Clock Regulation Research Articles

The circadian clock and extracellular matrix homeostasis in aging and age-related diseases.

The extracellular matrix (ECM) is the noncellular scaffolding component present within all tissues and organs. It provides crucial biochemical and biomechanical cues to instruct cellular behavior and has been shown to be under circadian clock regulation, a highly conserved cell-intrinsic timekeeping mechanism that has evolved with the 24-hour rhythmic environment. Aging is a major risk factor for many diseases, including cancer, fibrosis, and neurodegenerative disorders. Both aging and our modern 24/7 society disrupt circadian rhythms, which could contribute to altered ECM homeostasis. Understanding the daily dynamics of ECM and how this mechanism changes with age will have a profound impact on tissue health, disease prevention, and improving treatments. Maintaining rhythmic oscillations has been proposed as a hallmark of health. On the other hand, many hallmarks of aging turn out to be key regulators of circadian timekeeping mechanisms. In this review, we summarize new work linking the ECM with circadian clocks and tissue aging. We discuss how the changes in the biomechanical and biochemical properties of ECM during aging may contribute to circadian clock dysregulation. We also consider how the dampening of clocks with age could compromise the daily dynamic regulation of ECM homeostasis in matrix-rich tissues. This review aims to encourage new concepts and testable hypotheses about the two-way interactions between circadian clocks and ECM in the context of aging.

1773-P: Glucocorticoid Receptor Signaling Regulates Pancreatic Beta-Cell Circadian Transcriptome and Function

Glucocorticoid (GC) signaling is an important regulator of glucose homeostasis. GC secretion also serves as an entrainment signal that coordinates circadian gene expression of peripheral circadian clocks. Pancreatic beta cells are key biological controllers of glucose homeostasis through the coordinated circadian release of insulin. However, the role of GC signaling in the regulation of beta cell circadian clocks and insulin secretion remains unknown. To address this, we first performed chromatin immunoprecipitation/sequencing (ChIP-seq) in beta cell lines (MIN6 and INS1) and identified >1,000 glucocorticoid receptor (GR)-bound regions (FDR<0.01) in response to the synthetic GC dexamethasone (DEX, 100nM). Annotation of GR-bound regions showed enrichment for pathways regulating insulin secretion and circadian rhythms. To further explore the effects of GC on circadian beta cell gene expression and function in vivo, we used C57BL/6 mice housed under either 12h/12h light-dark cycles (LD) or constant light-mediated circadian disruption (CD). Mice were subsequently administered daily intraperitoneal injections of DEX (2 mg/kg) or vehicle, designed to restore circadian secretion of GC disrupted in CD group. DEX treatment normalized circadian regulation of glucose tolerance (p<0.05 vs. vehicle) and glucose-stimulated insulin secretion (p<0.05 vs vehicle) in CD group. We next utilized RNA sequencing to assess circadian transcriptional profiles in islets isolated from LD, CD, and CD + DEX mice. Consistent with previous studies, CD resulted in abrogation of circadian islet mRNA expression, whereas DEX restored circadian rhythmicity and enhanced expression of core clock genes (e.g., Bmal1, Dbp, Nr1d1) and genes regulating beta cell identity (e.g., Ins1, Nkx6.1) and insulin secretion (e.g., Gck, Irs2). These data highlight the GC/GR axis as a direct regulator of beta cell circadian gene expression, insulin secretion and glucose tolerance. Disclosure S.K.Sen: None. L.Wang: None. M.Brown: None. J.Wu: None. Z.Wei: None. A.Matveyenko: None. Funding National Institutes of Health (DK098468)

The Clock-modulatory Activity of Nobiletin Suppresses Adipogenesis Via Wnt Signaling.

The circadian clock machinery exerts transcriptional control to modulate adipogenesis and its disruption leads to the development of obesity. Here, we report that Nobiletin, a circadian clock amplitude-enhancing molecule, displays antiadipogenic properties via activation of Wnt signaling pathway that is dependent on its clock modulation. Nobiletin augmented clock oscillatory amplitude with period lengthening in the adipogenic mesenchymal precursor cells and preadipocytes, accompanied by an induction of Bmal1 and clock components within the negative feedback arm. Consistent with its clock-modulatory activity, Nobiletin strongly inhibited the lineage commitment and terminal differentiation of adipogenic progenitors. Mechanistically, we show that Nobiletin induced the reactivation of Wnt signaling during adipogenesis via transcriptional up-regulation of key components within this pathway. Furthermore, Nobiletin administration in mice markedly reduced adipocyte hypertrophy, leading to a significant loss of fat mass and reduction of body weight. Last, Nobiletin inhibited the differentiation of primary preadipocytes, and this effect was dependent on a functional clock regulation. Collectively, our findings uncover a novel activity of Nobiletin in suppressing adipocyte development in a clock-dependent manner, implicating its potential application in countering obesity and associated metabolic consequences.

B-Box proteins BBX28 and BBX29 interplay with PSEUDO-RESPONSE REGULATORS to fine-tune circadian clock in Arabidopsis.

As an endogenous time-keeping mechanism, the circadian clock benefits plant fitness and adaptation to the rhythmically changed diel environments. The key components within the core oscillator of plant circadian clock have been extensively characterised, however, the fine-tuning circadian regulators are still less identified. Here, we demonstrated that BBX28 and BBX29, the two B-Box V subfamily members lacking DNA-binding motifs, are involved in the regulation of Arabidopsis circadian clock. Over-expressing either BBX28 or BBX29 significantly lengthened circadian period, whereas loss-of-function of BBX28 rather than BBX29 displayed a modestly long period in free-running condition. Mechanistically, BBX28 and BBX29 interacted with core clock components PRR5, PRR7 and PRR9 in nucleus to augment their transcriptional repressive activities. RNA-sequencing analysis further revealed that BBX28 and BBX29 shared 686 common differentially expressed genes (DEGs) including a subset of known direct transcriptional targets of PRR proteins within core oscillator, including CCA1, LHY, LNKs and RVE8 etc. Intriguingly, PRR proteins can feedback repress BBX28 and BBX29 transcription by associating with their promoters. Together, our findings unmasked an exquisite mechanism in which BBX28 and BBX29 interplay with PRR proteins to fine-tune the circadian pace.

Comprehensive profiling of clock genes expression in hepatocellular carcinoma (HCC).

4108 Background: The circadian clock mechanism controls the physiological homeostasis of the liver and plays a key role in hepatocarcinogenesis. Recent evidence unveiled core clock proteins as novel therapeutic targets in cancer. Our group showed that clock regulators BMAL1 and CLOCK can promote proliferation of liver cancer cells by modulating the cell cycle checkpoint kinase Wee1. Here we further evaluated the molecular landscape of clock pathway alterations in HCC leveraging multi-platform profiling of patient tumor samples. Methods: 780 HCC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) were analyzed. Clock gene Score (CS) was determined using expression of core clock genes Z scores (positives of CLOCK, ARNTL, RORA/B/C and negatives of repressors CRY1/2, PER1/2/3, REVERBA/B) stratified by quartiles. xCell was used to quantify cell infiltration in the tumor microenvironment (TME). Significance was determined as P-values and adjusted for multiple testing ( q) of < .05. Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy including the T cell inflamed score (TIS) and IFG score. Real world survival was obtained from insurance claims data and Kaplan-Meier estimates were calculated for comparison. Results: CS was higher in metastatic sites than primary tumors (median transcripts per million [TPM]: 0.81 vs 0.37, P < .05). No significant differences in patient age and sex were observed between CS Q1 (lowest) and Q4 (highest) cohorts, although a trend towards a higher frequency of males was observed in Q4 (76% vs 68%, Q4 vs Q1, P = .07). CS was positively associated with telomerase subunit TERT mutations (64% vs 52%, Q4 vs Q1, P = .04) and negatively correlated with FGF3 copy number amplification (2% vs 6%, P = .04) and WEE1 gene expression (median TPM: 15 vs 28, q < .05). No dMMR/MSI-H tumors were observed in our series and there were no significant associations with tumor mutational burden and PD-L1 protein expression. Expression of immune related genes was lower in tumors with high CS, including IDO1, CD80, PD-L1, LAG3, CD86, TIM3, PD-1 and PD-L2 (fold change: 0.57-0.67 q < .05). NK cell infiltration in the TME and the TIS score were also significantly lower in CS-high HCC ( q < .05). Individually, lower CLOCK and CRY1 tumor mRNA expression were associated with longer OS (Q1 vs Q4: CLOCK HR 0.71, 95%CI [0.51-0.98], P = .04 and CRY1 HR 0.70 [0.51-0.95], P = .02, respectively). Conclusions: This is the most extensive profiling study to investigate the expression of clock genes in HCC. Our data show that clock genes expression impacts patient survival and is associated with alterations in immune-related gene expression and TIS score which suggest a role in the modulation of anti-tumor immunity. These results support the clock pathway role as a oncogenic driver and its potential as a therapeutic target in HCC.

Circadian clock regulation via biomaterials for nucleus pulposus.

Circadian clock disorder during tissue degeneration has been considered the potential pathogenesis for various chronic diseases, such as intervertebral disc degeneration (IVDD). In this study, circadian clock-regulating biomaterials (ClockMPs) that could effectively activate the intrinsic circadian clock of nucleus pulposus cells (NPCs) in IVDD and improve the physiological function of NPCs for disc regeneration were fabricated via air-microfluidic technique and the chemical cross-linking between polyvinyl alcohol and modified-phenylboronic acid. In vitro experiments verified that ClockMPs could scavenge reactive oxygen species to maintain a stable microenvironment for the circadian clock by promoting the binding of BMAL1 and CLOCK proteins. ClockMPs could regulate the expression of core circadian clock genes by activating the PI3K-AKT pathway in NPCs to remodel the intrinsic circadian clock and promote extracellular matrix synthesis. Furthermore, in vivo experiments of IVDD treated with ClockMPs proved that ClockMPs could promote disc regeneration by regulating the circadian clock of NPCs. In conclusion, ClockMPs provided a novel and promising strategy for circadian clock regulation during tissue regeneration. This article is protected by copyright. All rights reserved.

From the archives: Circadian clock regulation of phyA, regulation of Z-ring positioning in chloroplast division, and ADP-glucose pyrophosphorylase subunit interactions.

From the archives: Circadian clock regulation of phyA, regulation of Z-ring positioning in chloroplast division, and ADP-glucose pyrophosphorylase subunit interactions.

Molecular clocks, satellite cells, and skeletal muscle regeneration.

Skeletal muscle comprises approximately 50% of individual body mass and plays vital roles in locomotion, heat production, and whole body metabolic homeostasis. This tissue exhibits a robust diurnal rhythm that is under control of the suprachiasmatic nucleus (SCN) region of the hypothalamus. The SCN acts as a "central" coordinator of circadian rhythms, while cell-autonomous "peripheral" clocks are located within almost all other tissues/organs in the body. Synchronization of the peripheral clocks in muscles (and other tissues) together with the central clock is crucial to ensure temporally coordinated physiology across all organ systems. By virtue of its mass, human skeletal muscle contains the largest collection of peripheral clocks, but within muscle resides a local stem cell population, satellite cells (SCs), which have their own functional molecular clock, independent of the numerous muscle clocks. Skeletal muscle has a daily turnover rate of 1%-2%, so the regenerative capacity of this tissue is important for whole body homeostasis/repair and depends on successful SC myogenic progression (i.e., proliferation, differentiation, and fusion). Emerging evidence suggests that SC-mediated muscle regeneration may, in part, be regulated by molecular clocks involved in SC-specific diurnal transcription. Here we provide insights on molecular clock regulation of muscle regeneration/repair and provide a novel perspective on the interplay between SC-specific molecular clocks, myogenic programs, and cell cycle kinetics that underpin myogenic progression.

PO-01-244 BMAL1 REGULATES THE CIRCADIAN RHYTHM OF RYR2 AND CONTRIBUTES TO CIRCADIAN VENTRICULAR ARRHYTHMIA IN CHRONIC HEART FAILURE AFTER MYOCARDIAL INFARCTION

Ventricular arrhythmia (VAs) is the major cause of sudden cardiac death in chronic heart failure (CHF) after myocardial infarction (MI), and previous studies have reported that VAs exist circadian oscillation in CHF. Peripheral circadian clock can regulate expression and function of ion channels in cardiomyocytes, which is associated with circadian rhythm of VAs in CHF. However, the regulation of peripheral circadian clock on Ryanodine Receptor 2 (RYR2), a calcium channel on cardiac sarcoplasmic reticulum, remain to be fully understood. We aimed to explore the regulation of peripheral circadian clock on RYR2 and VA events in CHF. CHF was induced by ligation of coronary artery of mice, and RT-PCR were performed to investigate the circadian rhythm of RYR2 in CHF myocardial tissue. Amplitude of calcium transient of ISO-induced cardiomyocytes at different time was measured to study the circadian rhythm of RYR2 function. To confirm the circadian oscillation of VAs, VAs incidences at different time of CHF wild type mice and cardiomyocyte-specific Bmal1 knockout (CKO) mice were investigated by programmed electrical stimulation. Infections of Ad-CLOCK and/or Ad-BMAL1 were applied to overexpress CLOCK, BMAL1 and CLOCK-BMAL1 in cardiomyocytes, and expression of RYR2 was measured by Western blot. Chromatin immunoprecipitation assay (ChIP) and luciferase (LUC) were applied to determine whether CLOCK-BMAL1 transcriptionally regulate RYR2 expression. CLOCK, BMAL1, and RYR2 exhibited notable circadian rhythm in CHF mice (Figure A, P<0.05). Amplitude of calcium transient at CT36 was lower than CT24 (Figure B, P<0.05). Compared to CT36, VAs had higher incidence at CT24(Figure C, P<0.05), and incidences of VAs had no significant difference between CT24 and CT36 in CKO Mice or using Dantrolene, an antagonist of RYR2. Ad-CLOCK and Ad-BMAL1 co-infection resulted in overexpression of RYR2 (Figure D, P<0.05), and BMAL1 bound to the enhancer of RYR2 gene and upregulated RYR2 expression transcriptionally (Figure E). In conclusion, BMAL1, as peripheral circadian clock, could contribute to circadian oscillation of VAs in CHF by regulating the circadian rhythm of RYR2.

Adipose tissue macrophages as potential targets for obesity and metabolic diseases.

Macrophage infiltration into adipose tissue is a key pathological factor inducing adipose tissue dysfunction and contributing to obesity-induced inflammation and metabolic disorders. In this review, we aim to present the most recent research on macrophage heterogeneity in adipose tissue, with a focus on the molecular targets applied to macrophages as potential therapeutics for metabolic diseases. We begin by discussing the recruitment of macrophages and their roles in adipose tissue. While resident adipose tissue macrophages display an anti-inflammatory phenotype and promote the development of metabolically favorable beige adipose tissue, an increase in pro-inflammatory macrophages in adipose tissue has negative effects on adipose tissue function, including inhibition of adipogenesis, promotion of inflammation, insulin resistance, and fibrosis. Then, we presented the identities of the newly discovered adipose tissue macrophage subtypes (e.g. metabolically activated macrophages, CD9+ macrophages, lipid-associated macrophages, DARC+ macrophages, and MFehi macrophages), the majority of which are located in crown-like structures within adipose tissue during obesity. Finally, we discussed macrophage-targeting strategies to ameliorate obesity-related inflammation and metabolic abnormalities, with a focus on transcriptional factors such as PPARγ, KLF4, NFATc3, and HoxA5, which promote macrophage anti-inflammatory M2 polarization, as well as TLR4/NF-κB-mediated inflammatory pathways that activate pro-inflammatory M1 macrophages. In addition, a number of intracellular metabolic pathways closely associated with glucose metabolism, oxidative stress, nutrient sensing, and circadian clock regulation were examined. Understanding the complexities of macrophage plasticity and functionality may open up new avenues for the development of macrophage-based treatments for obesity and other metabolic diseases.

Multiomics reveals multilevel control of renal and systemic metabolism by the renal tubular circadian clock.

Circadian rhythmicity in renal function suggests rhythmic adaptations in renal metabolism. To decipher the role of the circadian clock in renal metabolism, we studied diurnal changes in renal metabolic pathways using integrated transcriptomic, proteomic, and metabolomic analysis performed on control mice and mice with an inducible deletion of the circadian clock regulator Bmal1 in the renal tubule (cKOt). With this unique resource, we demonstrated that approximately 30% of RNAs, approximately 20% of proteins, and approximately 20% of metabolites are rhythmic in the kidneys of control mice. Several key metabolic pathways, including NAD+ biosynthesis, fatty acid transport, carnitine shuttle, and β-oxidation, displayed impairments in kidneys of cKOt mice, resulting in perturbed mitochondrial activity. Carnitine reabsorption from primary urine was one of the most affected processes with an approximately 50% reduction in plasma carnitine levels and a parallel systemic decrease in tissue carnitine content. This suggests that the circadian clock in the renal tubule controls both kidney and systemic physiology.

Influence of Fasting until Noon (Extended Postabsorptive State) on Clock Gene mRNA Expression and Regulation of Body Weight and Glucose Metabolism.

The trend of fasting until noon (omission or delayed breakfast) is increasingly prevalent in modern society. This eating pattern triggers discordance between endogenous circadian clock rhythms and the feeding/fasting cycle and is associated with an increased incidence of obesity and T2D. Although the underlying mechanism of this association is not well understood, growing evidence suggests that fasting until noon, also known as an "extended postabsorptive state", has the potential to cause a deleterious effect on clock gene expression and to disrupt regulation of body weight, postprandial and overall glycemia, skeletal muscle protein synthesis, and appetite, and may also lead to lower energy expenditure. This manuscript overviews the clock gene-controlled glucose metabolism during the active and resting phases and the consequences of postponing until noon the transition from postabsorptive to fed state on glucose metabolism, weight control, and energy expenditure. Finally, we will discuss the metabolic advantages of shifting more energy, carbohydrates (CH), and proteins to the early hours of the day.

Tendon circadian clock and diurnal regulation of collagen homeostasis as novel aetiological and therapeutic binoculars in chronic tendinopathy.

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Metabolic regulation of the circadian clock in classically and alternatively activated macrophages.

Macrophages exhibit a range of functional pro- and anti-inflammatory states that induce changes in their cellular metabolism. We aimed to elucidate whether these changes affect the molecular properties of their circadian clock focusing on their anti-inflammatory phenotype. Primary cell cultures of bone marrow-derived macrophages (BMDMs; nonpolarized M0 BMDM) from PER2::LUC (fusion protein of PERIOD2 and LUCIFERASE) mice were polarized into the M1 (proinflammatory) or M2 (anti-inflammatory) phenotype, and PER2-driven bioluminescence was recorded in real-time at the cell-population and single-cell levels. Viability, clock gene expression profiles, polarization plasticity and peroxisome proliferator-activated receptor γ (PPARγ) protein levels were analyzed. The effects of pharmacological activation/inhibition of PPARγ (rosiglitazone/GW9662) and inhibition of fatty acid oxidation (FAO) by etomoxir in M2 BMDM cell cultures were examined. The parameters of PER2-driven bioluminescence rhythms differed between M0, M1 and M2 BMDM cultures at cell-population and single-cell levels. Compared with M0, polarization to M2 did not change the period but increased amplitude, mean bioluminescence level and rhythm persistence. Polarization to M1 shortened the period but had no effect on the amplitude of the rhythm. The same period changes were observed after a bidirectional switch between M1- and M2-polarized states in the same culture. Both PPARγ activation/inhibition and FAO inhibition modulated the clock in M2 BMDMs, suggesting metabolic regulation of the M2 clock. Our results indicate that bidirectional changes in the properties of BMDM circadian clocks in response to their actual polarization are mediated via changes in their metabolic state. They provide new information on the interrelationship between the BMDM polarization, their circadian clock and cellular metabolism.

Identification and characterization of the CDK1-BMAL1-UHRF1 pathway driving tumor progression

The abnormal regulation of BMAL1 could lead to the occurrence and progression of various tumors. However, the mechanism of phosphorylation regulation of BMAL1 in tumorigenesis remains poorly understood. In this study, we report a previously unrecognized BMAL1 dephosphorylation pathway that promotes tumor progression. BMAL1 accelerates cell proliferation, migration, and invasion of HT1080 and Calu1 cells. CDK1 binds to BMAL1 through a conserved domain and regulates the dephosphorylation of BMAL1 on Ser42 residues, but not on Ser78 or Thr224, thereby enhancing the oncogenic activity of BMAL1. Dephosphorylation of BMAL1 Ser42 promotes tumor growth and metastasis in mouse subcutaneous transplantation tumor and lung metastatic tumor models. Moreover, UHRF1 is recognized as an important target gene of BMAL1 in cancer cells. Consequently, UHRF1 depletion mimics BMAL1 deficiency with respect to tumor suppression, whereas transfection-enforced re-expression of UHRF1 restores tumor growth in BMAL1-deficient cells. These findings suggest a link between the circadian clock regulator and cancer progression.

XAP5 CIRCADIAN TIMEKEEPER regulates RNA splicing and the circadian clock by genetically separable pathways.

The circadian oscillator allows organisms to synchronize their cellular and physiological activities with diurnal environmental changes. In plants, the circadian clock is primarily composed of multiple transcriptional-translational feedback loops. Regulators of post-transcriptional events, such as precursor messenger RNAs (pre-mRNA) splicing factors, are also involved in controlling the pace of the clock. However, in most cases the underlying mechanisms remain unclear. We have previously identified XAP5 CIRCADIAN TIMEKEEPER (XCT) as an Arabidopsis thaliana circadian clock regulator with uncharacterized molecular functions. Here, we report that XCT physically interacts with components of the spliceosome, including members of the Nineteen Complex (NTC). PacBio Iso-Seq data show that xct mutants have transcriptome-wide pre-mRNA splicing defects, predominantly aberrant 3' splice site selection. Expression of a genomic copy of XCT fully rescues those splicing defects, demonstrating that functional XCT is important for splicing. Dawn-expressed genes are significantly enriched among those aberrantly spliced in xct mutants, suggesting that the splicing activity of XCT may be circadian regulated. Furthermore, we show that loss-of-function mutations in PRP19A or PRP19B, 2 homologous core NTC components, suppress the short circadian period phenotype of xct-2. However, we do not see rescue of the splicing defects of core clock genes in prp19 xct mutants. Therefore, our results suggest that XCT may regulate splicing and the clock function through genetically separable pathways.

Euglena gracilis-derived β-glucan paramylon entrains the peripheral circadian clocks in mice.

Paramylon, a β-1,3-glucan storage polysaccharide derived from Euglena gracilis, has various health benefits, such as anti-obesity effects and modulation of immune function. However, whether paramylon intake affects the circadian clock remains unknown. In this study, we examined the effect of paramylon intake on the circadian clock. The results showed that the paramylon intake regulated peripheral clocks in mice. Furthermore, cecal pH and short-chain fatty acid concentrations after paramylon intake were measured. The correlation between changes in the expression of clock-related genes and alterations in the intestinal environment was confirmed. In addition, peripheral clock entrainment by paramylon intake was not observed in antibiotic-treated mice whose gut microbiota was weakened. These findings suggest that the regulation of the circadian clock by paramylon intake was mediated by changes in gut microbiota. In addition, the entraining effect of paramylon intake was also confirmed in mice bred under conditions mimicking social jetlag, which implies that paramylon intake may contribute to recovery from social jetlag. Thus, the appropriate consumption of paramylon may have a beneficial effect on health from a chrono-nutritional perspective.

Silmitasertib (CX-4945), a Clinically Used CK2-Kinase Inhibitor with Additional Effects on GSK3β and DYRK1A Kinases: A Structural Perspective.

A clinical casein kinase 2 inhibitor, CX-4945 (silmitasertib), shows significant affinity toward the DYRK1A and GSK3β kinases, involved in down syndrome phenotypes, Alzheimer's disease, circadian clock regulation, and diabetes. This off-target activity offers an opportunity for studying the effect of the DYRK1A/GSK3β kinase system in disease biology and possible line extension. Motivated by the dual inhibition of these kinases, we solved and analyzed the crystal structures of DYRK1A and GSK3β with CX-4945. We built a quantum-chemistry-based model to rationalize the compound affinity for CK2α, DYRK1A, and GSK3β kinases. Our calculations identified a key element for CK2α's subnanomolar affinity to CX-4945. The methodology is expandable to other kinase selectivity modeling. We show that the inhibitor limits DYRK1A- and GSK3β-mediated cyclin D1 phosphorylation and reduces kinase-mediated NFAT signaling in the cell. Given the CX-4945's clinical and pharmacological profile, this inhibitory activity makes it an interesting candidate with potential for application in additional disease areas.

Reciprocal regulation of flower induction by ELF3α and ELF3β generated via alternative promoter usage.

Flowering is critical for sexual reproduction and fruit production. Several pear (Pyrus sp.) varieties produce few flower buds, but the underlying mechanisms are unknown. The circadian clock regulator EARLY FLOWERING3 (ELF3) serves as a scaffold protein in the evening complex that controls flowering. Here, we report that the absence of a 58-bp sequence in the 2nd intron of PbELF3 is genetically associated with the production of fewer flower buds in pear. From rapid amplification of cDNA ends sequencing results, we identified a short, previously unknown transcript from the PbELF3 locus, which we termed PbELF3β, whose transcript level was significantly lower in pear cultivars that lacked the 58-bp region. The heterologous expression of PbELF3β in Arabidopsis (Arabidopsis thaliana) accelerated flowering, whereas the heterologous expression of the full-length transcript PbELF3α caused late flowering. Notably, ELF3β was functionally conserved in other plants. Deletion of the 2nd intron reduced AtELF3β expression and caused delayed flowering time in Arabidopsis. AtELF3β physically interacted with AtELF3α, disrupting the formation of the evening complex and consequently releasing its repression of flower induction genes such as GIGANTEA (GI). AtELF3β had no effect in the absence of AtELF3α, supporting the idea that AtELF3β promotes flower induction by blocking AtELF3α function. Our findings show that alternative promoter usage at the ELF3 locus allows plants to fine-tune flower induction.

Heat shock factor 1 induces a short burst of transcription of the clock gene Per2 during interbout arousal in mammalian hibernation

During winter hibernation, a diverse range of small mammals can enter prolonged torpor. They spend the nonhibernation season as a homeotherm but the hibernation season as a heterotherm. In the hibernation season, chipmunks (Tamias asiaticus) cycle regularly between 5 and 6days-long deep torpor with a body temperature (Tb) of 5 to 7 °C and interbout arousal of ∼20h, during which, their Tb returns to the normothermic level. Here, we investigated Per2 expression in the liver to elucidate the regulation of the peripheral circadian clock in a mammalian hibernator. In the nonhibernation season, as in mice, heat shock factor 1, activated by elevated Tb during the wake period, activated Per2 transcription in the liver, which contributed to synchronizing the peripheral circadian clock to the Tb rhythm. In the hibernation season, we determined that the Per2 mRNA was at low levels during deep torpor, but Per2 transcription was transiently activated by heat shock factor 1, which was activated by elevated Tb during interbout arousal. Nevertheless, we found that the mRNA from the core clock gene Bmal1 exhibited arrhythmic expression during interbout arousal. Since circadian rhythmicity is dependent on negative feedback loops involving the clock genes, these results suggest that the peripheral circadian clock in the liver is nonfunctional in the hibernation season.