Insulin/IGF-1 Drives PERIOD Synthesis to Entrain Circadian Rhythms with Feeding Time.

In mammals, endogenous circadian clocks sense and respond to daily feeding and lighting cues, adjusting internal ∼24 h rhythms to resonate with, and anticipate, external cycles of day and night. The mechanism underlying circadian entrainment to feeding time is critical for understanding why mistimed feeding, as occurs during shift work, disrupts circadian physiology, a state that is associated with increased incidence of chronic diseases such as type 2 (T2) diabetes. We show that feeding-regulated hormones insulin and insulin-like growth factor 1 (IGF-1) reset circadian clocks in vivo and in vitro by induction of PERIOD proteins, and mistimed insulin signaling disrupts circadian organization of mouse behavior and clock gene expression. Insulin and IGF-1 receptor signaling is sufficient to determine essential circadian parameters, principally via increased PERIOD protein synthesis. This requires coincident mechanistic target of rapamycin (mTOR) activation, increased phosphoinositide signaling, and microRNA downregulation. Besides its well-known homeostatic functions, we propose insulin and IGF-1 are primary signals of feeding time to cellular clocks throughout the body.

Meet the authors: Jonathan Philpott and Carrie L. Partch.

Here, Molecular Cell talks to first author Jonathan Philpott and co-corresponding author Carrie Partch about their paper, "PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period" (in this issue of Molecular Cell) and their scientific journeys until now.

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.

Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation.

Period (PER) proteins are essential components of the mammalian circadian clock. They form complexes with cryptochromes (CRY), which negatively regulate CLOCK/BMAL1-dependent transactivation of clock and clock-controlled genes. To define the roles of mammalian CRY/PER complexes in the circadian clock, we have determined the crystal structure of a complex comprising the photolyase homology region of mouse CRY1 (mCRY1) and a C-terminal mouse PER2 (mPER2) fragment. mPER2 winds around the helical mCRY1 domain covering the binding sites of FBXL3 and CLOCK/BMAL1, but not the FAD binding pocket. Our structure revealed an unexpected zinc ion in one interface, which stabilizes mCRY1-mPER2 interactions in vivo. We provide evidence that mCRY1/mPER2 complex formation is modulated by an interplay of zinc binding and mCRY1 disulfide bond formation, which may be influenced by the redox state of the cell. Our studies may allow for the development of circadian and metabolic modulators.

The phosphorylation switch that regulates ticking of the circadian clock.

In our 24/7 well-lit world, it's easy to skip or delay sleep to work, study, and play. However, our circadian rhythms are not easily fooled; the consequences of jet lag and shift work are many and severe, including metabolic, mood, and malignant disorders. The internal clock that keeps track of time has at its heart the reversible phosphorylation of the PERIOD proteins, regulated by isoforms of casein kinase 1 (CK1). In-depth biochemical, genetic, and structural studies of these kinases, their mutants, and their splice variants have combined over the past several years to provide a robust understanding of how the core clock is regulated by a phosphoswitch whereby phosphorylation of a stabilizing site on PER blocks phosphorylation of a distant phosphodegron. The recent structure of a circadian mutant form of CK1 implicates an internal activation loop switch that regulates this phosphoswitch and points to new approaches to regulation of the clock.

Natural antisense transcript of Period2, Per2AS, regulates the amplitude of the mouse circadian clock.

In mammals, a set of core clock genes form transcription-translation feedback loops to generate circadian oscillations. We and others recently identified a novel transcript at the Period2 (Per2) locus that is transcribed from the antisense strand of Per2 This transcript, Per2AS, is expressed rhythmically and antiphasic to Per2 mRNA, leading to our hypothesis that Per2AS and Per2 mutually inhibit each other's expression and form a double negative feedback loop. By perturbing the expression of Per2AS, we found that Per2AS transcription, but not transcript, represses Per2 However, Per2 does not repress Per2AS, as Per2 knockdown led to a decrease in the Per2AS level, indicating that Per2AS forms a single negative feedback loop with Per2 and maintains the level of Per2 within the oscillatory range. Per2AS also regulates the amplitude of the circadian clock, and this function cannot be solely explained through its interaction with Per2, as Per2 knockdown does not recapitulate the phenotypes of Per2AS perturbation. Overall, our data indicate that Per2AS is an important regulatory molecule in the mammalian circadian clock machinery. Our work also supports the idea that antisense transcripts of core clock genes constitute a common feature of circadian clocks, as they are found in other organisms.

RNA-methylation-dependent RNA processing controls the speed of the circadian clock.

The eukaryotic biological clock involves a negative transcription-translation feedback loop in which clock genes regulate their own transcription and that of output genes of metabolic significance. While around 10% of the liver transcriptome is rhythmic, only about a fifth is driven by de novo transcription, indicating mRNA processing is a major circadian component. Here, we report that inhibition of transmethylation reactions elongates the circadian period. RNA sequencing then reveals methylation inhibition causes widespread changes in the transcription of the RNA processing machinery, associated with m(6)A-RNA methylation. We identify m(6)A sites on many clock gene transcripts and show that specific inhibition of m(6)A methylation by silencing of the m(6)A methylase Mettl3 is sufficient to elicit circadian period elongation and RNA processing delay. Analysis of the circadian nucleocytoplasmic distribution of clock genes Per2 and Arntl then revealed an uncoupling between steady-state pre-mRNA and cytoplasmic mRNA rhythms when m(6)A methylation is inhibited.

Blood-borne circadian signal stimulates daily oscillations in actin dynamics and SRF activity.

In peripheral tissues circadian gene expression can be driven either by local oscillators or by cyclic systemic cues controlled by the master clock in the brain's suprachiasmatic nucleus. In the latter case, systemic signals can activate immediate early transcription factors (IETFs) and thereby control rhythmic transcription. In order to identify IETFs induced by diurnal blood-borne signals, we developed an unbiased experimental strategy, dubbed Synthetic TAndem Repeat PROMoter (STAR-PROM) screening. This technique relies on the observation that most transcription factor binding sites exist at a relatively high frequency in random DNA sequences. Using STAR-PROM we identified serum response factor (SRF) as an IETF responding to oscillating signaling proteins present in human and rodent sera. Our data suggest that in mouse liver SRF is regulated via dramatic diurnal changes of actin dynamics, leading to the rhythmic translocation of the SRF coactivator Myocardin-related transcription factor-B (MRTF-B) into the nucleus.

Structures of Drosophila cryptochrome and mouse cryptochrome1 provide insight into circadian function.

Drosophila cryptochrome (dCRY) is a FAD-dependent circadian photoreceptor, whereas mammalian cryptochromes (CRY1/2) are integral clock components that repress mCLOCK/mBMAL1-dependent transcription. We report crystal structures of full-length dCRY, a dCRY loop deletion construct, and the photolyase homology region of mouse CRY1 (mCRY1). Our dCRY structures depict Phe534 of the regulatory tail in the same location as the photolesion in DNA-repairing photolyases and reveal that the sulfur loop and tail residue Cys523 plays key roles in the dCRY photoreaction. Our mCRY1 structure visualizes previously characterized mutations, an NLS, and MAPK and AMPK phosphorylation sites. We show that the FAD and antenna chromophore-binding regions, a predicted coiled-coil helix, the C-terminal lid, and charged surfaces are involved in FAD-independent mPER2 and FBXL3 binding and mCLOCK/mBMAL1 transcriptional repression. The structure of a mammalian cryptochrome1 protein may catalyze the development of CRY chemical probes and the design of therapeutic metabolic modulators.

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.

Circadian period is compensated for repressor protein turnover rates in single cells.

Most mammalian cells have molecular circadian clocks that generate widespread rhythms in transcript and protein abundance. While circadian clocks are robust to fluctuations in the cellular environment, little is known about the mechanisms by which the circadian period compensates for fluctuating metabolic states. Here, we exploit the heterogeneity of single cells both in circadian period and a metabolic parameter-protein stability-to study their interdependence without the need for genetic manipulation. We generated cells expressing key circadian proteins (CRYPTOCHROME1/2 (CRY1/2) and PERIOD1/2 (PER1/2)) as endogenous fusions with fluorescent proteins and simultaneously monitored circadian rhythms and degradation in thousands of single cells. We found that the circadian period compensates for fluctuations in the turnover rates of circadian repressor proteins and uncovered possible mechanisms using a mathematical model. In addition, the stabilities of the repressor proteins are circadian phase dependent and correlate with the circadian period in a phase-dependent manner, in contrast to the prevailing model.

Phosphorylation of DNA-binding domains of CLOCK-BMAL1 complex for PER-dependent inhibition in circadian clock of mammalian cells.

In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E-box sequences and activates transcription of target genes, including Period (Per). Translated PER proteins then bind to the CLOCK-BMAL1 complex to inhibit its transcriptional activity. However, the molecular mechanism and the impact of this PER-dependent inhibition on the circadian clock oscillation remain elusive. We previously identified Ser38 and Ser42 in a DNA-binding domain of CLOCK as phosphorylation sites at the PER-dependent inhibition phase. In this study, knockout rescue experiments showed that nonphosphorylatable (Ala) mutations at these sites shortened circadian period, whereas their constitutive-phospho-mimetic (Asp) mutations completely abolished the circadian rhythms. Similarly, we found that nonphosphorylatable (Ala) and constitutive-phospho-mimetic (Glu) mutations at Ser78 in a DNA-binding domain of BMAL1 also shortened the circadian period and abolished the rhythms, respectively. The mathematical modeling predicted that these constitutive-phospho-mimetic mutations weaken the DNA binding of the CLOCK-BMAL1 complex and that the nonphosphorylatable mutations inhibit the PER-dependent displacement (reduction of DNA-binding ability) of the CLOCK-BMAL1 complex from DNA. Biochemical experiments supported the importance of these phosphorylation sites for displacement of the complex in the PER2-dependent inhibition. Our results provide direct evidence that phosphorylation of CLOCK-Ser38/Ser42 and BMAL1-Ser78 plays a crucial role in the PER-dependent inhibition and the determination of the circadian period.

Bmal1 integrates circadian function and temperature sensing in the suprachiasmatic nucleus.

Circadian regulation and temperature dependency are important orchestrators of molecular pathways. How the integration between these two drivers is achieved, is not understood. We monitored circadian- and temperature-dependent effects on transcription dynamics of cold-response protein RNA Binding Motif 3 (Rbm3). Temperature changes in the mammalian master circadian pacemaker, the suprachiasmatic nucleus (SCN), induced Rbm3 transcription and regulated its circadian periodicity, whereas the core clock gene Per2 was unaffected. Rbm3 induction depended on a full Brain And Muscle ARNT-Like Protein 1 (Bmal1) complement: reduced Bmal1 erased Rbm3 responses and weakened SCN circuit resilience to temperature changes. By focusing on circadian and temperature dependency, we highlight weakened transmission between core clock and downstream pathways as a potential route for reduced circadian resilience.

Clock-dependent chromatin accessibility rhythms regulate circadian transcription.

Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.

NEMO/NLK phosphorylates PERIOD to initiate a time-delay phosphorylation circuit that sets circadian clock speed.

The speed of circadian clocks in animals is tightly linked to complex phosphorylation programs that drive daily cycles in the levels of PERIOD (PER) proteins. Using Drosophila, we identify a time-delay circuit based on hierarchical phosphorylation that controls the daily downswing in PER abundance. Phosphorylation by the NEMO/NLK kinase at the "per-short" domain on PER stimulates phosphorylation by DOUBLETIME (DBT/CK1δ/ɛ) at several nearby sites. This multisite phosphorylation operates in a spatially oriented and graded manner to delay progressive phosphorylation by DBT at other more distal sites on PER, including those required for recognition by the F box protein SLIMB/ß-TrCP and proteasomal degradation. Highly phosphorylated PER has a more open structure, suggesting that progressive increases in global phosphorylation contribute to the timing mechanism by slowly increasing PER susceptibility to degradation. Our findings identify NEMO as a clock kinase and demonstrate that long-range interactions between functionally distinct phospho-clusters collaborate to set clock speed.

Endogenous circadian reporters reveal functional differences of PERIOD paralogs and the significance of PERIOD:CK1 stable interaction.

Adverse consequences from having a faulty circadian clock include compromised sleep quality and poor performance in the short-term, and metabolic diseases and cancer in the long-term. However, our understanding of circadian disorders is limited by the incompleteness of our molecular models and our dearth of defined mutant models. Because it would be prohibitively expensive to develop live animal models to study the full range of complicated clock mechanisms, we developed PER1-luc and PER2-luc endogenous circadian reporters in a validated clock cell model, U-2 OS, where the genome can be easily manipulated, and functional consequences of mutations can be accurately studied. When major clock genes were knocked out in these cells, circadian rhythms were modulated similarly compared with corresponding mutant mice, validating the platform for genetics studies. Using these reporter cells, we uncovered critical differences between two paralogs of PER. Although PER1 and PER2 are considered redundant and either one can serve as a pacemaker alone, they were dramatically different in biochemical parameters such as stability and phosphorylation kinetics. Consistently, circadian phase was dramatically different between PER1 and PER2 knockout reporter cells. We further showed that the stable binding of casein kinase1δ/ε to PER is not required for PER phosphorylation itself, but is critical for delayed timing of phosphorylation. Our system can be used as an efficient platform to study circadian disorders associated with pathogenic mutations and their underlying molecular mechanisms.

Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation.

Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.

Circadian ribosome profiling reveals a role for the Period2 upstream open reading frame in sleep.

Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered posttranscriptionally. We used ribosome profiling to examine posttranscriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). An increase in the number of uORFs in the 5'UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic reporter constructs. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF cells. Mutation of the Per2 uORF in mice increased Per2 mRNA expression, enhanced ribosome binding on Per2, and reduced total sleep time compared to that in wild-type mice. These results suggest that uORFs affect mRNA posttranscriptionally, which can impact physiological rhythms and sleep.

Hierarchical and scaffolded phosphorylation of two degrons controls PER2 stability.

The duration of the transcription-repression cycles that give rise to mammalian circadian rhythms is largely determined by the stability of the PERIOD (PER) protein, the rate-limiting components of the molecular clock. The degradation of PERs is tightly regulated by multisite phosphorylation by casein kinase 1 (CK1δ/ε). In this phosphoswitch, phosphorylation of a PER2 degron [degron 2 (D2)] causes degradation, while phosphorylation of the PER2 familial advanced sleep phase (FASP) domain blocks CK1 activity on the degron, stabilizing PER2. However, this model and many other studies of PER2 degradation do not include the second degron of PER2 that is conserved in PER1, termed degron 1 (D1). We examined how these two degrons contribute to PER2 stability, affect the balance of the phosphoswitch, and how they are differentiated by CK1. Using PER2-luciferase fusions and real-time luminometry, we investigated the contribution of both D2 and of CK1-PER2 binding. We find that D1, like D2, is a substrate of CK1 but that D1 plays only a 'backup' role in PER2 degradation. Notably, CK1 bound to a PER1:PER2 dimer protein can phosphorylate PER1 D1 in trans. This scaffolded phosphorylation provides additional levels of control to PER stability and circadian rhythms.