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.

Acid Suspends the Circadian Clock in Hypoxia through Inhibition of mTOR.

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

Physiology Flies with Time.

The 2017 Nobel Prize in Medicine or Physiology has been awarded to Jeffrey Hall, Michael Rosbash, and Michael Young for elucidating molecular mechanisms of the circadian clock. From studies beginning in fruit flies, we now know that circadian regulation pervades most biological processes and has strong ties to human health and disease.

Core Circadian Clock Genes Regulate Leukemia Stem Cells in AML.

Leukemia stem cells (LSCs) have the capacity to self-renew and propagate disease upon serial transplantation in animal models, and elimination of this cell population is required for curative therapies. Here, we describe a series of pooled, in vivo RNAi screens to identify essential transcription factors (TFs) in a murine model of acute myeloid leukemia (AML) with genetically and phenotypically defined LSCs. These screens reveal the heterodimeric, circadian rhythm TFs Clock and Bmal1 as genes required for the growth of AML cells in vitro and in vivo. Disruption of canonical circadian pathway components produces anti-leukemic effects, including impaired proliferation, enhanced myeloid differentiation, and depletion of LSCs. We find that both normal and malignant hematopoietic cells harbor an intact clock with robust circadian oscillations, and genetic knockout models reveal a leukemia-specific dependence on the pathway. Our findings establish a role for the core circadian clock genes in AML.

Interactive features of proteins composing eukaryotic circadian clocks.

Research into the molecular mechanisms of eukaryotic circadian clocks has proceeded at an electrifying pace. In this review, we discuss advances in our understanding of the structures of central molecular players in the timing oscillators of fungi, insects, and mammals. A series of clock protein structures demonstrate that the PAS (Per/Arnt/Sim) domain has been used with great variation to formulate the transcriptional activators and repressors of the clock. We discuss how posttranslational modifications and external cues, such as light, affect the conformation and function of core clock components. Recent breakthroughs have also revealed novel interactions among clock proteins and new partners that couple the clock to metabolic and developmental pathways. Overall, a picture of clock function has emerged wherein conserved motifs and structural platforms have been elaborated into a highly dynamic collection of interacting molecules that undergo orchestrated changes in chemical structure, conformational state, and partners.

Partitioning circadian transcription by SIRT6 leads to segregated control of cellular metabolism.

Circadian rhythms are intimately linked to cellular metabolism. Specifically, the NAD(+)-dependent deacetylase SIRT1, the founding member of the sirtuin family, contributes to clock function. Whereas SIRT1 exhibits diversity in deacetylation targets and subcellular localization, SIRT6 is the only constitutively chromatin-associated sirtuin and is prominently present at transcriptionally active genomic loci. Comparison of the hepatic circadian transcriptomes reveals that SIRT6 and SIRT1 separately control transcriptional specificity and therefore define distinctly partitioned classes of circadian genes. SIRT6 interacts with CLOCK:BMAL1 and, differently from SIRT1, governs their chromatin recruitment to circadian gene promoters. Moreover, SIRT6 controls circadian chromatin recruitment of SREBP-1, resulting in the cyclic regulation of genes implicated in fatty acid and cholesterol metabolism. This mechanism parallels a phenotypic disruption in fatty acid metabolism in SIRT6 null mice as revealed by circadian metabolome analyses. Thus, genomic partitioning by two independent sirtuins contributes to differential control of circadian metabolism.

Impact of circadian nuclear receptor REV-ERBα on midbrain dopamine production and mood regulation.

The circadian nature of mood and its dysfunction in affective disorders is well recognized, but the underlying molecular mechanisms are still unclear. Here, we show that the circadian nuclear receptor REV-ERBα, which is associated with bipolar disorder, impacts midbrain dopamine production and mood-related behavior in mice. Genetic deletion of the Rev-erbα gene or pharmacological inhibition of REV-ERBα activity in the ventral midbrain induced mania-like behavior in association with a central hyperdopaminergic state. Also, REV-ERBα repressed tyrosine hydroxylase (TH) gene transcription via competition with nuclear receptor-related 1 protein (NURR1), another nuclear receptor crucial for dopaminergic neuronal function, thereby driving circadian TH expression through a target-dependent antagonistic mechanism. In conclusion, we identified a molecular connection between the circadian timing system and mood regulation, suggesting that REV-ERBα could be targeting in the treatment of circadian rhythm-related affective disorders.

Cooperation between bHLH transcription factors and histones for DNA access.

The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2,3). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry-exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A-H2B acidic patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.

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.

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.

Competing E3 ubiquitin ligases govern circadian periodicity by degradation of CRY in nucleus and cytoplasm.

Period determination in the mammalian circadian clock involves the turnover rate of the repressors CRY and PER. We show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss of function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals.

Reprogramming of the circadian clock by nutritional challenge.

Circadian rhythms and cellular metabolism are intimately linked. Here, we reveal that a high-fat diet (HFD) generates a profound reorganization of specific metabolic pathways, leading to widespread remodeling of the liver clock. Strikingly, in addition to disrupting the normal circadian cycle, HFD causes an unexpectedly large-scale genesis of de novo oscillating transcripts, resulting in reorganization of the coordinated oscillations between coherent transcripts and metabolites. The mechanisms underlying this reprogramming involve both the impairment of CLOCK:BMAL1 chromatin recruitment and a pronounced cyclic activation of surrogate pathways through the transcriptional regulator PPARγ. Finally, we demonstrate that it is specifically the nutritional challenge, and not the development of obesity, that causes the reprogramming of the clock and that the effects of the diet on the clock are reversible.

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.

A role for alternative splicing in circadian control of exocytosis and glucose homeostasis.

The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic ß cells that are perturbed in Clock-/- and Bmal1-/- ß-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant ß cells, including transcripts encoding Cask (calcium/calmodulin-dependent serine protein kinase) and Madd (MAP kinase-activating death domain). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd, and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in ß-cell function across the sleep/wake cycle.

Ion Channels Controlling Circadian Rhythms in Suprachiasmatic Nucleus Excitability.

Animals synchronize to the environmental day-night cycle by means of an internal circadian clock in the brain. In mammals, this timekeeping mechanism is housed in the suprachiasmatic nucleus (SCN) of the hypothalamus and is entrained by light input from the retina. One output of the SCN is a neural code for circadian time, which arises from the collective activity of neurons within the SCN circuit and comprises two fundamental components: 1) periodic alterations in the spontaneous excitability of individual neurons that result in higher firing rates during the day and lower firing rates at night, and 2) synchronization of these cellular oscillations throughout the SCN. In this review, we summarize current evidence for the identity of ion channels in SCN neurons and the mechanisms by which they set the rhythmic parameters of the time code. During the day, voltage-dependent and independent Na+ and Ca2+ currents, as well as several K+ currents, contribute to increased membrane excitability and therefore higher firing frequency. At night, an increase in different K+ currents, including Ca2+-activated BK currents, contribute to membrane hyperpolarization and decreased firing. Layered on top of these intrinsically regulated changes in membrane excitability, more than a dozen neuromodulators influence action potential activity and rhythmicity in SCN neurons, facilitating both synchronization and plasticity of the neural code.

Pairing of segmentation clock genes drives robust pattern formation.

Gene expression is an inherently stochastic process1,2; however, organismal development and homeostasis require cells to coordinate the spatiotemporal expression of large sets of genes. In metazoans, pairs of co-expressed genes often reside in the same chromosomal neighbourhood, with gene pairs representing 10 to 50% of all genes, depending on the species3-6. Because shared upstream regulators can ensure correlated gene expression, the selective advantage of maintaining adjacent gene pairs remains unknown6. Here, using two linked zebrafish segmentation clock genes, her1 and her7, and combining single-cell transcript counting, genetic engineering, real-time imaging and computational modelling, we show that gene pairing boosts correlated transcription and provides phenotypic robustness for the formation of developmental patterns. Our results demonstrate that the prevention of gene pairing disrupts oscillations and segmentation, and the linkage of her1 and her7 is essential for the development of the body axis in zebrafish embryos. We predict that gene pairing may be similarly advantageous in other organisms, and our findings could lead to the engineering of precise synthetic clocks in embryos and organoids.

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.

Mutations of the circadian clock genes Cry, Per, or Bmal1 have different effects on the transcribed and nontranscribed strands of cycling genes.

One important goal of circadian medicine is to apply time-of-day dosing to improve the efficacy of chemotherapy. However, limited knowledge of how the circadian clock regulates DNA repair presents a challenge to mechanism-based clinical application. We studied time-series genome-wide nucleotide excision repair in liver and kidney of wild type and three different clock mutant genotypes (Cry1-/-Cry2-/-, Per1-/-Per2-/-, and Bmal1-/-). Rhythmic repair on the nontranscribed strand was lost in all three clock mutants. Conversely, rhythmic repair of hundreds of genes on the transcribed strand (TSs) persisted in the livers of Cry1-/-Cry2-/- and Per1-/-Per2-/- mice. We identified a tissue-specific, promoter element-driven repair mode on TSs of collagen and angiogenesis genes in the absence of clock activators or repressors. Furthermore, repair on TSs of thousands of genes was altered when the circadian clock is disrupted. These data contribute to a better understanding of the regulatory role of the circadian clock on nucleotide excision repair in mammals and may be invaluable toward the design of time-aware platinum-based interventions in cancer.

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.