Teruya Tamaru

CLOCK-mediated acetylation of BMAL1 controls circadian function

Regulation of circadian physiology relies on the interplay of interconnected transcriptional–translational feedback loops. The CLOCK–BMAL1 complex activates clock-controlled genes, including cryptochromes (Crys), the products of which act as repressors by interacting directly with CLOCK–BMAL1. We have demonstrated that CLOCK possesses intrinsic histone acetyltransferase activity and that this enzymatic function contributes to chromatin-remodelling events implicated in circadian control of gene expression. Here we show that CLOCK also acetylates a non-histone substrate: its own partner, BMAL1, is specifically acetylated on a unique, highly conserved Lys 537 residue. BMAL1 undergoes rhythmic acetylation in mouse liver, with a timing that parallels the downregulation of circadian transcription of clock-controlled genes. BMAL1 acetylation facilitates recruitment of CRY1 to CLOCK–BMAL1, thereby promoting transcriptional repression. Importantly, ectopic expression of a K537R-mutated BMAL1 is not able to rescue circadian rhythmicity in a cellular model of peripheral clock. These findings reveal that the enzymatic interplay between two clock core components is crucial for the circadian machinery.

CK2|[alpha]| phosphorylates BMAL1 to regulate the mammalian clock

Clock proteins govern circadian physiology and their function is regulated by various mechanisms. Here we demonstrate that Casein kinase (CK)-2α phosphorylates the core circadian regulator BMAL1. Gene silencing of CK2α or mutation of the highly conserved CK2-phosphorylation site in BMAL1, Ser90, result in impaired nuclear BMAL1 accumulation and disruption of clock function. Notably, phosphorylation at Ser90 follows a rhythmic pattern. These findings reveal that CK2 is an essential regulator of the mammalian circadian system.

Nucleocytoplasmic shuttling and phosphorylation of BMAL1 are regulated by circadian clock in cultured fibroblasts

Background:  Recent discoveries of clock proteins have unveiled an important part of the mammalian circadian clock mechanism. However, the molecular clockwork that cause these fundamental feedback loops to stably oscillate with a ∼24 h‐periodicity remain unclear.

Synchronization of Circadian Per2 Rhythms and HSF1- BMAL1:CLOCK Interaction in Mouse Fibroblasts after Short-Term Heat Shock Pulse

Circadian rhythms are the general physiological processes of adaptation to daily environmental changes, such as the temperature cycle. A change in temperature is a resetting cue for mammalian circadian oscillators, which are possibly regulated by the heat shock (HS) pathway. The HS response (HSR) is a universal process that provides protection against stressful conditions, which promote protein-denaturation. Heat shock factor 1 (HSF1) is essential for HSR. In the study presented here, we investigated whether a short-term HS pulse can reset circadian rhythms. Circadian Per2 rhythm and HSF1-mediated gene expression were monitored by a real-time bioluminescence assay for mPer2 promoter-driven luciferase and HS element (HSE; HSF1-binding site)-driven luciferase activity, respectively. By an optimal duration HS pulse (43°C for approximately 30 minutes), circadian Per2 rhythm was observed in the whole mouse fibroblast culture, probably indicating the synchronization of the phases of each cell. This rhythm was preceded by an acute elevation in mPer2 and HSF1-mediated gene expression. Mutations in the two predicted HSE sites adjacent (one of them proximally) to the E-box in the mPer2 promoter dramatically abolished circadian mPer2 rhythm. Circadian Per2 gene/protein expression was not observed in HSF1-deficient cells. These findings demonstrate that HSF1 is essential to the synchronization of circadian rhythms by the HS pulse. Importantly, the interaction between HSF1 and BMAL1:CLOCK heterodimer, a central circadian transcription factor, was observed after the HS pulse. These findings reveal that even a short-term HS pulse can reset circadian rhythms and cause the HSF1-BMAL1:CLOCK interaction, suggesting the pivotal role of crosstalk between the mammalian circadian and HSR systems.

ROS stress resets circadian clocks to coordinate pro-survival signals.

Dysfunction of circadian clocks exacerbates various diseases, in part likely due to impaired stress resistance. It is unclear how circadian clock system responds toward critical stresses, to evoke life-protective adaptation. We identified a reactive oxygen species (ROS), H2O2 -responsive circadian pathway in mammals. Near-lethal doses of ROS-induced critical oxidative stress (cOS) at the branch point of life and death resets circadian clocks, synergistically evoking protective responses for cell survival. The cOS-triggered clock resetting and pro-survival responses are mediated by transcription factor, central clock-regulatory BMAL1 and heat shock stress-responsive (HSR) HSF1. Casein kinase II (CK2) –mediated phosphorylation regulates dimerization and function of BMAL1 and HSF1 to control the cOS-evoked responses. The core cOS-responsive transcriptome includes CK2-regulated crosstalk between the circadian, HSR, NF-kappa-B-mediated anti-apoptotic, and Nrf2-mediated anti-oxidant pathways. This novel circadian-adaptive signaling system likely plays fundamental protective roles in various ROS-inducible disorders, diseases, and death.

CRY Drives Cyclic CK2-Mediated BMAL1 Phosphorylation to Control the Mammalian Circadian Clock.

Intracellular circadian clocks, composed of clock genes that act in transcription-translation feedback loops, drive global rhythmic expression of the mammalian transcriptome and allow an organism to anticipate to the momentum of the day. Using a novel clock-perturbing peptide, we established a pivotal role for casein kinase (CK)-2-mediated circadian BMAL1-Ser90 phosphorylation (BMAL1-P) in regulating central and peripheral core clocks. Subsequent analysis of the underlying mechanism showed a novel role of CRY as a repressor for protein kinase. Co-immunoprecipitation experiments and real-time monitoring of protein–protein interactions revealed that CRY-mediated periodic binding of CK2β to BMAL1 inhibits BMAL1-Ser90 phosphorylation by CK2α. The FAD binding domain of CRY1, two C-terminal BMAL1 domains, and particularly BMAL1-Lys537 acetylation/deacetylation by CLOCK/SIRT1, were shown to be critical for CRY-mediated BMAL1–CK2β binding. Reciprocally, BMAL1-Ser90 phosphorylation is prerequisite for BMAL1-Lys537 acetylation. We propose a dual negative-feedback model in which a CRY-dependent CK2-driven posttranslational BMAL1–P-BMAL1 loop is an integral part of the core clock oscillator.

Periodically fluctuating protein kinases phosphorylate CLOCK, the putative target in the suprachiasmatic nucleus.

Abstract: We studied nuclear protein phosphorylation in the rat suprachiasmatic nucleus (SCN) and found that a nuclear fraction of the SCN contained histone H1 kinase activity that periodically fluctuated with a diurnal rhythm, reaching a maximum at the midpoint of the light phase and a minimum at the midpoint of the dark phase. A p13suc1‐bound fraction from the SCN nuclear fraction also exhibited diurnally fluctuating histone H1 kinase activity. Using in situ kinase assay, three histone H1 kinases, p45PFK, p100PFK, and p200PFK (termed periodically fluctuating protein kinases, or PFKs) were found in the p13suo1‐bound fractions. p45PFK exhibited the highest level of light/dark cycle phosphorylation activity fluctuation. p45PFK highly phosphorylated the Ser‐Pro‐rich region of CLOCK, the putative physiological target. These results suggest that PFKs, especially p45PFK, are involved in circadian clock‐related signal transduction and gene expression, through the phosphorylation of target proteins such as CLOCK.

Immunochemical assessment of neural visinin-like calcium-binding protein 3 expression in rat brain

Expression of neural visinin-like calcium-binding protein 3 (NVP3) was assessed by immunoblot and immunohistochemical analyses in rat brain. NVP3 was markedly expressed in the cerebellum, at a concentration of 9.5microM. On SDS-PAGE, native NVP3 migrated at 23kDa, identical to the recombinant myristoyl-form, but somewhat faster than the non-myristoyl-form. Both forms bound 3 moles of calcium. The myristoyl-form exhibited a cooperativity in binding calcium and calcium-dependent membrane-binding, but the non-myristoyl-form did not. At 3 months, NVP3 was primarily localized in the Purkinje cells, with intense staining in the cell bodies, dendrites and axons. The cerebellar granule cells and basal nuclear neurons were faintly stained. During development of the cerebellum, NVP3-positive Purkinje cells first appeared on post-natal day 14 (P14). The staining intensity then increased and plateaued on P28. Labeling showed a tendency to accumulate in the dendrites and nerve terminals in a fine granular pattern. During aging process, NVP3 levels decreased by 43% at 12 months and 68% at 24 months, while the levels of NVP1, synaptophysin and drebrin were preferentially preserved. These results suggest that NVP3 is involved in dendritic arborization and postsynaptic function in cerebellar Purkinje cells and that presynaptic nerve terminals are another functional site of the protein.

Circadian expression of hnRNP U, a nuclear multi-potent regulatory protein, in the murine suprachiasmatic nucleus

Heterogeneous ribonuclear protein U (hnRNP U/SAF-A) is a nuclear multi-potent regulatory protein. We investigated whether hnRNP U protein and transcript levels undergo circadian changes by immunoblot and quantitative RT-PCR analyses. In the suprachiasmatic nucleus (SCN), hnRNP U immunoreactivity (ir) changed in a robust circadian pattern as it showed a peak at late nighttime in both light/dark and constant dark conditions. hnRNP U transcript levels in the SCN changed in a similar circadian pattern. In the hippocampus, hnRNP transcript levels also showed a peak at late nighttime but hnRNP U-ir showed an opposite pattern as it peaked at late daytime. These findings suggest that hnRNP U participates in nuclear regulatory events that are involved in mammalian central and peripheral circadian clocks.

Circadian adaptation to cell injury stresses: a crucial interplay of BMAL1 and HSF1.

The circadian clock system confers daily anticipatory physiological processes with the ability to be reset by environmental cues. This “circadian adaptation system” (CAS), driven by cell-autonomous molecular clocks, orchestrates various rhythmic physiological processes in the entire body. Hence, the dysfunction of these clocks exacerbates various diseases, which may partially be due to the impairment of protective pathways. If this is the case, how does the CAS respond to cell injury stresses that are critical in maintaining health and life by evoking protective pathways? To address this question, here we review and discuss recent evidence revealing life-protective (pro-survival) molecular networks between clock (e.g., BMAL1, CLOCK, and PER2) and adaptation (e.g., HSF1, Nrf2, NF-κB, and p53) pathways, which are evoked by various cell injury stresses (e.g., heat, reactive oxygen species, and UV). The CK2 protein kinase-integrated interplay of the BMAL1 (clock) and HSF1 (heat-shock response) pathways is one of the crucial events in CAS.