Jun Hirayama

The NAD+-Dependent Deacetylase SIRT1 Modulates CLOCK-Mediated Chromatin Remodeling and Circadian Control

Circadian rhythms govern a large array of metabolic and physiological functions. The central clock protein CLOCK has HAT properties. It directs acetylation of histone H3 and of its dimerization partner BMAL1 at Lys537, an event essential for circadian function. We show that the HDAC activity of the NAD(+)-dependent SIRT1 enzyme is regulated in a circadian manner, correlating with rhythmic acetylation of BMAL1 and H3 Lys9/Lys14 at circadian promoters. SIRT1 associates with CLOCK and is recruited to the CLOCK:BMAL1 chromatin complex at circadian promoters. Genetic ablation of the Sirt1 gene or pharmacological inhibition of SIRT1 activity lead to disturbances in the circadian cycle and in the acetylation of H3 and BMAL1. Finally, using liver-specific SIRT1 mutant mice we show that SIRT1 contributes to circadian control in vivo. We propose that SIRT1 functions as an enzymatic rheostat of circadian function, transducing signals originated by cellular metabolites to the circadian clock.

Circadian regulator CLOCK is a histone acetyltransferase.

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.

PER2 controls lipid metabolism by direct regulation of PPARγ

Accumulating evidence highlights intriguing interplays between circadian and metabolic pathways. We show that PER2 directly and specifically represses PPARγ, a nuclear receptor critical in adipogenesis, insulin sensitivity, and inflammatory response. PER2-deficient mice display altered lipid metabolism with drastic reduction of total triacylglycerol and nonesterified fatty acids. PER2 exerts its inhibitory function by blocking PPARγ recruitment to target promoters and thereby transcriptional activation. Whole-genome microarray profiling demonstrates that PER2 dictates the specificity of PPARγ transcriptional activity. Indeed, lack of PER2 results in enhanced adipocyte differentiation of cultured fibroblasts. PER2 targets S112 in PPARγ, a residue whose mutation has been associated with altered lipid metabolism. Lipidomic profiling demonstrates that PER2 is necessary for normal lipid metabolism in white adipocyte tissue. Our findings support a scenario in which PER2 controls the proadipogenic activity of PPARγ by operating as its natural modulator, thereby revealing potential avenues of pharmacological and therapeutic intervention.

Circadian control by the reduction/oxidation pathway: catalase represses light-dependent clock gene expression in the zebrafish.

Light is the key entraining stimulus for the circadian clock, but several features of the signaling pathways that convert the photic signal to clock entrainment remain to be deciphered. Here, we show that light induces the production of hydrogen peroxide (H2O2) that acts as the second messenger coupling photoreception to the zebrafish circadian clock. Treatment of light-responsive Z3 cells with H2O2 triggers the induction of zCry1a and zPer2 genes and the subsequent circadian oscillation of zPer1. Remarkably, the induction kinetics and oscillation profile in response to H2O2 are identical to those initiated by light. Catalase (Cat), an antioxidant enzyme degrading H2O2, shows an oscillating pattern of expression and activity, antiphasic to zCry1a and zPer2. Interestingly, overexpression of zCAT results in a reduced light-dependent zCry1a and zPer2 gene induction. In contrast, inhibition of zCAT function enhances light-mediated inducibility of these clock genes. These findings implicate the enzymatic function of zCAT enzyme in the negative regulation of light-dependent clock gene transcriptional activation. Our findings provide an attractive link between the regulation of the cellular reduction/oxidation (redox) state and the photic signaling pathways implicated in circadian control.

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.

Common light signaling pathways controlling DNA repair and circadian clock entrainment in zebrafish.

UV radiation causes a number of harmful events including growth delay, cell death and ultimately cancer. The reversal of such effects by concomitant exposure to visible light is a conserved mechanism which has been uncovered in many multi-cellular organisms. Here we show that light-dependent UV-tolerance is a cell autonomous phenomenon in zebrafish. In addition, we provide several lines of evidence indicating that light induction of 64PHR, a DNA repair enzyme, and the subsequent light-dependent DNA repair mediated by this enzyme are prerequisites for light-mediated UV tolerance. 64PHR is evolutionary related to and has a high degree of structural similarity to animal CRY, an essential circadian regulator. The zebrafish circadian clock is controlled by a cell-autonomous and light-dependent oscillator, where zCRY1a functions as an important mediator of light entrainment of the circadian clock. In this study, we show that light directly activates MAPK signaling cascades in zebrafish cells and we provide evidence that light-induced activation of these pathways controls the expression of two evolutionary-related genes, z64Phr and zCry1a, revealing that light-dependent DNA repair and the entrainment of circadian clock share common regulatory pathways.

Linking Oxygen to Time: The Bidirectional Interaction Between the Hypoxic Signaling Pathway and the Circadian Clock

The circadian clock and the hypoxic signaling pathway play critical roles in physiological homeostasis as well as in tumorgenesis. Interactions between both pathways have repeatedly been reported for mammals during the last decade, the molecular basis, though, has not been identified so far. Expression levels of oxygen-regulated and circadian clock genes in zebrafish larvae (Danio rerio) and zebrafish cell lines were significantly altered under hypoxic conditions. Thus, long-term hypoxic incubation of larvae resulted in a dampening of the diurnal oscillation amplitude of the period1 gene expression starting only several hours after start of the hypoxic incubation. A significant decrease in the amplitude of the period1 circadian oscillation in response to hypoxia and in response to the hypoxic mimic CoCl2 was also observed using a zebrafish luciferase reporter cell line in constant darkness. In addition, activity measurements of zebrafish larvae using an infrared-sensitive camera demonstrated the loss of their usual circadian activity pattern under hypoxic conditions. To explore the functional basis of the observed cross-talk between both signaling pathways ChIP assays were performed. Increasing with the duration of hypoxia, a nearly 4-fold occupancy of hypoxia-inducible factor 1 (Hif-1α) at two specific E-box binding sites located in the period1 gene control region was shown, demonstrating therewith the transcriptional co-regulation of the core clock gene by the major transcription factor of the hypoxic pathway. On the other hand, circadian transgenic zebrafish cells, simulating a repressed or an overstimulated circadian clock, modified gene transcription levels of oxygen-regulated genes such as erythropoietin and vascular endothelial growth factor 165 and altered the hypoxia-induced increase in Hif-1α protein concentration. In addition, the amount of Hif-1α protein accumulated during the hypoxic response was shown to depend on the time of the day, with one maximum during the light phase and a second one during the dark phase. The direct binding of Hif-1α to the period1 gene control region provides a mechanistic explanation for the repeatedly observed interaction between hypoxia and the circadian clock. The cross-talk between both major signaling pathways was shown for the first time to be bidirectional and may provide the advantage of orchestrating a broad range of genes and metabolic pathways to cope with altered oxygen availabilities. (Author correspondence: margit.egg@uibk.ac.at)

p38 Mitogen-Activated Protein Kinase Controls a Switch Between Cardiomyocyte and Neuronal Commitment of Murine Embryonic Stem Cells by Activating Myocyte Enhancer Factor 2C-Dependent Bone Morphogenetic Protein 2 Transcription

Many studies have shown that it is possible to use culture conditions to direct the differentiation of murine embryonic stem (ES) cells into a variety of cell types, including cardiomyocytes and neurons. However, the molecular mechanisms that control lineage commitment decisions by ES cells remain poorly understood. In this study, we investigated the role of the 3 major mitogen-activated protein kinases (MAPKs: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) in ES cell lineage commitment and showed that the p38 MAPK-specific inhibitor SB203580 blocks the spontaneous differentiation of ES cells into cardiomyocytes and instead induces the differentiation of these ES cells into neurons. Robust p38 MAPK activity between embryoid body culture days 3 and 4 is crucial for cardiomyogenesis of ES cells, and specific inhibition of p38 MAPK activity at this time results in ES cell differentiation into neurons rather than cardiomyocytes. At the molecular level, inhibition of p38 MAPK activity suppresses the expression of bmp-2 mRNA, whereas treatment of ES cells with bone morphogenetic protein 2 (BMP-2) inhibits the neurogenesis induced by SB203580. Further, luciferase reporter assays and chromatin immunoprecipitation experiments showed that BMP-2 expression in ES cells is regulated directly by the transcription factor myocyte enhancer factor 2C, a well-known substrate of p38 MAPK. Our findings reveal the molecular mechanism by which p38 MAPK activity in ES cells drives their commitment to differentiate preferentially into cardiomyocytes, and the conditions under which these same cells might develop into neurons.

The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAPs functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAPs co-activation of TEAD-mediated CTGF transcription.

A Novel Acetylation Cycle of Transcription Co-activator Yes-associated Protein That Is Downstream of Hippo Pathway Is Triggered in Response to SN2 Alkylating Agents

Background: YAP is a target molecule of the Hippo pathway. Results: YAP acetylation and deacetylation were mediated by CBP/p300 acetyltransferase and SIRT1 deacetylase, respectively. Conclusion: A YAP acetylation/deacetylation cycle is located downstream of the Hippo pathway. Significance: The discovery that YAP undergoes an acetylation cycle advances our understanding of YAP functions. Yes-associated protein (YAP) is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes. Although cytoplasmic retention of YAP is known to be mediated by Hippo pathway-dependent phosphorylation, post-translational modifications that regulate YAP in the nucleus remain unclear. Here we report the discovery of a novel cycle of acetylation/deacetylation of nuclear YAP induced in response to SN2 alkylating agents. We show that after treatment of cells with the SN2 alkylating agent methyl methanesulfonate, YAP phosphorylation mediated by the Hippo pathway is markedly reduced, leading to nuclear translocation of YAP and its acetylation. This YAP acetylation occurs on specific and highly conserved C-terminal lysine residues and is mediated by the nuclear acetyltransferases CBP (CREB binding protein) and p300. Conversely, the nuclear deacetylase SIRT1 is responsible for YAP deacetylation. Intriguingly, we found that YAP acetylation is induced specifically by SN2 alkylating agents and not by other DNA-damaging stimuli. These results identify a novel YAP acetylation cycle that occurs in the nucleus downstream of the Hippo pathway. Intriguingly, our findings also indicate that YAP acetylation is involved in responses to a specific type of DNA damage.