BMAL1-Driven Tissue Clocks Respond Independently to Light to Maintain Homeostasis.

Circadian rhythms control organismal physiology throughout the day. At the cellular level, clock regulation is established by a self-sustained Bmal1-dependent transcriptional oscillator network. However, it is still unclear how different tissues achieve a synchronized rhythmic physiology. That is, do they respond independently to environmental signals, or require interactions with each other to do so? We show that unexpectedly, light synchronizes the Bmal1-dependent circadian machinery in single tissues in the absence of Bmal1 in all other tissues. Strikingly, light-driven tissue autonomous clocks occur without rhythmic feeding behavior and are lost in constant darkness. Importantly, tissue-autonomous Bmal1 partially sustains homeostasis in otherwise arrhythmic and prematurely aging animals. Our results therefore support a two-branched model for the daily synchronization of tissues: an autonomous response branch, whereby light entrains circadian clocks without any commitment of other Bmal1-dependent clocks, and a memory branch using other Bmal1-dependent clocks to "remember" time in the absence of external cues.

Defining the Independence of the Liver Circadian Clock.

Mammals rely on a network of circadian clocks to control daily systemic metabolism and physiology. The central pacemaker in the suprachiasmatic nucleus (SCN) is considered hierarchically dominant over peripheral clocks, whose degree of independence, or tissue-level autonomy, has never been ascertained in vivo. Using arrhythmic Bmal1-null mice, we generated animals with reconstituted circadian expression of BMAL1 exclusively in the liver (Liver-RE). High-throughput transcriptomics and metabolomics show that the liver has independent circadian functions specific for metabolic processes such as the NAD+ salvage pathway and glycogen turnover. However, although BMAL1 occupies chromatin at most genomic targets in Liver-RE mice, circadian expression is restricted to ∼10% of normally rhythmic transcripts. Finally, rhythmic clock gene expression is lost in Liver-RE mice under constant darkness. Hence, full circadian function in the liver depends on signals emanating from other clocks, and light contributes to tissue-autonomous clock function.

Multiple Functions and Regulation of Mammalian Peroxiredoxins.

Peroxiredoxins (Prxs) constitute a major family of peroxidases, with mammalian cells expressing six Prx isoforms (PrxI to PrxVI). Cells produce hydrogen peroxide (H2O2) at various intracellular locations where it can serve as a signaling molecule. Given that Prxs are abundant and possess a structure that renders the cysteine (Cys) residue at the active site highly sensitive to oxidation by H2O2, the signaling function of this oxidant requires extensive and highly localized regulation. Recent findings on the reversible regulation of PrxI through phosphorylation at the centrosome and on the hyperoxidation of the Cys at the active site of PrxIII in mitochondria are described in this review as examples of such local regulation of H2O2 signaling. Moreover, their high affinity for and sensitivity to oxidation by H2O2 confer on Prxs the ability to serve as sensors and transducers of H2O2 signaling through transfer of their oxidation state to bound effector proteins.

Molecular mechanisms and physiological importance of circadian rhythms.

To accommodate daily recurring environmental changes, animals show cyclic variations in behaviour and physiology, which include prominent behavioural states such as sleep-wake cycles but also a host of less conspicuous oscillations in neurological, metabolic, endocrine, cardiovascular and immune functions. Circadian rhythmicity is created endogenously by genetically encoded molecular clocks, whose components cooperate to generate cyclic changes in their own abundance and activity, with a periodicity of about a day. Throughout the body, such molecular clocks convey temporal control to the function of organs and tissues by regulating pertinent downstream programmes. Synchrony between the different circadian oscillators and resonance with the solar day is largely enabled by a neural pacemaker, which is directly responsive to certain environmental cues and able to transmit internal time-of-day representations to the entire body. In this Review, we discuss aspects of the circadian clock in Drosophila melanogaster and mammals, including the components of these molecular oscillators, the function and mechanisms of action of central and peripheral clocks, their synchronization and their relevance to human health.

How the circadian nuclear orphan receptor REV-ERBα represses transcription: Temporal and spatial phase separation combined.

In this issue of Molecular Cell, Zhu et al.1 demonstrate that REV-ERBα and its co-repressor NCOR1 are assembled into daytime-dependent liquid droplets that constitute hubs in which the transcription of multiple REV-ERBα target genes is simultaneously repressed.

An intrinsically disordered region controlling condensation of a circadian clock component and rhythmic transcription in the liver.

Circadian gene transcription is fundamental to metabolic physiology. Here we report that the nuclear receptor REV-ERBα, a repressive component of the molecular clock, forms circadian condensates in the nuclei of mouse liver. These condensates are dictated by an intrinsically disordered region (IDR) located in the protein's hinge region which specifically concentrates nuclear receptor corepressor 1 (NCOR1) at the genome. IDR deletion diminishes the recruitment of NCOR1 and disrupts rhythmic gene transcription in vivo. REV-ERBα condensates are located at high-order transcriptional repressive hubs in the liver genome that are highly correlated with circadian gene repression. Deletion of the IDR disrupts transcriptional repressive hubs and diminishes silencing of target genes by REV-ERBα. This work demonstrates physiological circadian protein condensates containing REV-ERBα whose IDR is required for hub formation and the control of rhythmic gene expression.

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

PERIOD (PER) and Casein Kinase 1δ regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1δ phosphorylation of the familial advanced sleep phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortening circadian period in human cells. We found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

Circadian mechanisms in adipose tissue bioenergetics and plasticity.

The circadian clock plays an essential role in coordinating feeding and metabolic rhythms with the light/dark cycle. Disruption of clocks is associated with increased adiposity and metabolic disorders, whereas aligning feeding time with cell-autonomous rhythms in metabolism improves health. Here, we provide a comprehensive overview of recent literature in adipose tissue biology as well as our understanding of molecular mechanisms underlying the circadian regulation of transcription, metabolism, and inflammation in adipose tissue. We highlight recent efforts to uncover the mechanistic links between clocks and adipocyte metabolism, as well as its application to dietary and behavioral interventions to improve health and mitigate obesity.

Genetics of circadian rhythms and sleep in human health and disease.

Circadian rhythms and sleep are fundamental biological processes integral to human health. Their disruption is associated with detrimental physiological consequences, including cognitive, metabolic, cardiovascular and immunological dysfunctions. Yet many of the molecular underpinnings of sleep regulation in health and disease have remained elusive. Given the moderate heritability of circadian and sleep traits, genetics offers an opportunity that complements insights from model organism studies to advance our fundamental molecular understanding of human circadian and sleep physiology and linked chronic disease biology. Here, we review recent discoveries of the genetics of circadian and sleep physiology and disorders with a focus on those that reveal causal contributions to complex diseases.

Post-transcriptional gene regulation by mRNA modifications.

The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis.

PARP-1 drives slumber: A reciprocal relationship between sleep homeostasis and DNA damage repair.

Sleep-wake cycles are orchestrated by the circadian clock and a sleep homeostat that records accumulating sleep pressure. Zada et al. (2021) now show that DNA lesions, recognized by PARP-1, accumulate in neurons of zebrafish larvae and promote homeostatic sleep pressure.

Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity.

In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, we performed a systematic RNAi screen in human cells and identified protein phosphatase 4 (PPP4) with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals and Drosophila Genetic depletion of PPP4 shortens the circadian period, whereas overexpression lengthens it. PPP4 inhibits CLOCK/BMAL1 transactivation activity by binding to BMAL1 and counteracting its phosphorylation. This leads to increased CLOCK/BMAL1 DNA occupancy and decreased transcriptional activity, which counteracts the "kamikaze" properties of CLOCK/BMAL1. Through this mechanism, PPP4 contributes to the critical delay of negative feedback by retarding PER/CRY/CK1δ-mediated inhibition of CLOCK/BMAL1.

BMAL1 dephosphorylation determines the pace of the circadian clock.

In mammals, virtually all body cells harbor cell-autonomous and self-sustained circadian oscillators that rely on delayed negative feedback loops in gene expression. Transcriptional activation and repression play a major role in keeping these clocks ticking, but numerous post-translational mechanisms-and particularly the phosphorylation of core clock components by protein kinases-are also critically involved in setting the pace of these timekeepers. In this issue of Genes & Development, Klemz and colleagues (pp. 1161-1174) now show how dephosphorylation of BMAL1 by protein phosphatase 4 (PPP4) participates in the modulation of circadian timing.

Transcriptional rewiring of an evolutionarily conserved circadian clock.

Circadian clocks temporally coordinate daily organismal biology over the 24-h cycle. Their molecular design, preserved between fungi and animals, is based on a core-oscillator composed of a one-step transcriptional-translational-negative-feedback-loop (TTFL). To test whether this evolutionarily conserved TTFL architecture is the only plausible way for achieving a functional circadian clock, we adopted a transcriptional rewiring approach, artificially co-opting regulators of the circadian output pathways into the core-oscillator. Herein we describe one of these semi-synthetic clocks which maintains all basic circadian features but, notably, it also exhibits new attributes such as a "lights-on timer" logic, where clock phase is fixed at the end of the night. Our findings indicate that fundamental circadian properties such as period, phase and temperature compensation are differentially regulated by transcriptional and posttranslational aspects of the clockworks.

NAD+ Controls Circadian Reprogramming through PER2 Nuclear Translocation to Counter Aging.

Disrupted sleep-wake and molecular circadian rhythms are a feature of aging associated with metabolic disease and reduced levels of NAD+, yet whether changes in nucleotide metabolism control circadian behavioral and genomic rhythms remains unknown. Here, we reveal that supplementation with the NAD+ precursor nicotinamide riboside (NR) markedly reprograms metabolic and stress-response pathways that decline with aging through inhibition of the clock repressor PER2. NR enhances BMAL1 chromatin binding genome-wide through PER2K680 deacetylation, which in turn primes PER2 phosphorylation within a domain that controls nuclear transport and stability and that is mutated in human advanced sleep phase syndrome. In old mice, dampened BMAL1 chromatin binding, transcriptional oscillations, mitochondrial respiration rhythms, and late evening activity are restored by NAD+ repletion to youthful levels with NR. These results reveal effects of NAD+ on metabolism and the circadian system with aging through the spatiotemporal control of the molecular clock.

Senescence of Timing Reverted: NAD+ Rejuvenates the Circadian Clock.

The amplitude of circadian rhythms dampens with age, but Levine et al. (2020) now show that nicotinamide adenine dinucleotide (NAD+) can restore robust circadian gene expression and behavior in aged mice through SIRT1-dependent deacetylation of the core clock protein PER2.

Clocks at sea: the genome-editing tide is rising.

The coastline is a particularly challenging environment for its inhabitants. Not only do they have to cope with the solar day and the passing of seasons, but they must also deal with tides. In addition, many marine species track the phase of the moon, especially to coordinate reproduction. Marine animals show remarkable behavioral and physiological adaptability, using biological clocks to anticipate specific environmental cycles. Presently, we lack a basic understanding of the molecular mechanisms underlying circatidal and circalunar clocks. Recent advances in genome engineering and the development of genetically tractable marine model organisms are transforming how we study these timekeeping mechanisms and opening a novel era in marine chronobiology.

CONSTANS, a HUB for all seasons: How photoperiod pervades plant physiology regulatory circuits.

How does a plant detect the changing seasons and make important developmental decisions accordingly? How do they incorporate daylength information into their routine physiological processes? Photoperiodism, or the capacity to measure the daylength, is a crucial aspect of plant development that helps plants determine the best time of the year to make vital decisions, such as flowering. The protein CONSTANS (CO) constitutes the central regulator of this sensing mechanism, not only activating florigen production in the leaves but also participating in many physiological aspects in which seasonality is important. Recent discoveries place CO in the center of a gene network that can determine the length of the day and confer seasonal input to aspects of plant development and physiology as important as senescence, seed size, or circadian rhythms. In this review, we discuss the importance of CO protein structure, function, and evolutionary mechanisms that embryophytes have developed to incorporate annual information into their physiology.

Molecular circadian rhythms are robust in marine annelids lacking rhythmic behavior.

The circadian clock controls behavior and metabolism in various organisms. However, the exact timing and strength of rhythmic phenotypes can vary significantly between individuals of the same species. This is highly relevant for rhythmically complex marine environments where organismal rhythmic diversity likely permits the occupation of different microenvironments. When investigating circadian locomotor behavior of Platynereis dumerilii, a model system for marine molecular chronobiology, we found strain-specific, high variability between individual worms. The individual patterns were maintained for several weeks. A diel head transcriptome comparison of behaviorally rhythmic versus arrhythmic wild-type worms showed that 24-h cycling of core circadian clock transcripts is identical between both behavioral phenotypes. While behaviorally arrhythmic worms showed a similar total number of cycling transcripts compared to their behaviorally rhythmic counterparts, the annotation categories of their transcripts, however, differed substantially. Consistent with their locomotor phenotype, behaviorally rhythmic worms exhibit an enrichment of cycling transcripts related to neuronal/behavioral processes. In contrast, behaviorally arrhythmic worms showed significantly increased diel cycling for metabolism- and physiology-related transcripts. The prominent role of the neuropeptide pigment-dispersing factor (PDF) in Drosophila circadian behavior prompted us to test for a possible functional involvement of Platynereis pdf. Differing from its role in Drosophila, loss of pdf impacts overall activity levels but shows only indirect effects on rhythmicity. Our results show that individuals arrhythmic in a given process can show increased rhythmicity in others. Across the Platynereis population, rhythmic phenotypes exist as a continuum, with no distinct "boundaries" between rhythmicity and arrhythmicity. We suggest that such diel rhythm breadth is an important biodiversity resource enabling the species to quickly adapt to heterogeneous or changing marine environments. In times of massive sequencing, our work also emphasizes the importance of time series and functional tests.

Circadian autophagy drives iTRF-mediated longevity.

Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans1-5. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. Here, to exploit the genetic tools and well-characterized ageing markers of Drosophila, we developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. We found that iTRF enhanced circadian-regulated transcription and that iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension.