Time-course RNASeq of Camponotus floridanus forager and nurse ant brains indicate links between plasticity in the biological clock and behavioral division of labor

Background Circadian clocks allow organisms to anticipate daily fluctuations in their environment by driving rhythms in physiology and behavior. Inter-organismal differences in daily rhythms, called chronotypes, exist and can shift with age. In ants, age, caste-related behavior and chronotype appear to be linked. Brood-tending nurse ants are usually younger individuals and show “around-the-clock” activity. With age or in the absence of brood, nurses transition into foraging ants that show daily rhythms in activity. Ants can adaptively shift between these behavioral castes and caste-associated chronotypes depending on social context. We investigated how changes in daily gene expression could be contributing to such behavioral plasticity in Camponotus floridanus carpenter ants by combining time-course behavioral assays and RNA-Sequencing of forager and nurse brains. Results We found that nurse brains have three times fewer 24 h oscillating genes than foragers. However, several hundred genes that oscillated every 24 h in forager brains showed robust 8 h oscillations in nurses, including the core clock genes Period and Shaggy. These differentially rhythmic genes consisted of several components of the circadian entrainment and output pathway, including genes said to be involved in regulating insect locomotory behavior. We also found that Vitellogenin, known to regulate division of labor in social insects, showed robust 24 h oscillations in nurse brains but not in foragers. Finally, we found significant overlap between genes differentially expressed between the two ant castes and genes that show ultradian rhythms in daily expression. Conclusion This study provides a first look at the chronobiological differences in gene expression between forager and nurse ant brains. This endeavor allowed us to identify a putative molecular mechanism underlying plastic timekeeping: several components of the ant circadian clock and its output can seemingly oscillate at different harmonics of the circadian rhythm. We propose that such chronobiological plasticity has evolved to allow for distinct regulatory networks that underlie behavioral castes, while supporting swift caste transitions in response to colony demands. Behavioral division of labor is common among social insects. The links between chronobiological and behavioral plasticity that we found in C. floridanus, thus, likely represent a more general phenomenon that warrants further investigation.

EARLY FLOWERING 3 and Photoperiod Sensing in Brachypodium distachyon

The proper timing of flowering, which is key to maximize reproductive success and yield, relies in many plant species on the coordination between environmental cues and endogenous developmental programs. The perception of changes in day length is one of the most reliable cues of seasonal change, and this involves the interplay between the sensing of light signals and the circadian clock. Here, we describe a Brachypodium distachyon mutant allele of the evening complex protein EARLY FLOWERING 3 (ELF3). We show that the elf3 mutant flowers more rapidly than wild type plants in short days as well as under longer photoperiods but, in very long (20 h) days, flowering is equally rapid in elf3 and wild type. Furthermore, flowering in the elf3 mutant is still sensitive to vernalization, but not to ambient temperature changes. Molecular analyses revealed that the expression of a short-day marker gene is suppressed in elf3 grown in short days, and the expression patterns of clock genes and flowering time regulators are altered. We also explored the mechanisms of photoperiodic perception in temperate grasses by exposing B. distachyon plants grown under a 12 h photoperiod to a daily night break consisting of a mixture of red and far-red light. We showed that 2 h breaks are sufficient to accelerate flowering in B. distachyon under non-inductive photoperiods and that this acceleration of flowering is mediated by red light. Finally, we discuss advances and perspectives for research on the perception of photoperiod in temperate grasses.

Diurnal RNAPII-tethered chromatin interactions are associated with rhythmic gene expression in rice

Background The daily cycling of plant physiological processes is speculated to arise from the coordinated rhythms of gene expression. However, the dynamics of diurnal 3D genome architecture and their potential functions underlying the rhythmic gene expression remain unclear. Results Here, we reveal the genome-wide rhythmic occupancy of RNA polymerase II (RNAPII), which precedes mRNA accumulation by approximately 2 h. Rhythmic RNAPII binding dynamically correlates with RNAPII-mediated chromatin architecture remodeling at the genomic level of chromatin interactions, spatial clusters, and chromatin connectivity maps, which are associated with the circadian rhythm of gene expression. Rhythmically expressed genes within the same peak phases of expression are preferentially tethered by RNAPII for coordinated transcription. RNAPII-associated chromatin spatial clusters (CSCs) show high plasticity during the circadian cycle, and rhythmically expressed genes in the morning phase and non-rhythmically expressed genes in the evening phase tend to be enriched in RNAPII-associated CSCs to orchestrate expression. Core circadian clock genes are associated with RNAPII-mediated highly connected chromatin connectivity networks in the morning in contrast to the scattered, sporadic spatial chromatin connectivity in the evening; this indicates that they are transcribed within physical proximity to each other during the AM circadian window and are located in discrete “transcriptional factory” foci in the evening, linking chromatin architecture to coordinated transcription outputs. Conclusion Our findings uncover fundamental diurnal genome folding principles in plants and reveal a distinct higher-order chromosome organization that is crucial for coordinating diurnal dynamics of transcriptional regulation.

Disturbance of the Circadian System in Shift Work and Its Health Impact

The various non-standard schedules required of shift workers force abrupt changes in the timing of sleep and light-dark exposure. These changes result in disturbances of the endogenous circadian system and its misalignment with the environment. Simulated night-shift experiments and field-based studies with shift workers both indicate that the circadian system is resistant to adaptation from a day- to a night-oriented schedule, as determined by a lack of substantial phase shifts over multiple days in centrally controlled rhythms, such as those of melatonin and cortisol. There is evidence that disruption of the circadian system caused by night-shift work results not only in a misalignment between the circadian system and the external light-dark cycle, but also in a state of internal desynchronization between various levels of the circadian system. This is the case between rhythms controlled by the central circadian pacemaker and clock genes expression in tissues such as peripheral blood mononuclear cells, hair follicle cells, and oral mucosa cells. The disruptive effects of atypical work schedules extend beyond the expression profile of canonical circadian clock genes and affects other transcripts of the human genome. In general, after several days of living at night, most rhythmic transcripts in the human genome remain adjusted to a day-oriented schedule, with dampened group amplitudes. In contrast to circadian clock genes and rhythmic transcripts, metabolomics studies revealed that most metabolites shift by several hours when working nights, thus leading to their misalignment with the circadian system. Altogether, these circadian and sleep-wake disturbances emphasize the all-encompassing impact of night-shift work, and can contribute to the increased risk of various medical conditions. Here, we review the latest scientific evidence regarding the effects of atypical work schedules on the circadian system, sleep and alertness of shift-working populations, and discuss their potential clinical impacts.

Machine Learning and In-Vivo Expression Analyses Reveal Circadian Clock Features Predictive of Anxiety in Humans

Mood disorders, including anxiety, are associated with disruptions in circadian rhythms and are linked to polymorphisms in circadian clock genes. Molecular mechanisms underlying these connections may be direct—via transcriptional activity of clock genes on downstream mood pathways in the brain, or indirect—via clock gene influences on the phase and amplitude of circadian rhythms which, in turn, modulate physiological processes influencing mood. Employing machine learning combined with statistical approaches, we explored clock genotype combinations that predict risk for anxiety symptoms in a deeply phenotyped population. We identified multiple novel circadian genotypes predictive of anxiety, with the PER3B-AG/CRY1-CG genotype being the strongest predictor of anxiety risk in males. Molecular chronotyping, using clock gene expression oscillations, revealed that advanced circadian phase and robust circadian amplitudes are associated with high levels of anxiety symptoms. Further analyses revealed that individuals with advanced phases and pronounced circadian misalignment were at higher risk for severe anxiety symptoms. Our results support both direct and indirect influences of clock gene variants on mood: while sex-specific clock genotype combinations predictive of anxiety symptoms suggest direct effects on mood pathways, the mediation of PER3B effects on anxiety via diurnal preference measures and the association of circadian phase with anxiety symptoms provide evidence for indirect effects of the molecular clockwork on mood. Unraveling the complex molecular mechanisms underlying the links between circadian physiology and mood is essential to identifying the core clock genes to target in future functional studies, thereby advancing the development of non-invasive treatments for anxiety-related disorders.

GUN4 Affects the Circadian Clock and Seedlings Adaptation to Changing Light Conditions

The chloroplast is a key organelle for photosynthesis and perceiving environmental information. GENOME UNCOUPLED 4 (GUN4) has been shown to be required for the regulation of both chlorophyll synthesis, reactive oxygen species (ROS) homeostasis and plastid retrograde signaling. In this study, we found that growth of the gun4 mutant was significantly improved under medium strong light (200 μmol photons m−2s−1) compared to normal light (100 μmol photons m−2s−1), in marked contrast to wild-type (WT). Further analysis revealed that GUN4 interacts with SIGNAL RECOGNITION PARTICLE 54 KDA SUBUNIT (SRP43) and SRP54. RNA-seq analysis indicated that the expression of genes for light signaling and the circadian clock is altered in gun4 compared with (WT). qPCR analysis confirmed that the expression of the clock genes CLOCK-RELATED 1 (CCA1), LATE ELONGATION HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1) and PSEUDO RESPONSE REGULATOR 7 (PRR7) is significantly changed in the gun4 and srp54 mutants under normal and medium strong light conditions. These results suggest that GUN4 may coordinate the adaptation of plants to changing light conditions by regulating the biological clock, although it is not clear whether the effect is direct or indirect.

Expression Patterns of Clock Gene mRNAs and Clock Proteins in Human Psoriatic Skin Samples

Psoriasis is a systemic inflammatory skin disorder that can be associated with sleep disturbance and negatively influence the daily rhythm. The link between the pathomechanism of psoriasis and the circadian rhythm has been suggested by several previous studies. However, there are insufficient data on altered clock mechanisms in psoriasis to prove these theories. Therefore, we investigated the expression of the core clock genes in human psoriatic lesional and non-lesional skin and in human adult low calcium temperature (HaCaT) keratinocytes after stimulation with pro-inflammatory cytokines. Furthermore, we examined the clock proteins in skin biopsies from psoriatic patients by immunohistochemistry. We found that the clock gene transcripts were elevated in psoriatic lesions, especially in non-lesional psoriatic areas, except for rev-erbα, which was consistently downregulated in the psoriatic samples. In addition, the REV-ERBα protein showed a different epidermal distribution in non-lesional skin than in healthy skin. In cytokine-treated HaCaT cells, changes in the amplitude of the bmal1, cry1, rev-erbα and per1 mRNA oscillation were observed, especially after TNFα stimulation. In conclusion, in our study a perturbation of clock gene transcripts was observed in uninvolved and lesional psoriatic areas compared to healthy skin. These alterations may serve as therapeutic targets and facilitate the development of chronotherapeutic strategies in the future.