Components Of Circadian Clock Research Articles

The Circadian Clock Component RORA Increases Immunosurveillance in Melanoma by Inhibiting PD-L1 Expression.

Circadian clock perturbation frequently occurs in cancer and facilitates tumor progression by regulating malignant growth and shaping the immune microenvironment. Emerging evidence has indicated that clock genes are disrupted in melanoma and linked to immune escape. Here, we found that the expression of retinoic acid receptor-related orphan receptor-α (RORA) is downregulated in melanoma patients and that patients with higher RORA expression have a better prognosis after immunotherapy. Additionally, RORA was significantly positively correlated with T-cell infiltration and recruitment. Overexpression or activation of RORA stimulated cytotoxic T-cell-mediated antitumor responses. RORA bound to the CD274 promoter and formed an inhibitory complex with HDAC3 to suppress PD-L1 expression. In contrast, the DEAD-box helicase family member DDX3X competed with HDAC3 for binding to RORA, and DDX3X overexpression promoted RORA release from the suppressive complex and thereby increased PD-L1 expression to generate an inhibitory immune environment. The combination of a RORA agonist with an anti-CTLA4 antibody synergistically increased T-cell antitumor immunity in vivo. A score based on the combined expression of HDAC3, DDX3X and RORA correlated with immunotherapy response in melanoma patients. Together, this study elucidates a mechanism of clock component-regulated antitumor immunity, which will help inform the use of immunotherapy and lead to improved outcomes for melanoma patients receiving combined therapeutic treatments.

Appropriate induction of TOC1 ensures optimal MYB44 expression in ABA signaling and stress response in Arabidopsis.

Plants possess the remarkable ability to integrate the circadian clock with various signalling pathways, enabling them to quickly detect and react to both external and internal stress signals. However, the interplay between the circadian clock and biological processes in orchestrating responses to environmental stresses remains poorly understood. TOC1, a core component of the plant circadian clock, plays a vital role in maintaining circadian rhythmicity and participating in plant defences. Here, our study reveals a direct interaction between TOC1 and the promoter region of MYB44, a key gene involved in plant defence. TOC1 rhythmically represses MYB44 expression, thereby ensuring elevated MYB44 expression at dawn to help the plant in coping with lowest temperatures during diurnal cycles. Additionally, both TOC1 and MYB44 can be induced by cold stress in an Abscisic acid (ABA)-dependent and independent manner. TOC1 demonstrates a rapid induction in response to lower temperatures compared to ABA treatment, suggesting timely flexible regulation of TOC1-MYB44 regulatory module by the circadian clock in ensuring a proper response to diverse stresses and maintaining a balance between normal physiological processes and energy-consuming stress responses. Our study elucidates the role of TOC1 in effectively modulating expression of MYB44, providing insights into the regulatory network connecting the circadian clock, ABA signalling, and stress-responsive genes.

Circadian Rhythms in NLRP3 Inflammasome Regulation: Possible Implications for the Nighttime Risk of Gout Flares

Gout flares more frequently start late at night or in the early morning compared to during the day. The reasons for this are unknown. Activation of the NLRP3 inflammasome in monocytes/macrophages is central to initiation of gout flares. Here, we review the mechanisms by which circadian clocks control the NLRP3 inflammasome and the implications of this for the nighttime pattern of gout flares. Several hormones involved in inflammation regulation, e.g., glucocorticoids, melatonin and melanocortins, are under circadian control, with both circulating hormone levels as well as the expression of their receptors on target tissues showing time-of day differences. In addition, the NLRP3 inflammasome is also under the control of the macrophage circadian clock, leading to time-of-day differences in expression of NLRP3 inflammasome components and susceptibility to inflammasome-activating stimuli. MSU crystal exposure leads to altered expression of circadian clock components in macrophages, leading to time-of-day-specific loss of repression of NLRP3 inflammasome activity. Taken together, there is clear evidence that circadian clocks regulate the NLRP3 inflammasome and that this regulation may be compromised by MSU crystal exposure in gout. Circadian control of the inflammasome may be one of the factors contributing to nighttime susceptibility to gout flares.

Cryptochrome 1 regulates ovarian granulosa cell senescence through NCOA4-mediated ferritinophagy

Age-associated decreases in follicle number and oocyte quality result in a decline in female fertility, which is associated with increased infertility. Granulosa cells play a major role in oocyte development and maturation both in vivo and in vitro. However, it is unclear whether a reduction in cryptochrome 1 (Cry1) expression contributes to granulosa cell senescence, and further exploration is needed to understand the underlying mechanisms. In this study, we investigated the role of Cry1, a core component of the molecular circadian clock, in the regulation of senescence in ovarian granulosa cells. Western blotting and qRT-PCR showed that Cry1 expression was downregulated in aged human ovarian granulosa cells and was correlated with age and anti-Müllerian hormone (AMH) levels. RNA-seq analysis suggested that ferritinophagy was increased after Cry1 knockdown in KGN cells. MDA, iron, and reactive oxygen species (ROS) assays were used to detect cellular ferritinophagy levels. Ferroptosis inhibitors, iron chelators, autophagy inhibitors, and nuclear receptor coactivator 4 (NCOA4) knockdown alleviated KGN cell senescence induced by Cry1 knockdown. Immunofluorescence, immunoprecipitation, and ubiquitination assays indicated that Cry1 affected NCOA4 ubiquitination and degradation through HERC2, thereby affecting NCOA4-mediated ferritinophagy and causing granulosa cell senescence. KL201, a Cry1 stabilizer, enhanced ovarian function in naturally aged mice by reducing ferritinophagy. Our study reveals the potential mechanisms of action of Cry1 during ovarian aging and provides new insights for the clinical treatment of age-related fertility decline.

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.

Abstract 669: Interrogating circadian clock compounds as an adjuvant to chemoradiation against GBM

Abstract Despite the extensive efforts of researchers and physicians over the past two decades, glioblastoma (GBM) remains the most prevalent brain tumor type with a grim average survival time of 8 months following diagnosis irrespective of treatment. Additionally, the current standard-of-care has not changed from maximal surgical resection followed by concurrent chemoradiation from its establishment in 2005. One of the major challenges that limits progress in treating GBM is the presence of GBM stem cells (GSCs) that are highly invasive, reaching far past the tumor margins, are resistant to both chemotherapy and radiation, and can recapitulate the primary tumor following resection. Recent studies have found that GSCs, however, are highly sensitive to targeting of core circadian clock components, particularly positive regulators of the clock Brain and Muscle ARNTL-Like 1 (BMAL1) and Circadian Locomoter Output Cycles Protein Kaput (CLOCK). Here we employ the use of small molecule compounds that upregulate the activity of the negative regulators of the clock in order to suppress BMAL1::CLOCK transcriptional activity or repress BMAL1 transcription. We find that these compounds not only have a much more potent effect against GSCs compared to temozolomide (TMZ) chemotherapy but also, they display combination effect against both GSCs and differentiated GSCs (DGCs) when combined with TMZ. These results suggest that the blood brain barrier (BBB) penetrant circadian clock compounds can be utilized as an adjuvant to current GBM therapies as they can effectively target both the primary tumor cells and GSCs. Citation Format: Priscilla Chan, Qiulian Wu, Anahit Hovsepyan, Seda Mkhitaryan, Gevorg Karapetyan, Khalid Shah, Hiroaki Wakimoto, Theodore Kamenecka, Laura A. Solt, Jamie Cope, Tsuyoshi Hirota, Rex A. Moats, Jeremy N. Rich, Steve A. Kay. Interrogating circadian clock compounds as an adjuvant to chemoradiation against GBM [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 669.

The interplay between the PI3K/AKT pathway and circadian clock in physiologic and cancer‐related pathologic conditions

AbstractThe circadian clock is responsible for the regulation of different cellular processes, and its disturbance has been linked to the development of different diseases, such as cancer. The main molecular mechanism for this issue has been linked to the crosstalk between core clock regulators and intracellular pathways responsible for cell survival. The PI3K/AKT signalling pathway is one of the most known intracellular pathways in the case of cancer initiation and progression. This pathway regulates different aspects of cell survival including proliferation, apoptosis, metabolism, and response to environmental stimuli. Accumulating evidence indicates that there is a link between the PI3K/AKT pathway activity and circadian rhythm in physiologic and cancer‐related pathogenesis. Different classes of PI3Ks and AKT isoforms are involved in regulating circadian clock components in a transcriptional and functional manner. Reversely, core clock components induce a rhythmic fashion in PI3K and AKT activity in physiologic and pathogenic conditions. The aim of this review is to re‐examine the interplay between this pathway and circadian clock components in normal condition and cancer pathogenesis, which provides a better understanding of how circadian rhythms may be involved in cancer progression.

Complex epistatic interactions between ELF3, PRR9, and PRR7 regulate the circadian clock and plant physiology.

Circadian clocks are endogenous timekeeping mechanisms that coordinate internal physiological responses with the external environment. EARLY FLOWERING3 (ELF3), PSEUDO RESPONSE REGULATOR (PRR9), and PRR7 are essential components of the plant circadian clock and facilitate entrainment of the clock to internal and external stimuli. Previous studies have highlighted a critical role for ELF3 in repressing the expression of PRR9 and PRR7. However, the functional significance of activity in regulating circadian clock dynamics and plant development is unknown. To explore this regulatory dynamic further, we first employed mathematical modeling to simulate the effect of the prr9/prr7 mutation on the elf3 circadian phenotype. These simulations suggested that simultaneous mutations in prr9/prr7 could rescue the elf3 circadian arrhythmia. Following these simulations, we generated all Arabidopsis elf3/prr9/prr7 mutant combinations and investigated their circadian and developmental phenotypes. Although these assays could not replicate the results from the mathematical modeling, our results have revealed a complex epistatic relationship between ELF3 and PRR9/7 in regulating different aspects of plant development. ELF3 was essential for hypocotyl development under ambient and warm temperatures, while PRR9 was critical for root thermomorphogenesis. Finally, mutations in prr9 and prr7 rescued the photoperiod-insensitive flowering phenotype of the elf3 mutant. Together, our results highlight the importance of investigating the genetic relationship among plant circadian genes.

Zika Virus Infection Alters the Circadian Clock Expression in Human Neuronal Monolayer and Neurosphere Cultures.

Rhythmic regulations are virtually described in all physiological processes, including central nervous system development and immunologic responses. Zika virus (ZIKV), a neurotropic arbovirus, has been recently linked to a series of birth defects and neurodevelopmental disorders. Given the well-characterized role of the intrinsic cellular circadian clock within neurogenesis, cellular metabolism, migration, and differentiation among other processes, this study aimed to characterize the influence of ZIKV infection in the circadian clock expression in human neuronal cells. For this, in vitro models of human-induced neuroprogenitor cells (hiNPCs) and neuroblastoma cell line SH-SY5Y, cultured as monolayer and neurospheres, were infected by ZIKV, followed by RNA-Seq and RT-qPCR investigation, respectively. Targeted circadian clock components presented mRNA oscillations only after exogenous synchronizing stimuli (Forskolin) in SH-SY5Y monolayer culture. Interestingly, when these cells were grown as 3D-arranged neurospheres, an intrinsic oscillatory expression pattern was observed for some core clock components without any exogenous stimulation. The ZIKV infection significantly disturbed the mRNA expression pattern of core clock components in both neuroblastoma cell culture models, which was also observed in hiNPCs infected with different strains of ZIKV. The ZIKV-mediated desynchronization of the circadian clock expression in human cells might further contribute to the virus impairment of neuronal metabolism and function observed in adults and ZIKV-induced congenital syndrome. In vitro models of Zika virus (ZIKV) neuronal infection. Human neuroprogenitor cells were cultured as monolayer and neurospheres and infected by ZIKV. Monolayer-cultured cells received forskolin (FSK) as a coupling factor for the circadian clock rhythmicity, while 3D-arranged neurospheres showed an intrinsic oscillatory pattern in the circadian clock expression. The ZIKV infection affected the mRNA expression pattern of core clock components in both cell culture models. The ZIKV-mediated desynchronization of the circadian clock machinery might contribute to the impairment of neuronal metabolism and function observed in both adults (e.g., Guillain-Barré syndrome) and ZIKV-induced congenital syndrome (microcephaly). The graphical abstract has beencreated with Canva at the canva.com website.

Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation.

Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.

Circadian Clock REV-ERBs Agonist SR9009 Induces Synergistic Antitumor Activity in Multiple Myeloma by Suppressing Glucose-Regulated Protein 78-Dependent Autophagy and Lipogenesis.

Proteasome inhibitors, such as bortezomib, have demonstrated efficacy in the therapeutic management of multiple myeloma (MM). However, it is important to note that these inhibitors also elicit endoplasmic reticulum stress, which subsequently triggers the unfolded protein response (UPR) and autophagy, which have been shown to facilitate the survival of tumor cells. The disruption of the circadian clock is considered a characteristic feature of cancer. However, how disrupted circadian clock intertwines with tumor metabolism and drug resistance is not clearly clarified. This work explores the antitumor effectiveness of bortezomib and the circadian clock agonist SR9009, elucidating their impact on glucose-regulated protein 78 (GRP78), the autophagy process, and lipogenesis. The antitumor effects of bortezomib and SR9009 were evaluated using human MM cell lines (RPMI8226 and U266) in vitro and in vivo nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft MM model. The assessment of cell viability was conducted using the cell counting kit-8 (CCK8) method, whereas the measurement of cell proliferation was performed with the inclusion of EdU (5-ethynyl-2'-deoxyuridine). Apoptosis was assessed by flow cytometry. The cells were transduced using adenovirus-tf-LC3, which was labeled with dual fluorescence. Subsequently, confocal imaging was employed to observe and examine the autophagosomes. REV-ERBα knockdown leads to upregulation of ATG5 and BENC1 at the protein level with immunoblot. Changes in the expression levels of GRP78, LC3, stearoyl-CoA desaturase 1 (SCD1), and fatty acid synthase (FASN) were assessed through the utilization of quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Our results showed that both bortezomib and circadian clock REV-ERBs agonist SR9009 decreased MM viability, proliferation rate and induced an apoptotic response in a dose-dependent manner in vitro. However, the two differ greatly in their mechanisms of action. Bortezomib upregulated GRP78 and autophagy LC3, while circadian clock agonist SR9009 inhibited GRP78 and autophagy LC3. Combined SR9009 with bortezomib induced synergistic cytotoxicity against MM cells. REV-ERBα knockdown lead to upregulation of ATG5, BENC1 and significant upregulation of FASN, and SCD1. Mechanically, SR9009 inhibited the core autophagy gene ATG5 and BECN1, and two essential enzymes for de novo lipogenesis FASN and SCD1. SR9009 had synergistic effect with bortezomib and slowed down murine xenograft models of human MM tumor growth in vivo. Taken together, these results demonstrated that the circadian clock component REV-ERBs agonist SR9009 could inhibit GRP78-induced autophagy and de novo lipogenesis processes and had a synergistic effect with proteasome inhibitors in both in vitro and in vivo models of MM. Our findings shed light on how a disrupted circadian clock interacts with metabolic mechanisms to shape proteasome inhibitor drug resistance and suggest that SR9009 may be able to overcome the inherent drug resistance of proteasome inhibitors.

Effects of Pinealectomy on the Daily Profile of Circadian Clock and Apoptosis Components in Rat Retroperitoneal Adipose Tissue

Effects of Pinealectomy on the Daily Profile of Circadian Clock and Apoptosis Components in Rat Retroperitoneal Adipose Tissue

Cardiomyocyte-specific disruption of the circadian BMAL1–REV-ERBα/β regulatory network impacts distinct miRNA species in the murine heart

Circadian disruption increases cardiovascular disease (CVD) risk, through poorly understood mechanisms. Given that small RNA species are critical modulators of cardiac physiology/pathology, we sought to determine the extent to which cardiomyocyte circadian clock (CCC) disruption impacts cardiac small RNA species. Accordingly, we collected hearts from cardiomyocyte-specific Bmal1 knockout (CBK; a model of CCC disruption) and littermate control (CON) mice at multiple times of the day, followed by small RNA-seq. The data reveal 47 differentially expressed miRNAs species in CBK hearts. Subsequent bioinformatic analyses predict that differentially expressed miRNA species in CBK hearts influence processes such as circadian rhythmicity, cellular signaling, and metabolism. Of the induced miRNAs in CBK hearts, 7 are predicted to be targeted by the transcriptional repressors REV-ERBα/β (integral circadian clock components that are directly regulated by BMAL1). Similar to CBK hearts, cardiomyocyte-specific Rev-erbα/β double knockout (CM-RevDKO) mouse hearts exhibit increased let-7c-1-3p, miR-23b-5p, miR-139-3p, miR-5123, and miR-7068-3p levels. Importantly, 19 putative targets of these 5 miRNAs are commonly repressed in CBK and CM-RevDKO heart (of which 16 are targeted by let-7c-1-3p). These observations suggest that disruption of the circadian BMAL1–REV-ERBα/β regulatory network in the heart induces distinct miRNAs, whose mRNA targets impact critical cellular functions.

Functional characterization of the CRY2 circadian clock component variant p.Ser420Phe revealed a new degradation pathway for CRY2

Cryptochromes (CRYs) are essential components of the circadian clock, playing a pivotal role as transcriptional repressors. Despite their significance, the precise mechanisms underlying CRYs' involvement in the circadian clock remain incompletely understood. In this study, we identified a rare CRY2 variant, p.Ser420Phe, from the 1000 Genomes Project and Ensembl database that is located in the functionally important coiled-coil-like helix (CC−helix) region. Functional characterization of this variant at the cellular level revealed that p.Ser420Phe CRY2 had reduced repression activity on CLOCK:BMAL1−driven transcription due to its reduced affinity to the core clock protein PER2 and defective translocation into the nucleus. Intriguingly, the CRY2 variant exhibited an unexpected resistance to degradation via the canonical proteasomal pathway, primarily due to the loss of interactions with E3 ligases (FBXL3 and FBXL21), which suggests Ser-420 of CRY2 is required for the interaction with E3 ligases. Further studies revealed that wild type and CRY2 variant are degraded by the lysosomal-mediated degradation pathway, a mechanism not previously associated with CRY2. Surprisingly, our complementation study with Cry1−/−Cry2−/− double knockout mouse embryonic fibroblast cells indicated that CRY2 variant caused 7 hours shorter circadian period length in contrast to the observed prolonged period length in CRY2−/− cell lines. In summary, this study reveals a hitherto unknown degradation pathway for CRY2, shedding new light on the regulation of circadian rhythm period length.

Assessing the impacts of genetic defects on starch metabolism in Arabidopsis plants using the carbon homeostasis model.

Starch serves as an important carbon storage mechanism for many plant species, facilitating their adaptation to the cyclic variations in the light environment, including day-night cycles as well as seasonal changes in photoperiod. By dynamically adjusting starch accumulation and degradation rates, plants maintain carbon homeostasis, enabling continuous growth under fluctuating environmental conditions. To understand dynamic nature of starch metabolism at the molecular level, it is necessary to integrate empirical knowledge from genetic defects in specific regulatory pathways into the dynamical system of starch metabolism. To achieve this, we evaluated the impact of genetic defects in the circadian clock, sugar sensing and starch degradation pathways using the carbon homeostasis model that encompasses the interplay between these pathways. Through the collection of starch metabolism data from 10 Arabidopsis mutants, we effectively fitted the experimental data to the model. The system-level assessment revealed that genetic defects in both circadian clock components and sugar sensing pathway hindered the appropriate adjustment of the starch degradation rate, particularly under long-day conditions. These findings not only confirmed the previous empirical findings but also provide the novel insights into the role of each gene within the gene regulatory network on the emergence of carbon homeostasis.

The Molecular Circadian Clock Is a Target of Anti-cancer Translation Inhibitors.

Circadian-paced biological processes are key to physiology and required for metabolic, immunologic, and cardiovascular homeostasis. Core circadian clock components are transcription factors whose half-life is precisely regulated, thereby controlling the intrinsic cellular circadian clock. Genetic disruption of molecular clock components generally leads to marked pathological events phenotypically affecting behavior and multiple aspects of physiology. Using a transcriptional signature similarity approach, we identified anti-cancer protein synthesis inhibitors as potent modulators of the cardiomyocyte molecular clock. Eukaryotic protein translation inhibitors, ranging from translation initiation (rocaglates, 4-EGI1, etc.) to ribosomal elongation inhibitors (homoharringtonine, puromycin, etc.), were found to potently ablate protein abundance of REV-ERBα, a repressive nuclear receptor and component of the molecular clock. These inhibitory effects were observed both in vitro and in vivo and could be extended to PER2, another component of the molecular clock. Taken together, our observations suggest that the activity spectrum of protein synthesis inhibitors, whose clinical use is contemplated not only in cancers but also in viral infections, must be extended to circadian rhythm disruption, with potential beneficial or iatrogenic effects upon acute or prolonged administration.

BMAL1 modulates senescence programming via AP-1.

Cellular senescence and circadian dysregulation are biological hallmarks of aging. Whether they are coordinately regulated has not been thoroughly studied. We hypothesize that BMAL1, a pioneer transcription factor and master regulator of the molecular circadian clock, plays a role in the senescence program. Here, we demonstrate BMAL1 is significantly upregulated in senescent cells and has altered rhythmicity compared to non-senescent cells. Through BMAL1-ChIP-seq, we show that BMAL1 is uniquely localized to genomic motifs associated with AP-1 in senescent cells. Integration of BMAL1-ChIP-seq data with RNA-seq data revealed that BMAL1 presence at AP-1 motifs is associated with active transcription. Finally, we showed that BMAL1 contributes to AP-1 transcriptional control of key features of the senescence program, including altered regulation of cell survival pathways, and confers resistance to drug-induced apoptosis. Overall, these results highlight a previously unappreciated role of the core circadian clock component BMAL1 on the molecular phenotype of senescent cells.

An intrinsically disordered region controlling condensation of a circadian clock component and rhythmic transcription in the liver

Circadian gene transcription is fundamental to metabolic physiology. Here we report that the nuclear receptor REV-ERBα, a repressive component of the molecular clock, forms circadian condensates in the nuclei of mouse liver. These condensates are dictated by an intrinsically disordered region (IDR) located in the protein's hinge region which specifically concentrates nuclear receptor corepressor 1 (NCOR1) at the genome. IDR deletion diminishes the recruitment of NCOR1 and disrupts rhythmic gene transcription invivo. REV-ERBα condensates are located at high-order transcriptional repressive hubs in the liver genome that are highly correlated with circadian gene repression. Deletion of the IDR disrupts transcriptional repressive hubs and diminishes silencing of target genes by REV-ERBα. This work demonstrates physiological circadian protein condensates containing REV-ERBα whose IDR is required for hub formation and the control of rhythmic gene expression.

Circadian disruption does not alter tumorigenesis in a mouse model of lymphoma.

Background: Disruption of natural light cycles, as experienced by shift workers, is linked to enhanced cancer incidence. Several mouse models of cancer develop more severe disease when exposed to irregular light/dark cycles, supporting the connection between circadian disruption and increased cancer risk. Cryptochrome 2 (CRY2), a repressive component of the molecular circadian clock, facilitates turnover of the oncoprotein c-MYC, one mechanism that may link the molecular clock to tumorigenesis. In Eμ-MYC mice, which express transgenic c-MYC in B cells and develop aggressive lymphomas and leukemia, global Cry2 deletion reduces survival and enhances tumor formation. Lighting conditions that mimic the disruption experienced by shift workers dampen Cry2 transcripts in peripheral tissues of C57BL/6J mice. Although it is milder than homozygous deletion of Cry2, we hypothesized that reduced Cry2 rhythmicity could alter MYC protein accumulation and contribute to enhanced cancer risk caused by circadian disruption. We tested this hypothesis in MYC-driven lymphoma. Methods: We housed Eμ-MYC mice in light-tight boxes set to either control (continuous cycles of 12-hours of light followed by 12-hours of dark, LD12:12) or chronic jetlag (eight-hour light phase advances every two to three days, CJL) lighting conditions and assessed the impact of disrupted light cycles on survival and tumor formation in Eμ-MYC mice. Results: Environmental disruption of circadian rhythms did not alter tumor location, tumor growth, or survival in Eμ-MYC mice. Conclusions: Dampened rhythms of Cry2 following disruption of circadian light exposures is milder than deletion of Cry2. The lack of phenotype caused by altered circadian gene expression in contrast to enhanced tumorigenesis caused by homozygous deletion of Cry2 suggests that CRY2 dosage impacts this model. Importantly, these findings indicate that increased cancer risk associated with circadian disruption arises from one or more mechanisms that are not recapitulated here, and may be different in distinct tumor types.

The deleterious effects and potential therapeutic strategy of fluorene-9-bisphenol on circadian activity and liver diseases in zebrafish and mice

Increasing evidence indicates that disturbance of the clock genes, which leads to systemic endocrine perturbation, plays a crucial role in the pathogenesis of metabolic and liver diseases. Fluorene-9-bisphenol (BHPF) is utilized in the manufacturing of plastic materials but its biological effects on liver homeostasis remain unknown. The impacts and involved mechanisms of BHPF on the liver diseases, metabolism, and circadian clock were comprehensively studied by zebrafish and mouse models. The therapeutic effect of melatonin (MT) was also verified. Zebrafish and mouse models with either acute exposure (0.5 and 1 µmol/L, 1-4 days post-fertilization) or chronic oral exposure (0.5 and 50 mg/(kg·2 days), 30 days) were established with various BHPF concentrations. Herein, we identified a crucial role for estrogenic regulation in liver development and circadian locomotor rhythms damaged by BHPF in a zebrafish model. BHPF mice showed chaos in circadian activity through the imbalance of circadian clock component Brain and Muscle Aryl hydrocarbon receptor nuclear translocator-like 1 in the liver and brain. The liver sexual dimorphic alteration along with reduced growth hormone and estrogens played a critical role in damaged glucose metabolism, hepatic inflammation, and fibrosis induced by BHPF. Besides, sleep improvement by exogenous MT alleviated BHPF‐related glucose metabolism and liver injury in mice. We proposed the pathogenesis of metabolic and liver disease resulting from BHPF and promising targeted therapy for liver metabolism disorders associated with endocrine perturbation chemicals. These results might play a warning role in the administration of endocrine-disrupting chemicals in everyday life and various industry applications.