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.

The molecular machinery that governs circadian rhythmicity comprises proteins whose interplay generates time-specific transcription of clock genes. The role of chromatin remodeling in a physiological setting such as the circadian clock is yet unclear. We show that the protein CLOCK, a central component of the circadian pacemaker, has histone acetyltransferase (HAT) activity. CLOCK shares homology with acetyl-coenzyme A binding motifs within the MYST family of HATs. CLOCK displays high sequence similarity to ACTR, a member of SRC family of HATs, with which it shares also enzymatic specificity for histones H3 and H4. BMAL1, the heterodimerization partner of CLOCK, enhances HAT function. The HAT activity of CLOCK is essential to rescue circadian rhythmicity and activation of clock genes in Clock mutant cells. Identification of CLOCK as a novel type of DNA binding HAT reveals that chromatin remodeling is crucial for the core clock mechanism and identifies unforeseen links between histone acetylation and cellular physiology.

Malfunction of the circadian clock has been linked to the pathogenesis of a variety of diseases. We show that mice lacking the core clock components Cryptochrome-1 (Cry1) and Cryptochrome-2 (Cry2) (Cry-null mice) show salt-sensitive hypertension due to abnormally high synthesis of the mineralocorticoid aldosterone by the adrenal gland. An extensive search for the underlying cause led us to identify type VI 3beta-hydroxyl-steroid dehydrogenase (Hsd3b6) as a new hypertension risk factor in mice. Hsd3b6 is expressed exclusively in aldosterone-producing cells and is under transcriptional control of the circadian clock. In Cry-null mice, Hsd3b6 messenger RNA and protein levels are constitutively high, leading to a marked increase in 3beta-hydroxysteroid dehydrogenase-isomerase (3beta-HSD) enzymatic activity and, as a consequence, enhanced aldosterone production. These data place Hsd3b6 in a pivotal position through which circadian clock malfunction is coupled to the development of hypertension. Translation of these findings to humans will require clinical examination of human HSD3B1 gene, which we found to be functionally similar to mouse Hsd3b6.

Jet-lag symptoms arise from temporal misalignment between the internal circadian clock and external solar time. We found that circadian rhythms of behavior (locomotor activity), clock gene expression, and body temperature immediately reentrained to phase-shifted light-dark cycles in mice lacking vasopressin receptors V1a and V1b (V1a(-/-)V1b(-/-)). Nevertheless, the behavior of V1a(-/-)V1b(-/-) mice was still coupled to the internal clock, which oscillated normally under standard conditions. Experiments with suprachiasmatic nucleus (SCN) slices in culture suggested that interneuronal communication mediated by V1a and V1b confers on the SCN an intrinsic resistance to external perturbation. Pharmacological blockade of V1a and V1b in the SCN of wild-type mice resulted in accelerated recovery from jet lag, which highlights the potential of vasopressin signaling as a therapeutic target for management of circadian rhythm misalignment, such as jet lag and shift work.

SummaryNocturnal enuresis in children and nocturia in the elderly are two highly prevalent clinical conditions characterized by a mismatch between urine production rate in the kidneys and storage in the urinary bladder during the sleep phase. Here we demonstrate, using a novel method for automated recording of mouse micturition, that connexin43 (Cx43), a bladder gap junction protein, is a negative regulator of functional bladder capacity. Bladder Cx43 levels and functional capacity show circadian oscillations in wild-type mice, but such rhythms are completely lost in Cry-null mice having a dysfunctional biological clock. Bladder muscle cells have an internal clock, and show oscillations of Cx43 and gap junction function. A clock regulator, Rev-erbα, upregulates Cx43 transcription as a co-factor of Sp1 using Sp1 cis-elements of the promoter. Therefore, circadianoscillation of Cx43 is associated with the biological clock and contributes to diurnal changes in bladder capacity, which avoids disturbance of sleep by micturition.

The mammalian clock genes, Period and Cryptochrome (Cry), regulate circadian rhythm. We show that circadian rhythmicity and rhythmic expression of Period in the nuclei of inflammatory synovial cells and spleen cells are disturbed in mouse models of experimental arthritis. Expressions of other clock genes, Bmal1 and Dbp, are also disturbed in spleen cells by arthritis induction. Deletion of Cry1 and Cry2 results in an increase in the number of activated CD3+ CD69+ T cells and a higher production of TNF-α from spleen cells. When arthritis is induced, Cry1−/−Cry2−/− mice develop maximal exacerbation of joint swelling, and upregulation of essential mediators of arthritis, including TNF-α, IL-1β and IL-6, and matrix metalloproteinase-3. Wee-1 kinase is solely upregulated in Cry1−/−Cry2−/− mice, in line with upregulation of c-Fos and Wee-1 kinase in human rheumatoid arthritis. The treatment with anti–TNF-α Ab significantly reduced the severity and halted the progression of the arthritis of Cry1−/−Cry2−/− mice and vice versa, ectopic expression of Cry1 in the mouse embryonic fibroblast from Cry1−/−Cry2−/− mice significantly reduced the trans activation of TNF-α gene. Thus, the biological clock and arthritis influence each other, and this interplay can influence human health and disease.

Synchronous oscillations of thousands of cellular clocks in the suprachiasmatic nucleus (SCN), the circadian centre, are coordinated by precisely timed cell–cell communication, the principle of which is largely unknown. Here we show that the amount of RGS16 (regulator of G protein signalling 16), a protein known to inactivate Gαi, increases at a selective circadian time to allow time-dependent activation of intracellular cyclic AMP signalling in the SCN. Gene ablation of Rgs16 leads to the loss of circadian production of cAMP and as a result lengthens circadian period of behavioural rhythm. The temporally precise regulation of the cAMP signal by clock-controlled RGS16 is needed for the dorsomedial SCN to maintain a normal phase-relationship to the ventrolateral SCN. Thus, RGS16-dependent temporal regulation of intracellular G protein signalling coordinates the intercellular synchrony of SCN pacemaker neurons and thereby defines the 24 h rhythm in behaviour.

PERIOD proteins are central components of the Drosophila and mammalian circadian clock. Their function is controlled by daily changes in synthesis, cellular localization, phosphorylation, degradation, as well as specific interactions with other clock components. Here we present the crystal structure of a Drosophila PERIOD (dPER) fragment comprising two tandemly organized PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B) and two additional C-terminal alpha helices (alphaE and alphaF). Our analysis reveals a noncrystallographic dPER dimer mediated by intermolecular interactions of PAS-A with PAS-B and helix alphaF. We show that alphaF is essential for dPER homodimerization and that the PAS-A-alphaF interaction plays a crucial role in dPER clock function, as it is affected by the 29 hr long-period perL mutation.