Per2 Promoter Research Articles

Circadian control of histone turnover during cardiac development and growth

During postnatal cardiac hypertrophy, cardiomyocytes undergo mitotic exit, relying on DNA replication-independent mechanisms of histone turnover to maintain chromatin organization and gene transcription. In other tissues, circadian oscillations in nucleosome occupancy influence clock-controlled gene expression, suggesting a role for the circadian clock in temporal control of histone turnover and coordinated cardiomyocyte gene expression. To elucidate roles for the master circadian transcription factor, Bmal1, in histone turnover, chromatin organization, and myocyte-specific gene expression and cell growth in the neonatal period. Bmal1 knockdown in neonatal rat ventricular myocytes (NRVM) decreased myocyte size, total cellular protein synthesis, and transcription of the fetal hypertrophic gene Nppb following treatment with serum or the α-adrenergic agonist phenylephrine (PE). Depletion of Bmal1 decreased expression of clock-controlled genes Per2 and Tcap, as well as Sik1, a Bmal1 target upregulated in adult versus embryonic hearts. Bmal1 knockdown impaired Per2 and Sik1 promoter accessibility as measured by MNase-qPCR and impaired histone turnover as measured by metabolic labeling of acid-soluble chromatin fractions. Sik1 knockdown in turn decreased myocyte size, while simultaneously inhibiting Nppb transcription and activating Per2 transcription. Linking these changes to chromatin remodeling, depletion of the replication-independent histone variant H3.3a inhibited myocyte hypertrophy and prevented PE-induced changes in clock-controlled gene transcription. Bmal1 is required for neonatal myocyte growth, replication-independent histone turnover, and chromatin organization at the Sik1 promoter. Sik1 represents a novel clock-controlled gene that coordinates myocyte growth with hypertrophic and clock-controlled gene transcription. Replication-independent histone turnover is required for transcriptional remodeling of clock-controlled genes in cardiac myocytes in response to growth stimuli.

Abstract 1840: MYBBP1A AMPylated by SELO to influence its function

Abstract Introduction: MYBBP1A, named Myb-binding protein 1A, is a protein that mainly locates in nucleolar and shuttles between the nucleus and cytoplasm, nuclear import may be mediated by KPNA2, however export depend partially on XPO1/CRM1. It activates or represses transcription via interactions with sequence specific DNA-binding proteins. Repression may be mediated at least in part by histone deacetylase activity (HDAC activity). Acts as a corepressor and in concert with CRY1, represses the transcription of the core circadian clock component PER2. Preferentially binds to di-methylated histone H3 'Lys-9' (H3K9me2) on the PER2 promoter. MYBBP1A overexpressed in tumor than normal and have a significantly negative correlation with patient overall survival. We aim to explore the role of MYBBP1A in PDAC progression and underlying mechanism. In our research, we find and prove the novel AMPylating enzyme SELO-AMPylate MYBBP1A inhibits PDAC progression and modified site in S1353 and further influence downstream relative RNA, such as 18s rRNA, 28S rRNA. Methods and Materials: 1. The function of MYBBP1A in tumorigenesis at the organismal and cellular levels. MYBBP1A is widely expressed in different tissues. Firstly, we explored the correlation between MYBBP1A expression level and PDAC through TCGA database and Kaplan-Meier plotter. We then established MYBBP1A gene knockout mice mating with PDX/P53K172H/KRASG12C to induced a primary PDAC model to analyze the progression of pancreatic cancer. The effect of MYBBP1A on tumor cell growth and proliferation was explored through cell proliferation analysis and a nude mouse tumor xenograft model. 2. The MYBBP1A AMPylated by SELO influence MYBBP1A function in cell proliferation was investigated at the molecular and cellular levels. AMPylation-specific antibodies were used to confirmed MYBBP1A AMPylated by SELO in both cell lysates and purified proteins through experiments such as CO-IP and WB. Stably expressed MYBBP1A WT/S1353A were constructed to verify the function in cell proliferation or migration by SELO. The signaling molecules were further explored through CO-IP and WB to identify the changed molecular by SELO-AMPylate-MYBBP1A, revealing the entire signaling pathways of AMPylation-mediated regulation of PDAC proliferation. Conclusion: We have demonstrated the function of MYBBP1A in the formation of pancreatic cancer, and further revealed SELO-AMPylate-MYBBP1A importance in the process of reversing MYBBP1A’s role, which has played a certain significance for the treatment of pancreatic cancer and the exploration of related therapeutic targets. Citation Format: Wu Li. MYBBP1A AMPylated by SELO to influence its function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1840.

The Opioid Receptor Influences Circadian Rhythms in Human Keratinocytes through the β-Arrestin Pathway.

The recent emphasis on circadian rhythmicity in critical skin cell functions related to homeostasis, regeneration and aging has shed light on the importance of the PER2 circadian clock gene as a vital antitumor gene. Furthermore, delta-opioid receptors (DOPrs) have been identified as playing a crucial role in skin differentiation, proliferation and migration, which are not only essential for wound healing but also contribute to cancer development. In this study, we propose a significant association between cutaneous opioid receptor (OPr) activity and circadian rhythmicity. To investigate this link, we conducted a 48 h circadian rhythm experiment, during which RNA samples were collected every 5 h. We discovered that the activation of DOPr by its endogenous agonist Met-Enkephalin in N/TERT-1 keratinocytes, synchronized by dexamethasone, resulted in a statistically significant 5.6 h delay in the expression of the core clock gene PER2. Confocal microscopy further confirmed the simultaneous nuclear localization of the DOPr-β-arrestin-1 complex. Additionally, DOPr activation not only enhanced but also induced a phase shift in the rhythmic binding of β-arrestin-1 to the PER2 promoter. Furthermore, we observed that β-arrestin-1 regulates the transcription of its target genes, including PER2, by facilitating histone-4 acetylation. Through the ChIP assay, we determined that Met-Enkephalin enhances β-arrestin-1 binding to acetylated H4 in the PER2 promoter. In summary, our findings suggest that DOPr activation leads to a phase shift in PER2 expression via β-arrestin-1-facilitated chromatin remodeling. Consequently, these results indicate that DOPr, much like its role in wound healing, may also play a part in cancer development by influencing PER2.

Level of constitutively expressed BMAL1 affects the robustness of circadian oscillations

The circadian rhythm is a biological oscillation of physiological activities with a period of approximately 24 h, that is driven by a cell-autonomous oscillator called the circadian clock. The current model of the mammalian circadian clock is based on a transcriptional-translational negative feedback loop in which the protein products of clock genes accumulate in a circadian manner and repress their own transcription. However, several studies have revealed that constitutively expressed clock genes can maintain circadian oscillations. To understand the underlying mechanism, we expressed Bmal1 in Bmal1-disrupted cells using a doxycycline-inducible promoter and monitored Bmal1 and Per2 promoter activity using luciferase reporters. Although the levels of BMAL1 and other clock proteins, REV-ERBα and CLOCK, showed no obvious rhythmicity, robust circadian oscillation in Bmal1 and Per2 promoter activities with the correct phase relationship was observed, which proceeded in a doxycycline-concentration-dependent manner. We applied transient response analysis to the Bmal1 promoter activity in the presence of various doxycycline concentrations. Based on the obtained transfer functions, we suggest that, at least in our experimental system, BMAL1 is not directly involved in the oscillatory process, but modulates the oscillation robustness by regulating basal clock gene promoter activity.

Real time monitoring of cold Ca2+ dependent transcription and its modulation by NCX inhibitors

Real-time monitoring of cellular temperature responses is an important technique in thermal biology and drug development. Recent study identified that Na+/Ca2+ exchanger (NCX)-dependent Ca2+ influx transduces cold signals to circadian clock in mammalian cultured cells. The finding raised an idea that cellular responses to the cold signals can be analyzed by monitoring of clock gene expression. We found that Per1 and Per2 were up-regulated after culture at 27 °C compared to 37 °C in Rat-1 fibroblasts. In order to monitor cold-Ca2+-dependent transcription in living cells, we developed a luciferase-based real-time reporting system by using Per1 promoter, Per2 promoter, Ca2+/cAMP-response elements (CRE) or NFAT-binding elements. We found that benzyloxyphenyl NCX inhibitor KB-R7943 and SN-6, but not SEA-0400 or YM-244769 inhibited the cold induction of Per2. Our study established a real-time monitoring system for cold Ca2+ signaling which can be applied to evaluation of drugs.

Stabilization of hypoxia-inducible factor-1α alleviates osteoarthritis via interacting with Per2 and resetting the circadian clock

ObjectiveThe study aimed to establish whether HIF1α entrains the core clock in chondrocytes and how the HIF1α affects the circadian rhythm. MethodsWe subjected primary chondrocytes to chronic circadian desynchrony (CCD) and subsequently treated with oxygen rhythm and dimethyloxalylglycine (DMOG). Circadian oscillations were analyzed with a real-time monitoring system of Per2 promoter activity and qPCR. Chondrocytes were assayed core clock genes expression patterns and extracellular matrix metabolism. HIF1α siRNA was used to knock down HIF1α. ChIP-qPCR and dual-luciferase reporter assay were used to validate that Per2 was the target gene of HIF1α. The surgical model of osteoarthritis was induced by destabilization of the medial meniscus (DMM). The chronic jet lag model was established light/dark cycle advance shift. Joint degeneration was measured using histological staining, immunological assays, and micro-CT scanning. ResultsWe report that different patterns of clock genes expression between healthy and osteoarthritic cartilage tissues and oxygen rhythms and DMOG reset the molecular clockwork which was dampened after CCD culture in a HIF1α-dependent manner. HIF1α increased the amplitude of oscillation by directly binding to the HRE-like and E-box-like elements located on the Per2 promoter. The rhythmic circadian clock significantly enhanced extracellular matrix production. Our study also demonstrates that DMOG ameliorated the increased OA severity caused by jet lag in the DMM model. ConclusionsThis study shows that circadian clock resetting caused by DMOG is at least partially mediated by the HIF1α through interaction with the Per2 promoter and proposes DMOG as supportive therapy for OA.

LncRNA TPTEP1 inhibits the migration and invasion of gastric cancer cells through miR-548d-3p/KLF9/PER1 axis

BackgroundDespite the development of many methods and new therapeutic agents, the survival and prognosis of patients with gastric cancer are still poor. The role of TPTEP1 in gastric cancer has not been reported. MethodsWound healing assay and transwell assay analysis TPTEP1/miR-548d-3p/KLF9/PER1 effect on migration and invasiveness of gastric cells. Western blot and RT-qPCR certificate TPTEP1/miR-548d-3p/KLF9/PER1transcription and expression of migration and invasion related genes. Luciferase assay was used to determine the adsorption of miR-548d-3p by TPTEP1 sponge, the targeting of miR-548d-3p to KLF9, and the binding of KLF9 to the promoter of PER1. immunohistochemical assay and H&E staining prove the function of TPTEP1 and miR-548d-3p in nude mice model of gastric cancer. ResultsTPTEP1 inhibited its expression by sponge adsorption of miR-548d-3p. miR-548d-3p targets KLF9 3′UTR to inhibit its expression, and KLF9 binds to the PER1 promoter to promote its expression.TPTEP1/KLF9/PER1 inhibits gastric cancer cell migration and invasion, and miR-548d-3p does the opposite. ConclusionsOur data suggest that TPTEP1 affects gastric cancer progression by regulating the miR-548d-3p/KLF9/PER1 axis. Targeting this pathway may provide new therapeutic opportunities for gastric cancer.

The Natural Janus Kinase Inhibitor Agerarin Downregulates Interleukin-4-Induced PER2 Expression in HaCaT Keratinocytes.

The circadian clock system is closely associated with inflammatory responses. Dysregulation of the circadian clock genes in the skin impairs the skin barrier function and affects the pathophysiology of atopic dermatitis. Interleukin 4 (IL-4) is a proinflammatory cytokine derived from T-helper type 2 cells; it plays a critical role in the pathogenesis of atopic dermatitis. Agerarin (6,7-dimethoxy-2,2-dimethyl-2H-chromene) is a natural JAK1/2/3 inhibitor isolated from Ageratum houstonianum that has a protective effect on the epidermal skin barrier. However, it remains unclear whether agerarin affects the circadian clock system. The aim of this study is to investigate the effect of agerarin on IL-4-induced PER2 gene expression in human keratinocytes through reverse transcription (RT)-PCR, quantitative real-time PCR (qPCR), immunoblotting, immunofluorescence microscopic analysis, and real-time bioluminescence analysis. We found that agerarin reduced IL-4-induced PER2 mRNA expression by suppressing the JAK-STAT3 pathway. In addition, real-time bioluminescence analysis in PER2:luc2p promoter-reporter cells revealed that agerarin restored the oscillatory rhythmicity of PER2 promoter activity altered by IL-4. These findings suggest that agerarin may be useful as a cosmeceutical agent against inflammatory skin conditions associated with disrupted circadian rhythms, such as atopic dermatitis.

A highland-adaptation mutation of the Epas1 protein increases its stability and disrupts the circadian clock in the plateau pika

The Qinghai-Tibet Plateau (QTP) harbors hundreds of species well adapted to its extreme conditions, including its low-oxygen (hypoxic) atmosphere. Here, we show that the plateau pika-a keystone mammal of the QTP-lacks robust circadian rhythms. The major form of the plateau pika Epas1 protein includes a 24-residue insert caused by a point mutation at the 5' juncture site of Intron14 and is more stable than other mammalian orthologs. Biochemical studies reveal that an Epas1-Bmal1 complex with lower trans-activation activity occupies the E1/E2 motifs at the promoter of the core-clock gene Per2, thus explaining how an Epas1 mutation-selected in the hypoxic conditions of the QTP-disrupts the molecular clockwork. Importantly, experiments with hypoxic chambers show that mice expressing the plateau pika Epas1 ortholog in their suprachiasmatic nucleus have dysregulated central clocks, and pika Epas1 knockin mice reared in hypoxic conditions exhibit dramatically reduced heart damage compared with wild-type animals.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Transcription Factor EGR1 Regulates the Expression of the Clock Gene PER2 under IL-4 Stimulation in Human Keratinocytes

PER2 is a core circadian clock gene that regulates circadian rhythms. IL-4 plays a critical role in the pathogenesis of skin inflammation, including atopic dermatitis. IL-4 enhances PER2 expression, suggesting a relationship between inflammation and the circadian clock. However, little is known about the molecular and cellular mechanisms regulating PER2 expression by inflammatory cytokines. This study showed that transcription factor EGR1 interacted with the PER2 promoter and promoted IL-4‒induced transcriptional activation of the PER2, as revealed by promoter‒reporter assay, electrophoretic mobility shift assay, DNA affinity precipitation assay, and chromatin immunoprecipitation analysis. We also found that IL-4 can use both MAPK and Jak signaling pathways to induce EGR1-mediated PER2 expression, and c-Jun N-terminal kinase 1/2 can augment IL-4‒induced activation of the Jak‒signal transducer and activator of transcription 3 pathway. Consistently, Per2 expression was reduced in dinitrochlorobenzene-induced atopic dermatitis‒like skin lesions in Egr1‒/‒ mice compared with that in Egr1+/+ mice. In addition, using a real-time bioluminescence assay, we observed that EGR1 is required for rhythmic oscillation of PER2 expression under IL-4 exposure. These findings provide further insight into the role of EGR1 in regulating PER2 expression in impaired circadian rhythm in skin inflammation.

Effect of combination of residual glucose concentration and subsequent increment by temporal glucose feeding on oscillation of clock gene Per2 expression.

With the aim of regulating clock gene expression to control cell activities in cell processing engineering, the effect of the combination of residual glucose concentration and subsequent increment by temporal glucose feeding on the oscillation of the expression of clock gene Per2 was investigated employing rat Mesenchymal stem cell (MSC)-like cells having Per2 promoter gene with a destabilized luciferase gene (Per2-dLuc). Two experiments with several initial glucose concentrations and different times of cultures (2 and 5days) before temporal glucose feeding (0.9g/L) were employed to realize various concentrations of residual glucose in the medium before the feeding. In these experiments, the lower residual glucose concentrations (0.002-0.02g/L) before temporal glucose feeding tended to induce the larger amplitude of oscillation of Per2 expression than the higher ones (0.55-0.74g/L). When the residual glucose concentration before glucose feeding was low (0.014-0.038g/L), the higher temporal glucose concentration (0.23-0.9g/L) feeding tended to induce the larger amplitude of oscillation of Per2 expression than the lower ones (0.012-0.023g/L). Taken together, we found that the amplitude of oscillation of the expression of clock gene Per2 could be controlled by the combination of residual glucose concentration and glucose concentration of subsequent temporal feeding.

Insulin Directly Regulates the Circadian Clock in Adipose Tissue.

Adipose tissue (AT) is a key metabolic organ which functions are rhythmically regulated by an endogenous circadian clock. Feeding is a "zeitgeber" aligning the clock in AT with the external time, but mechanisms of this regulation remain largely unclear. We tested the hypothesis that postprandial changes of the hormone insulin directly entrain circadian clocks in AT and investigated a transcriptional-dependent mechanism of this regulation. We analyzed gene expression in subcutaneous AT (SAT) of obese subjects collected before and after the hyperinsulinemic-euglycemic clamp or control saline infusion (SC). The expressions of core clock genes PER2, PER3, and NR1D1 in SAT were differentially changed upon insulin and saline infusion, suggesting insulin-dependent clock regulation. In human stem cell-derived adipocytes, mouse 3T3-L1 cells, and AT explants from mPer2Luc knockin mice, insulin induced a transient increase of the Per2 mRNA and protein expression, leading to the phase shift of circadian oscillations, with similar effects for Per1 Insulin effects were dependent on the region between -64 and -43 in the Per2 promoter but not on CRE and E-box elements. Our results demonstrate that insulin directly regulates circadian clocks in AT and isolated adipocytes, thus representing a primary mechanism of feeding-induced AT clock entrainment.

Transgenic rats expressing dominant negative BMAL1 showed circadian clock amplitude reduction and rapid recovery from jet lag.

The circadian rhythms are endogenous rhythms of about 24h, and are driven by the circadian clock. The clock centre locates in the suprachiasmatic nucleus. Light signals from the retina shift the circadian rhythm in the suprachiasmatic nucleus, but there is a robust part of the suprachiasmatic nucleus that causes jet lag after an abrupt shift of the environmental lighting condition. To examine the effect of attenuated circadian rhythm on the duration of jet lag, we established a transgenic rat expressing BMAL1 dominant negative form under control by mouse Prnp-based transcriptional regulation cassette [BMAL1 DN (+)]. The transgenic rats became active earlier than controls, just after light offset. Compared to control rats, BMAL1 DN (+) rats showed smaller circadian rhythm amplitudes in both behavioural and Per2 promoter driven luciferase activity rhythms. A light pulse during the night resulted in a larger phase shift of behavioural rhythm. Furthermore, at an abrupt shift of the light-dark cycle, BMAL1 DN (+) rat showed faster entrainment to the new light-dark cycle compared to controls. The circadian rhythm has been regarded as a limit cycle phenomenon, and our results support the hypothesis that modification of the amplitude of the circadian limit cycle leads to alteration in the length of the phase shift.

The circadian rhythm and core gene Period2 regulate the chemotherapy effect and multidrug resistance of ovarian cancer through the PI3K signaling pathway.

Background: Ovarian cancer is the most lethal cancer in the female reproductive system. It has been shown that ‘time chemotherapy’ of ovarian cancer has an important impact on the chemotherapy effect and prognosis of patients, but the specific mechanism is not known. Methods: We designed a case–control study in strict accordance with epidemiological principles. We collected resection samples of ovarian cancer patients who worked night-shifts and those who did not, and analyzed the differences in protein expression. Through construction of a normal/circadian-rhythm disorder model of ovarian cancer in nude mice, we explored the molecular mechanism of a ‘biological clock’ rhythm on treatment of ovarian cancer. Results: Expression of interleukin (IL)-6, programmed cell death receptor-1 (PD-1) and programmed death ligand 1 (PD-L1) increased, and expression of tumor necrosis factor (TNF)-α, Period 1 (Per1) and Period 2 (Per2) decreased in the night-shift group. Methylation of CpG islands in the promoter of Per2 could result in its decreased expression in SKOV3/DDP (Cisplatin) cells. Dysrhythmia of the circadian clock: (i) had a negative effect on the chemotherapy effect against ovarian cancer; (ii) affected expression of immune factors and the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Conclusion: The Per2 gene can affect the drug resistance of ovarian cancer by inhibiting the PI3K/Akt signaling pathway and then acting on its downstream drug-resistance factors, thereby providing a new target for ovarian cancer treatment.

Contraction influences Per2 gene expression in skeletal muscle through a calcium-dependent pathway.

Key points Exercising at different times of day elicits different effects on exercise performance and metabolic health. However, the specific signals driving the observed time‐of‐day specific effects of exercise have not been fully identified.Exercise influences the skeletal muscle circadian clock, although the relative contribution of muscle contraction and extracellular signals is unknown.Here, we show that contraction acutely increases the expression of the core circadian clock gene Period Circadian Regulator 2 (Per2) and phase‐shifts Per2 rhythmicity in muscle cells. This contraction effect on core clock genes is mediated through a calcium‐dependant mechanism;The results obtained in the present study suggest that a proportion of the ability of exercise to entrain the skeletal muscle clock is driven directly by muscle contraction. Contraction interventions may be used to mimic some time‐of‐day specific effects of exercise on metabolism and muscle performance. Exercise entrains the central and peripheral circadian clocks, although the mechanism by which exercise modulates expression of skeletal muscle clock genes is unclear. The present study aimed to determine whether skeletal muscle contraction alone could directly influence circadian rhythmicity and uncover the underlying mechanism by which contraction modulates clock gene expression. We investigated the expression of core clock genes in human skeletal muscle after acute exercise, as well as following in vitro contraction in mouse soleus muscle and cultured C2C12 skeletal muscle myotubes. Additionally, we interrogated the molecular pathways by which skeletal muscle contraction could influence clock gene expression. Contraction acutely increased the expression of the core circadian clock gene Period Circadian Regulator 2 (Per2) and phase‐shifted Per2 rhythmicity in C2C12 myotubes in vitro. Further investigation revealed that pharmacologically increasing cytosolic calcium concentrations by ionomycin treatment mimicked the effect of contraction on Per2 expression. Similarly, treatment with a calcium channel blocker, nifedipine, blocked the effect of electric pulse stimulation‐induced contraction on Per2 expression. Increased calcium influx from contraction lead to binding of the phosphorylated form of cAMP response element‐binding protein (CREB) to the Per2 promoter, suggesting a role of CREB in contraction‐induced Per2 transcription. Thus, by dissociating the effect of muscle contraction alone from the whole effect of exercise, our investigations indicate that a proportion of the ability of exercise to entrain the skeletal muscle clock is driven directly by contraction.

CAMP response element induces Per1 in vivo

Light is an important cue for resetting the circadian clock. In mammals, light signals are thought to be transmitted to the cAMP response element (CRE) via a binding protein (CREB) to induce the expression of Per1 and Per2 genes in the mammalian circadian pacemaker, the suprachiasmatic nuclei (SCN). Several in vitro studies have suggested candidate CRE sites that contribute to the Per1 and Per2 induction by light, resulting in a phase shift of the circadian rhythm. However, it remains unclear whether the CREs are responsible for the light-induced Per1/2 induction. To address this question, we generated CRE-deleted mice in the Per1 and Per2 promoter regions. Deletion of a cAMP-responsive CRE in the Per1 promoter blunted light-induced Per1 expression in the SCN at night, while deletion of an ATF4 (CREB-2)-associated CRE in the Per2 promoter had no effect on its expression. These results suggested that the CRE in the Per1 promoter works for light induction but not CRE in the Per2 promoter. Behavioral rhythms observed under some light conditions were not affected by the CRE-deletion in Per1 promoter, suggesting that the attenuated Per1 induction did not affect the entrainment in some light conditions.

Novel insights into biological roles of inducible cAMP early repressor ICER

ICER corresponds to a group of alternatively spliced Inducible cAMP Early Repressors with high similarity, but multiple roles, including in circadian rhythm, and are involved in attenuation of cAMP-dependent gene expression. We present experimental and in silico data revealing biological differences between the isoforms with exon gamma (ICER) or without it (ICERγ). Both isoforms are expressed in the liver and the adrenal glands and can derive from differential splicing. In adrenals the expression is circadian, with maximum at ZT12 and higher amplitude of Icerγ. In the liver, the expression of Icerγ is lower than Icer in the 24 h time frame. Icer mRNA has a delayed early response to forskolin. The longer ICER protein binds to three DNA grooves of the Per1 promoter, while ICERγ only to two, as deduced by molecular modelling. This is in line with gel shift competition assays showing stronger binding of ICER to Per1 promotor. Only Icerγ siRNA provoked an increase of Per1 expression. In conclusion, we show that ICER and ICERγ have distinct biochemical properties in tissue expression, DNA binding, and response to forskolin. Data are in favour of ICERγ as the physiologically important form in hepatic cells where weaker binding of repressor might be preferred in guiding the cAMP-dependent response.

Programming effects of maternal stress on the circadian system of adult offspring

Maternal stress has long-lasting influences on the brain functions of offspring, and several brain regions have been proposed to mediate such programming. Although perinatal programming of crosstalk between the circadian and stress systems has been proposed, the functional consequences of prenatal stress on the circadian system and the underlying mechanisms remain largely unknown. Therefore, we investigated whether exposing pregnant mice to chronic restraint stress had prolonged effects on the suprachiasmatic nucleus (SCN), which bears the central pacemaker for mammalian circadian rhythms, of offspring. SCN explants from maternally stressed mice exhibited altered cyclic expression patterns of a luciferase reporter under control of the mouse Per1 promoter (mPer1::LUC), which manifested as a decreased amplitude and impaired stability of the rhythm. Bioluminescence imaging at the single-cell level subsequently revealed that impaired synchrony among individual cells was responsible for the impaired rhythmicity. These intrinsic defects appeared to persist during adulthood. Adult male offspring from stressed mothers showed advanced-phase behavioral rhythms with impaired stability as well as altered clock gene expression in the SCN. In addition to affecting the central rhythm, maternal stress also had prolonged influences on the circadian characteristics of the adrenal gland and liver, as determined by circulating corticosterone levels and hepatic glycogen content, and on canonical clock gene mRNA expression in those tissues. Taken together, our findings suggest that the SCN is a key target of the programming effects of maternal stress. The widespread effects of circadian disruptions caused by a misprogrammed clock may have further impacts on metabolic and mental health in later life.

The eIF2α Kinase GCN2 Modulates Period and Rhythmicity of the Circadian Clock by Translational Control of Atf4

The integrated stress response (ISR) is activated in response to diverse stress stimuli to maintain homeostasis in neurons. Central to this process is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). Here, we report a critical role for ISR in regulating the mammalian circadian clock. The eIF2α kinase GCN2 rhythmically phosphorylates eIF2α in the suprachiasmatic circadian clock. Increased eIF2α phosphorylation shortens the circadian period in both fibroblasts and mice, whereas reduced eIF2α phosphorylation lengthens the circadian period and impairs circadian rhythmicity in animals. Mechanistically, phosphorylation of eIF2α promotes mRNA translation of Atf4. ATF4 binding motifs are identified in multiple clock genes, including Per2, Per3, Cry1, Cry2, and Clock. ATF4 binds to the TTGCAGCA motif in the Per2 promoter and activates its transcription. Together, these results demonstrate a significant role for ISR in circadian physiology and provide a potential link between dysregulated ISR and circadian dysfunction in brain diseases.