep/wake cycles. Peripheral clocks present in other organs coordinate local and global physiology according to rhythmic signals from the SCN and via metabolic cues. The core circadian clockwork is identical in all cells. However, there is only a small amount of overlap of the circadian transcriptomes in different organs and tissues. A novel study by Beytebiere and colleagues (pp. 294-309) indicates that the regulation of tissue-specific rhythmic gene expression involves the cooperation of the circadian transcription factor (TF) BMAL1:CLOCK with tissue-specific TFs (ts-TFs) and correlates with the potential of BMAL1:CLOCK to facilitate rhythmic enhancer-enhancer interactions.

Thiolutin is a zinc chelator that inhibits the Rpn11 and other JAMM metalloproteases.

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 deubiquitinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome; AMSH, which regulates ubiquitin-dependent sorting of cell-surface receptors; and BRCC36, a K63-specific deubiquitinase of the BRCC36-containing isopeptidase complex and the BRCA1-BRCA2-containing complex. We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.

Circadian Clock, Cell Division, and Cancer: From Molecules to Organism.

As a response to environmental changes driven by the Earth's axial rotation, most organisms evolved an internal biological timer-the so called circadian clock-which regulates physiology and behavior in a rhythmic fashion. Emerging evidence suggests an intimate interplay between the circadian clock and another fundamental rhythmic process, the cell cycle. However, the precise mechanisms of this connection are not fully understood. Disruption of circadian rhythms has a profound impact on cell division and cancer development and, vice versa, malignant transformation causes disturbances of the circadian clock. Conventional knowledge attributes tumor suppressor properties to the circadian clock. However, this implication might be context-dependent, since, under certain conditions, the clock can also promote tumorigenesis. Therefore, a better understanding of the molecular links regulating the physiological balance between the two cycles will have potential significance for the treatment of cancer and associated disorders.

MYC/MIZ1-dependent gene repression inversely coordinates the circadian clock with cell cycle and proliferation.

The circadian clock and the cell cycle are major cellular systems that organize global physiology in temporal fashion. It seems conceivable that the potentially conflicting programs are coordinated. We show here that overexpression of MYC in U2OS cells attenuates the clock and conversely promotes cell proliferation while downregulation of MYC strengthens the clock and reduces proliferation. Inhibition of the circadian clock is crucially dependent on the formation of repressive complexes of MYC with MIZ1 and subsequent downregulation of the core clock genes BMAL1 (ARNTL), CLOCK and NPAS2. We show furthermore that BMAL1 expression levels correlate inversely with MYC levels in 102 human lymphomas. Our data suggest that MYC acts as a master coordinator that inversely modulates the impact of cell cycle and circadian clock on gene expression.

Non-circadian expression masking clock-driven weak transcription rhythms in U2OS cells.

U2OS cells harbor a circadian clock but express only a few rhythmic genes in constant conditions. We identified 3040 binding sites of the circadian regulators BMAL1, CLOCK and CRY1 in the U2OS genome. Most binding sites even in promoters do not correlate with detectable rhythmic transcript levels. Luciferase fusions reveal that the circadian clock supports robust but low amplitude transcription rhythms of representative promoters. However, rhythmic transcription of these potentially clock-controlled genes is masked by non-circadian transcription that overwrites the weaker contribution of the clock in constant conditions. Our data suggest that U2OS cells harbor an intrinsically rather weak circadian oscillator. The oscillator has the potential to regulate a large number of genes. The contribution of circadian versus non-circadian transcription is dependent on the metabolic state of the cell and may determine the apparent complexity of the circadian transcriptome.

Impaired glucocorticoid production and response to stress in Arntl-deficient male mice.

The basic helix-loop-helix transcription factor Aryl Hydrocarbon Receptor Nuclear Translocator-Like (ARNTL, also known as BMAL1 or MOP3) is a core component of the circadian timing system in mammals, which orchestrates 24-hour rhythms of physiology and behavior. Genetic ablation of Arntl in mice leads to behavioral and physiological arrhythmicity, including loss of circadian baseline regulation of glucocorticoids (GCs). GCs are important downstream regulators of circadian tissue clocks and have essential functions in the physiological adaptation to stress. The role of the clock machinery in the regulation of stress-induced GC release, however, is not well understood. Here we show that already under unstressed conditions Arntl-deficient mice suffer from hypocortisolism with impaired adrenal responsiveness to ACTH and down-regulated transcription of genes involved in cholesterol transport in adrenocortical cells. Under stress they show diminished GC and behavioral responses and develop behavioral resistance to acute and subchronic stressors, as shown using forced swim, tail suspension, and sucrose preference tests. These data suggest that the clock gene Arntl regulates circadian and acute secretion of GCs by the adrenal gland. Arntl disruption, probably via its effect on adrenal clock function, modulates stress axis activity and, thus, may promote resistance to both acute and repeated stress.

Circadian regulation of adipose function.

Adipose physiology shows prominent variation over the course of the day, responding to changing demands in energy metabolism. In the last years the tight interaction between the endogenous circadian timing system and metabolic function has been increasingly acknowledged. Recent work suggests that clock and adipose function go hand in hand, regulating each other to ensure optimal adaptation to environmental changes over the 24-h cycle. In this review we describe the current knowledge on the mechanistic basis of this interaction and summarize recent findings on the impact of clock dysfunction on adipose physiology and energy homeostasis.

High-fat diet-induced hyperinsulinemia and tissue-specific insulin resistance in Cry-deficient mice.

Perturbation of circadian rhythmicity in mammals, either by environmental influences such as shiftwork or by genetic manipulation, has been associated with metabolic disturbance and the development of obesity and diabetes. Circadian clocks are based on transcriptional/translational feedback loops, comprising positive and negative components. Whereas the metabolic effects of deletion of the positive arm of the clock gene machinery, as in Clock- or Bmal1-deficient mice, have been well characterized, inactivation of Period genes (Per1-3) as components of the negative arm have more complex, sometimes contradictory effects on energy homeostasis. The CRYPTOCHROMEs are critical interaction partners of PERs, and simultaneous deletion of Cry1 and -2 results in behavioral and molecular circadian arrhythmicity. We show that, when challenged with a high-fat diet, Cry1/2(-/-) mice rapidly gain weight and surpass that of wild-type mice, despite displaying hypophagia. Transcript analysis of white adipose tissue reveals upregulated expression of lipogenic genes, many of which are insulin targets. High-fat diet-induced hyperinsulinemia, as a result of potentiated insulin secretion, coupled with selective insulin sensitivity in adipose tissue of Cry1/2(-/-) mice, correlates with increased lipid uptake. Collectively, these data indicate that Cry deficiency results in an increased vulnerability to high-fat diet-induced obesity that might be mediated by increased insulin secretion and lipid storage in adipose tissues.

Circadian regulation of lipid mobilization in white adipose tissues.

In mammals, a network of circadian clocks regulates 24-h rhythms of behavior and physiology. Circadian disruption promotes obesity and the development of obesity-associated disorders, but it remains unclear to which extent peripheral tissue clocks contribute to this effect. To reveal the impact of the circadian timing system on lipid metabolism, blood and adipose tissue samples from wild-type, ClockΔ19, and Bmal1(-/-) circadian mutant mice were subjected to biochemical assays and gene expression profiling. We show diurnal variations in lipolysis rates and release of free fatty acids (FFAs) and glycerol into the blood correlating with rhythmic regulation of two genes encoding the lipolysis pacemaker enzymes, adipose triglyceride (TG) lipase and hormone-sensitive lipase, by self-sustained adipocyte clocks. Circadian clock mutant mice show low and nonrhythmic FFA and glycerol blood content together with decreased lipolysis rates and increased sensitivity to fasting. Instead circadian clock disruption promotes the accumulation of TGs in white adipose tissue (WAT), leading to increased adiposity and adipocyte hypertrophy. In summary, circadian modulation of lipolysis rates regulates the availability of lipid-derived energy during the day, suggesting a role for WAT clocks in the regulation of energy homeostasis.

Clock genes and sleep.

In most species--from cyanobacteria to humans--endogenous clocks have evolved that drive 24-h rhythms of behavior and physiology. In mammals, these circadian rhythms are regulated by a hierarchical network of cellular oscillators controlled by a set of clock genes organized in a system of interlocked transcriptional feedback loops. One of the most prominent outputs of the circadian system is the synchronization of the sleep-wake cycle with external (day-) time. Clock genes also have a strong impact on many other biological functions, such as memory formation, energy metabolism, and immunity. Remarkably, large overlaps exist between clock gene and sleep (loss) mediated effects on these processes. This review summarizes sleep clock gene interactions for these three phenomena, highlighting potential mediators linking sleep and/or clock function to physiological output in an attempt to better understand the complexity of diurnal adaptation and its consequences for health and disease.

Diurnal rhythm of circulating nicotinamide phosphoribosyltransferase (Nampt/visfatin/PBEF): impact of sleep loss and relation to glucose metabolism.

CONTEXT: Animal studies indicate that nicotinamide phosphoribosyltransferase [Nampt/visfatin/pre-B-cell colony-enhancing factor (PBEF)] contributes to the circadian fine-tuning of metabolic turnover. However, it is unknown whether circulating Nampt concentrations, which are elevated in type 2 diabetes and obesity, display a diurnal rhythm in humans. OBJECTIVE: Our objective was to examine the 24-h profile of serum Nampt in humans under conditions of sleep and sleep deprivation and relate the Nampt pattern to morning postprandial glucose metabolism. INTERVENTION: Fourteen healthy men participated in two 24-h sessions starting at 1800 h, including either regular 8-h-night sleep or continuous wakefulness. Serum Nampt and leptin were measured in 1.5- to 3-h intervals. In the morning, plasma glucose and serum insulin responses to standardized breakfast intake were determined. MAIN OUTCOME MEASURES: Under regular sleep-wake conditions, Nampt levels displayed a pronounced diurnal rhythm, peaking during early afternoon (P < 0.001) that was inverse to leptin profiles peaking in the early night. When subjects stayed awake, the Nampt rhythm was preserved but phase advanced by about 2 h (P < 0.05). Two-hour postprandial plasma glucose concentrations were elevated after sleep loss (P < 0.05), whereas serum insulin was not affected. The relative glucose increase due to sleep loss displayed a positive association with the magnitude of the Nampt phase shift (r = 0.54; P < 0.05). CONCLUSIONS: Serum Nampt concentrations follow a diurnal rhythm, peaking in the afternoon. Sleep loss induces a Nampt rhythm phase shift that is positively related to the impairment of postprandial glucose metabolism due to sleep deprivation, suggesting a regulatory impact of Nampt rhythmicity on glucose homeostasis.

Synaptotagmin10-Cre, a driver to disrupt clock genes in the SCN.

Surgical lesion of the suprachiasmatic nuclei (SCN) profoundly affects the circadian timing system. A complication of SCN ablations is the concomitant scission of SCN afferents and efferents. Genetic disruption of the molecular clockwork in the SCN provides a complementary, less invasive experimental approach. The authors report the generation and functional analysis of a new Cre recombinase driver mouse that evokes homologous recombination with high efficiency in the SCN. They inserted the Cre recombinase cDNA into the Synaptotagmin10 (Syt10) locus, a gene strongly expressed in the SCN. Heterozygous Synaptotagmin10-Cre (Syt10(Cre)) mice have no obvious circadian locomotor phenotype, and homozygous animals show slightly reduced light-induced phase delays. Crosses of Syt10(Cre) mice with ß-galactosidase reporter animals revealed strong Cre activity in the vast majority of SCN cells. Cre activity is not detected in nonneuronal tissues with the exception of the testis. The authors demonstrate that conditionally deleting the clock gene Bmal1 using the Syt10(Cre) driver renders animals arrhythmic.