Circadian Gene Transcription Research Articles

Molecular correlates of swarming behaviour in Aedes aegypti males.

Mosquitoes are the deadliest vectors of diseases. They impose a huge health burden on human populations spreading parasites as disparate as protozoans (malaria), viruses (yellow fever and more) and nematodes (filariasis) that cause life-threatening conditions. In recent years, mating has been proposed as a putative target for population control. Mosquitoes mate mid-air, in swarms initiated by males and triggered by a combination of internal and external stimuli. As the number of females in a swarm is limited, there is intense competition among males, and they 'retune' their physiology for this demanding behaviour. There is limited knowledge on the 'genetic reprogramming' required to enable swarming. Interestingly, recent evidence indicates that the upregulation of circadian clock genes may be involved in the swarming of malaria mosquitoes of the genus Anopheles. Here, we use whole-head RNA-seq to identify gene expression changes in Aedes aegypti males that are engaged in swarming in a laboratory setting. Our results suggest that in preparation to swarming, males tend to lower some housekeeping functions while increasing remodelling of the cytoskeleton and neuronal connectivity; the transcription of circadian clock genes is unaffected.

Circadian gene transcription plays a role in cellular metabolism in hibernating brown bears, Ursus arctos.

Hibernation is a highly seasonal physiological adaptation that allows brown bears (Ursus arctos) to survive extended periods of low food availability. Similarly, daily or circadian rhythms conserve energy by coordinating body processes to optimally match the environmental light/dark cycle. Brown bears express circadian rhythms in vivo and their cells do in vitro throughout the year, suggesting that these rhythms may play important roles during periods of negative energy balance. Here, we use time-series analysis of RNA sequencing data and timed measurements of ATP production in adipose-derived fibroblasts from active and hibernation seasons under two temperature conditions to confirm that rhythmicity was present. Culture temperature matching that of hibernation body temperature (34°C) resulted in a delay of daily peak ATP production in comparison with active season body temperatures (37°C). The timing of peaks of mitochondrial gene transcription was altered as were the amplitudes of transcripts coding for enzymes of the electron transport chain. Additionally, we observed changes in mean expression and timing of key metabolic genes such as SIRT1 and AMPK which are linked to the circadian system and energy balance. The amplitudes of several circadian gene transcripts were also reduced. These results reveal a link between energy conservation and a functioning circadian system in hibernation.

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.

Maternal developmental history alters transfer of circadian clock genes to offspring in Japanese quail (Coturnix japonica)

Maternal signals shape embryonic development, and in turn post-natal phenotypes. RNA deposition is one such method of maternal signalling and circadian rhythms are one trait thought to be maternally inherited, through this mechanism. These maternal circadian gene transcripts aid development of a functioning circadian system. There is increasing evidence that maternal signals can be modified, depending on prevailing environmental conditions to optimise offspring fitness. However, currently, it is unknown if maternal circadian gene transcripts, and consequently early embryonic gene transcription, are altered by maternal developmental conditions. Here, using avian mothers who experienced either pre-natal corticosterone exposure, and/or post-natal stress as juveniles we were able to determine the effects of the timing of stress on downstream circadian RNA deposition in offspring. We demonstrated that maternal developmental history does indeed affect transfer of offspring circadian genes, but the timing of stress was important. Avian mothers who experienced stress during the first 2 weeks of post-natal life increased maternally deposited transcript levels of two core circadian clock genes, BMAL1 and PER2. These differences in transcript levels were transient and disappeared at the point of embryonic genome transcription. Pre-natal maternal stress alone was found to elicit delayed changes in circadian gene expression. After activation of the embryonic genome, both BMAL1 and PER2 expression were significantly decreased. If both pre-natal and post-natal stress occurred, then initial maternal transcript levels of BMAL1 were significantly increased. Taken together, these results suggest that developmental stress differentially produces persistent transgenerational effects on offspring circadian genes.

Dynamic modulation of genomic enhancer elements in the suprachiasmatic nucleus, the site of the mammalian circadian clock.

The mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, synchronizes and maintains daily cellular and physiological rhythms across the body, in accordance with environmental and visceral cues. Consequently, the systematic regulation of spatiotemporal gene transcription in the SCN is vital for daily timekeeping. So far, the regulatory elements assisting circadian gene transcription have only been studied in peripheral tissues, lacking the critical neuronal dimension intrinsic to the role of the SCN as central brain pacemaker. By using histone-ChIP-seq, we identified SCN-enriched gene regulatory elements that associated with temporal gene expression. Based on tissue-specific H3K27ac and H3K4me3 marks, we successfully produced the first-ever SCN gene-regulatory map. We found that a large majority of SCN enhancers not only show robust 24-h rhythmic modulation in H3K27ac occupancy, peaking at distinct times of day, but also possess canonical E-box (CACGTG) motifs potentially influencing downstream cycling gene expression. To establish enhancer-gene relationships in the SCN, we conducted directional RNA-seq at six distinct times across the day and night, and studied the association between dynamically changing histone acetylation and gene transcript levels. About 35% of the cycling H3K27ac sites were found adjacent to rhythmic gene transcripts, often preceding the rise in mRNA levels. We also noted that enhancers encompass noncoding, actively transcribing enhancer RNAs (eRNAs) in the SCN, which in turn oscillate, along with cyclic histone acetylation, and correlate with rhythmic gene transcription. Taken together, these findings shed light on genome-wide pretranscriptional regulation operative in the central clock that confers its precise and robust oscillation necessary to orchestrate daily timekeeping in mammals.

Is obstructive sleep apnea a circadian rhythm disorder?

Obstructive sleep apnea is the most common sleep-related breathing disorder worldwide and remains underdiagnosed. Its multiple associated comorbidities contribute to a decreased quality of life and work performance as well as an increased risk of death. Standard treatment seems to have limited effects on cardiovascular and metabolic aspects of the disease, emphasising the need for early diagnosis and additional therapeutic approaches. Recent evidence suggests that the dysregulation of circadian rhythms, processes with endogenous rhythmicity that are adjusted to the environment through various cues, is involved in the pathogenesis of comorbidities. In patients with obstructive sleep apnea, altered circadian gene expression patterns have been demonstrated. Obstructive respiratory events may promote circadian dysregulation through the effects of sleep disturbance and intermittent hypoxia, with subsequent inflammation and disruption of neural and hormonal homeostasis. In this review, current knowledge on obstructive sleep apnea, circadian rhythm regulation, and circadian rhythm sleep disorders is summarised. Studies that connect obstructive sleep apnea to circadian rhythm abnormalities are critically evaluated. Furthermore, pathogenetic mechanisms that may underlie this association, most notably hypoxia signalling, are presented. A bidirectional relationship between obstructive sleep apnea and circadian rhythm dysregulation is proposed. Approaching obstructive sleep apnea as a circadian rhythm disorder may prove beneficial for the development of new, personalised diagnostic, therapeutic and prognostic tools. However, further studies are needed before the clinical approach to obstructive sleep apnea includes targeting the circadian system.

Myeloid deficiency of the intrinsic clock protein BMAL1 accelerates cognitive aging by disrupting microglial synaptic pruning

Aging is associated with loss of circadian immune responses and circadian gene transcription in peripheral macrophages. Microglia, the resident macrophages of the brain, also show diurnal rhythmicity in regulating local immune responses and synaptic remodeling. To investigate the interaction between aging and microglial circadian rhythmicity, we examined mice deficient in the core clock transcription factor, BMAL1. Aging Cd11bcre;Bmallox/lox mice demonstrated accelerated cognitive decline in association with suppressed hippocampal long-term potentiation and increases in immature dendritic spines. C1q deposition at synapses and synaptic engulfment were significantly decreased in aging Bmal1-deficient microglia, suggesting that BMAL1 plays a role in regulating synaptic pruning in aging. In addition to accelerated age-associated hippocampal deficits, Cd11bcre;Bmallox/lox mice also showed deficits in the sleep–wake cycle with increased wakefulness across light and dark phases. These results highlight an essential role of microglial BMAL1 in maintenance of synapse homeostasis in the aging brain.

Co-alterations of circadian clock gene transcripts in human placenta in preeclampsia

Pre-eclampsia (PE) is a hypertensive condition that occurs during pregnancy and complicates up to 4% of pregnancies. PE exhibits several circadian-related characteristics, and the placenta possesses a functioning molecular clock. We examined the associations of 17 core circadian gene transcripts in placenta with PE vs. non-PE (a mixture of pregnant women with term, preterm, small-for-gestational-age, or chorioamnionitis) using two independent gene expression datasets: GSE75010-157 (80 PE vs. 77 non-PE) and GSE75010-173 (77 PE and 96 non-PE). We found a robust difference in circadian gene expression between PE and non-PE across the two datasets, where CRY1 mRNA increases and NR1D2 and PER3 transcripts decrease in PE placenta. Gene set variation analysis revealed an interplay between co-alterations of circadian clock genes and PE with altered hypoxia, cell migration/invasion, autophagy, and membrane trafficking pathways. Using human placental trophoblast HTR-8 cells, we show that CRY1/2 and NR1D1/2 regulate trophoblast migration. A subgroup study including only term samples demonstrated that CLOCK, NR1D2, and PER3 transcripts were simultaneously decreased in PE placenta, a finding supported by CLOCK protein downregulation in an independent cohort of human term PE placenta samples. These findings provide novel insights into the roles of the molecular clock in the pathogenesis of PE.

Express and Purify Human CRY2 for Functional Analyses

The circadian rhythm is a biological clock responsible for regulating the daily cycles of essential physiological and metabolic processes, such as sleep wake cycles, by responding to light and dark changes. Every cell has its own circadian rhythm that is synchronized by a master clock in the brain. At the cellular level, circadian rhythms are controlled by a time‐delayed transcriptional‐translational feed‐back loop (TTFL), which consists of several core proteins. These proteins include circadian locomotor output cycles protein kaput (CLOCK), brain and muscle ARNT‐like 1 (BMAL1), cryptochrome (CRY), and period (PER). CLOCK and BMAL1 initiate the TTFL by activating the transcription of several circadian genes including CRYs and PERs that will be translated in the cytosol. With accumulation after a certain time, CRYs/PERs complexes will translocate back to the nucleus to negatively inhibit CLOCK/BMAL1 activities to finish the TTFL. There are two paralogs of CRY within the human genome: CRY1 and CRY2, both of which have essential roles in the negative feedback. The purpose of this project is to study the structure and function of human CRY2. Regardless of many advances in this field including available structure of CRYs, the detailed interaction between CRY2 to other circadian core proteins are still limited. Through this research we would be able to express and purify large quantities of hCRY2 that will be used for the future interaction studies. The results will deepen our understanding of the mechanisms behind circadian rhythm and its associated diseases, facilitating the development of prevention and treatment strategies.

Aging disrupts circadian gene regulation and function in macrophages.

Aging is characterized by an increased vulnerability to infection and the development of inflammatory diseases, such as atherosclerosis, frailty, cancer and neurodegeneration. Here, we find that aging is associated with the loss of diurnally rhythmic innate immune responses, including monocyte trafficking from bone marrow to blood, response to lipopolysaccharide and phagocytosis. This decline in homeostatic immune responses was associated with a striking disappearance of circadian gene transcription in aged compared to young tissue macrophages. Chromatin accessibility was significantly greater in young macrophages than in aged macrophages; however, this difference did not explain the loss of rhythmic gene transcription in aged macrophages. Rather, diurnal expression of Kruppel-like factor 4 (Klf4), a transcription factor (TF) well established in regulating cell differentiation and reprogramming, was selectively diminished in aged macrophages. Ablation of Klf4 expression abolished diurnal rhythms in phagocytic activity, recapitulating the effect of aging on macrophage phagocytosis. Examination of individuals harboring genetic variants of KLF4 revealed an association with age-dependent susceptibility to death caused by bacterial infection. Our results indicate that loss of rhythmic Klf4 expression in aged macrophages is associated with disruption of circadian innate immune homeostasis, a mechanism that may underlie age-associated loss of protective immune responses.

A spatio-temporal model to reveal oscillator phenotypes in molecular clocks: Parameter estimation elucidates circadian gene transcription dynamics in single-cells.

We propose a stochastic distributed delay model together with a Markov random field prior and a measurement model for bioluminescence-reporting to analyse spatio-temporal gene expression in intact networks of cells. The model describes the oscillating time evolution of molecular mRNA counts through a negative transcriptional-translational feedback loop encoded in a chemical Langevin equation with a probabilistic delay distribution. The model is extended spatially by means of a multiplicative random effects model with a first order Markov random field prior distribution. Our methodology effectively separates intrinsic molecular noise, measurement noise, and extrinsic noise and phenotypic variation driving cell heterogeneity, while being amenable to parameter identification and inference. Based on the single-cell model we propose a novel computational stability analysis that allows us to infer two key characteristics, namely the robustness of the oscillations, i.e. whether the reaction network exhibits sustained or damped oscillations, and the profile of the regulation, i.e. whether the inhibition occurs over time in a more distributed versus a more direct manner, which affects the cells’ ability to phase-shift to new schedules. We show how insight into the spatio-temporal characteristics of the circadian feedback loop in the suprachiasmatic nucleus (SCN) can be gained by applying the methodology to bioluminescence-reported expression of the circadian core clock gene Cry1 across mouse SCN tissue. We find that while (almost) all SCN neurons exhibit robust cell-autonomous oscillations, the parameters that are associated with the regulatory transcription profile give rise to a spatial division of the tissue between the central region whose oscillations are resilient to perturbation in the sense that they maintain a high degree of synchronicity, and the dorsal region which appears to phase shift in a more diversified way as a response to large perturbations and thus could be more amenable to entrainment.

In silico investigation of the mechanism of action of kojic acid effects via protein-protein interaction network

Purpose: To evaluate the molecular mechanism of kojic acid by network pharmacology.Methods: This study was conducted by designing a protein-protein interaction network through the STITCH database and analyzing biological processes via Cytoscape plugin ClueGO.Results: A total of 19 protein targets of kojic acid including TYR, NOS3, NOS2, and NOS1 were found. The PPI network helped to understand the mode of action of kojic acid at a molecular level. Gene Ontology (GO) analysis resulted in the retrieval of 104 GO terms which were related to variousphysiological processes. GO analysis revealed that kojic acid might be involved in the regulation of several biological processes such as circadian gene expression and transcription initiation of RNA polymerase 2.Conclusion: The findings from this study reveal that the retrieved GO pathways are known to be involved in several diseases such as inflammation, cancer, aging, pigmentation, and melisma. Furthermore, these pathways are directly or indirectly related to kojic acid. Thus, this study has contributed to a better understanding of the mode of action of kojic acid.

Low CLOCK and CRY2 in 2nd trimester human maternal blood and risk of preterm birth: a nested case-control study†.

Previous studies have observed an association between maternal circadian rhythm disruption and preterm birth (PTB). However, the underlying molecular mechanisms and the potential of circadian clock genes to serve as predictors of PTB remain unexplored. We examined the association of 10 core circadian transcripts in maternal blood with spontaneous PTB (sPTB) vs term births using a nested case-control study design. We used a public gene expression dataset (GSE59491), which was nested within the All Our Babies (AOB) study cohort in Canada. Maternal blood was sampled in Trimesters 2-3 from women with sPTB (n = 51) and term births (n = 106), matched for five demographic variables. In 2nd trimester maternal blood, only CLOCK and CRY2 transcripts were significantly lower in sPTB vs term (P = 0.02-0.03, false discovery rate (FDR) < 0.20). A change of PER3 mRNA from trimesters 2-3 was significantly associated with sPTB (decline in sPTB, P = 0.02, FDR < 0.20). When CLOCK and CRY2 were modeled together in 2nd trimester blood, the odds of being in the low level of both circadian gene transcripts was greater in sPTB vs term (OR = 4.86, 95%CI = (1.75,13.51), P < 0.01). Using GSVA and Pearson correlation, we identified 98 common pathways that were negatively or positively correlated with CLOCK and CRY2 expression (all P < 0.05, FDR < 0.10). The top three identified pathways were amyotrophic lateral sclerosis, degradation of extracellular matrix, and inwardly rectifying potassium channels. These three processes have previously been shown to be involved in neuron death, parturition, and uterine excitability during pregnancy, respectively.

The global and promoter-centric 3D genome organization temporally resolved during a circadian cycle

BackgroundCircadian gene expression is essential for organisms to adjust their physiology and anticipate daily changes in the environment. The molecular mechanisms controlling circadian gene transcription are still under investigation. In particular, how chromatin conformation at different genomic scales and regulatory elements impact rhythmic gene expression has been poorly characterized.ResultsHere we measure changes in the spatial chromatin conformation in mouse liver using genome-wide and promoter-capture Hi-C alongside daily oscillations in gene transcription. We find topologically associating domains harboring circadian genes that switch assignments between the transcriptionally active and inactive compartment at different hours of the day, while their boundaries stably maintain their structure over time. To study chromatin contacts of promoters at high resolution over time, we apply promoter capture Hi-C. We find circadian gene promoters displayed a maximal number of chromatin contacts at the time of their peak transcriptional output. Furthermore, circadian genes, as well as contacted and transcribed regulatory elements, reach maximal expression at the same timepoints. Anchor sites of circadian gene promoter loops are enriched in DNA binding sites for liver nuclear receptors and other transcription factors, some exclusively present in either rhythmic or stable contacts. Finally, by comparing the interaction profiles between core clock and output circadian genes, we show that core clock interactomes are more dynamic compared to output circadian genes.ConclusionOur results identify chromatin conformation dynamics at different scales that parallel oscillatory gene expression and characterize the repertoire of regulatory elements that control circadian gene transcription through rhythmic or stable chromatin configurations.

Disruption of Circadian Transcriptome in Lung by Acute Sleep Deprivation.

Inadequate sleep prevails in modern society and it impairs the circadian transcriptome. However, to what extent acute sleep deprivation (SD) has impact on the circadian rhythms of peripheral tissues is not clear. Here, we show that in mouse lung, a 10-h acute sleep deprivation can alter the circadian expression of approximately 3,000 genes. We found that circadian rhythm disappears in genes related to metabolism and signaling pathways regulating protein phosphorylation after acute sleep deprivation, while the core circadian regulators do not change much in rhythmicity. Importantly, the strong positive correlation between mean expression and amplitude (E-A correlation) of cycling genes has been validated in both control and sleep deprivation conditions, supporting the energetic cost optimization model of circadian gene expression. Thus, we reveal that acute sleep deprivation leads to a profound change in the circadian gene transcription that influences the biological functions in lung.

Molecular mechanism of the repressive phase of the mammalian circadian clock

The mammalian circadian clock consists of a transcription-translation feedback loop (TTFL) composed of CLOCK-BMAL1 transcriptional activators and CRY-PER transcriptional repressors. Previous work showed that CRY inhibits CLOCK-BMAL1-activated transcription by a "blocking"-type mechanism and that CRY-PER inhibits CLOCK-BMAL1 by a "displacement"-type mechanism. While the mechanism of CRY-mediated repression was explained by both in vitro and in vivo experiments, the CRY-PER-mediated repression in vivo seemed in conflict with the in vitro data demonstrating PER removes CRY from the CLOCK-BMAL1-E-box complex. Here, we show that CRY-PER participates in the displacement-type repression by recruiting CK1δ to the nucleus and mediating an increased local concentration of CK1δ at CLOCK-BMAL1-bound promoters/enhancers and thus promoting the phosphorylation of CLOCK and dissociation of CLOCK-BMAL1 along with CRY from the E-box. Our findings bring clarity to the role of PER in the dynamic nature of the repressive phase of the TTFL.

Using GRO-Seq to Measure Circadian Transcription and Discover Circadian Enhancers.

Circadian gene transcription transmits timing information and drives cyclic physiological processes across various tissues. Recent studies indicate that oscillating enhancer activity is a major driving force of rhythmic gene transcription. Functional circadian enhancers can be identified in an unbiased manner by correlation with the rhythms of nearby gene transcription.Global run-on sequencing (GRO-seq) measures nascent transcription of both pre-mRNAs and enhancer RNAs (eRNAs) at a genome-wide level, making it a unique tool for unraveling complex gene regulation mechanisms in vivo. Here, we describe a comprehensive protocol, ranging from wet lab to in silico analysis, for detecting and quantifying circadian transcription of genes and eRNAs. Moreover, using gene-eRNA correlation, we detail the steps necessary to identify functional enhancers and transcription factors (TFs) that control circadian gene expression in vivo. While we use mouse liver as an example, this protocol is applicable for multiple tissues.

The CBP KIX domain regulates long-term memory and circadian activity

BackgroundCREB-dependent transcription necessary for long-term memory is driven by interactions with CREB-binding protein (CBP), a multi-domain protein that binds numerous transcription factors potentially affecting expression of thousands of genes. Identifying specific domain functions for multi-domain proteins is essential to understand processes such as cognitive function and circadian clocks. We investigated the function of the CBP KIX domain in hippocampal memory and gene expression using CBPKIX/KIX mice with mutations that prevent phospho-CREB (Ser133) binding.ResultsWe found that CBPKIX/KIX mice were impaired in long-term memory, but not learning acquisition or short-term memory for the Morris water maze. Using an unbiased analysis of gene expression in the dorsal hippocampus after training in the Morris water maze or contextual fear conditioning, we discovered dysregulation of CREB, CLOCK, and BMAL1 target genes and downregulation of circadian genes in CBPKIX/KIX mice. Given our finding that the CBP KIX domain was important for transcription of circadian genes, we profiled circadian activity and phase resetting in CBPKIX/KIX mice. CBPKIX/KIX mice exhibited delayed activity peaks after light offset and longer free-running periods in constant dark. Interestingly, CBPKIX/KIX mice displayed phase delays and advances in response to photic stimulation comparable to wildtype littermates. Thus, this work delineates site-specific regulation of the circadian clock by a multi-domain protein.ConclusionsThese studies provide insight into the significance of the CBP KIX domain by defining targets of CBP transcriptional co-activation in memory and the role of the CBP KIX domain in vivo on circadian rhythms.Graphical abstract

ARID1A-dependent permissive chromatin accessibility licenses estrogen-receptor signaling to regulate circadian rhythms genes in endometrial cancer.

Estrogen receptor α (ER) acts as an oncogenic signal in endometrial endometrioid carcinoma. ER binding activity largely depends on chromatin remodeling and recruitment of transcription factors to estrogen response elements. A deeper understanding of these regulatory mechanisms may uncover therapeutic targets for ER-dependent endometrial cancers. We show that estrogen induces accessible chromatin and ER binding at a subset of enhancers, which form higher-order super enhancers that are vital for ER signaling. ER positively correlates with active enhancers in primary tumors, and tumors were effectively classified into molecular subtypes with chromatin accessibility dynamics and ER-dependent gene signature. ARID1A binds within ER-bound enhancers and regulates ER-dependent transcription. Knockdown of ARID1A or fulvestrant treatment profoundly affects the gene-expression program, and inhibits cell growth phenotype by affecting the chromatin environment. Importantly, we found dysregulated expression of circadian rhythms genes by estrogen in cancer cells and in primary tumors. Knockdown of ARID1A reduces the chromatin accessibility and ER binding at enhancers of the circadian gene ARNTL and BHLHE41, leading to a decreased expression of these genes. Altogether, we uncover a critical role for ARID1A in ER signaling and therapeutic target in ER-positive endometrial cancer.

Full moonlight-induced circadian clock entrainment in Coffea arabica

BackgroundIt is now well documented that moonlight affects the life cycle of invertebrates, birds, reptiles, and mammals. The lunisolar tide is also well-known to alter plant growth and development. However, although plants are known to be very photosensitive, few studies have been undertaken to explore the effect of moonlight on plant physiology.ResultsHere for the first time we report a massive transcriptional modification in Coffea arabica genes under full moonlight conditions, particularly at full moon zenith and 3 h later. Among the 3387 deregulated genes found in our study, the main core clock genes were affected.ConclusionsMoonlight also negatively influenced many genes involved in photosynthesis, chlorophyll biosynthesis and chloroplast machinery at the end of the night, suggesting that the full moon has a negative effect on primary photosynthetic machinery at dawn. Moreover, full moonlight promotes the transcription of major rhythmic redox genes and many heat shock proteins, suggesting that moonlight is perceived as stress. We confirmed this huge impact of weak light (less than 6 lx) on the transcription of circadian clock genes in controlled conditions mimicking full moonlight.