Rhythmic Transcription Research Articles

Dynamic Inventory of Intermediate Metabolites of Cyanobacteria in a Diurnal Cycle.

SummaryCyanobacteria are gaining importance both as hosts for photoautotrophic production of chemicals and as model systems for studies of diurnal lifestyle. The proteome and transcriptome of cyanobacteria have been closely examined under diurnal growth, whereas the downstream effects on the intermediary metabolism have not received sufficient attention. The present study focuses on identifying the cellular metabolites whose inventories undergo dramatic changes in a fast-growing cyanobacterium, Synechococcus elongatus PCC 11801. We identified and quantified 67 polar metabolites, whose inventory changes significantly during diurnal growth, with some metabolites changing by 100-fold. The Calvin-Benson-Bassham cycle intermediates peak at midday to support fast growth. The hitherto unexplored γ-glutamyl peptides act as reservoirs of amino acids. Interestingly, several storage molecules or their precursors accumulate during the dark phase, dispelling the notion that all biosynthetic activity takes place in the light phase. Our results will guide metabolic modeling and strain engineering of cyanobacteria.

MOSAIC: a joint modeling methodology for combined circadian and non-circadian analysis of multi-omics data.

Circadian rhythms are approximately 24-h endogenous cycles that control many biological functions. To identify these rhythms, biological samples are taken over circadian time and analyzed using a single omics type, such as transcriptomics or proteomics. By comparing data from these single omics approaches, it has been shown that transcriptional rhythms are not necessarily conserved at the protein level, implying extensive circadian post-transcriptional regulation. However, as proteomics methods are known to be noisier than transcriptomic methods, this suggests that previously identified arrhythmic proteins with rhythmic transcripts could have been missed due to noise and may not be due to post-transcriptional regulation. To determine if one can use information from less-noisy transcriptomic data to inform rhythms in more-noisy proteomic data, and thus more accurately identify rhythms in the proteome, we have created the Multi-Omics Selection with Amplitude Independent Criteria (MOSAIC) application. MOSAIC combines model selection and joint modeling of multiple omics types to recover significant circadian and non-circadian trends. Using both synthetic data and proteomic data from Neurospora crassa, we showed that MOSAIC accurately recovers circadian rhythms at higher rates in not only the proteome but the transcriptome as well, outperforming existing methods for rhythm identification. In addition, by quantifying non-circadian trends in addition to circadian trends in data, our methodology allowed for the recognition of the diversity of circadian regulation as compared to non-circadian regulation. MOSAIC's full interface is available at https://github.com/delosh653/MOSAIC. An R package for this functionality, mosaic.find, can be downloaded at https://CRAN.R-project.org/package=mosaic.find. Supplementary data are available at Bioinformatics online.

Chromatin Dynamics and Transcriptional Control of Circadian Rhythms in Arabidopsis.

Circadian rhythms pervade nearly all aspects of plant growth, physiology, and development. Generation of the rhythms relies on an endogenous timing system or circadian clock that generates 24-h oscillations in multiple rhythmic outputs. At its bases, the plant circadian function relies on dynamic interactive networks of clock components that regulate each other to generate rhythms at specific phases during the day and night. From the initial discovery more than 13 years ago of a parallelism between the oscillations in chromatin status and the transcriptional rhythms of an Arabidopsis clock gene, a number of studies have later expanded considerably our view on the circadian epigenome and transcriptome landscapes. Here, we describe the most recent identification of chromatin-related factors that are able to directly interact with Arabidopsis clock proteins to shape the transcriptional waveforms of circadian gene expression and clock outputs. We discuss how changes in chromatin marks associate with transcript initiation, elongation, and the rhythms of nascent RNAs, and speculate on future interesting research directions in the field.

Pharmacological activation of the circadian component REV-ERB inhibits HIV-1 replication

Human immunodeficiency virus 1 (HIV-1) is a life-threatening pathogen that still lacks a curative therapy or vaccine. Despite the reduction in AIDS-related deaths achieved by current antiretroviral therapies, drawbacks including drug resistance and the failure to eradicate infection highlight the need to identify new pathways to target the infection. Circadian rhythms are endogenous 24-h oscillations which regulate physiological processes including immune responses to infection, and there is an emerging role for the circadian components in regulating viral replication. The molecular clock consists of transcriptional/translational feedback loops that generate rhythms. In mammals, BMAL1 and CLOCK activate rhythmic transcription of genes including the nuclear receptor REV-ERBα, which represses BMAL1 and plays an essential role in sustaining a functional clock. We investigated whether REV-ERB activity regulates HIV-1 replication and found REV-ERB agonists inhibited HIV-1 promoter activity in cell lines, primary human CD4 T cells and macrophages, whilst antagonism or genetic disruption of REV-ERB increased promoter activity. The REV-ERB agonist SR9009 inhibited promoter activity of diverse HIV-subtypes and HIV-1 replication in primary T cells. This study shows a role for REV-ERB synthetic agonists to inhibit HIV-1 LTR promoter activity and viral replication, supporting a role for circadian clock components in regulating HIV-1 replication.

Daily rhythmic leaf movements and expression of circadian clock genes are differentially synchronized by light in Phaseolus vulgaris

Efforts of recent years have achieved significant progress towards the elucidation of the molecular mechanisms underlying the plant circadian clock function. Daily rhythmic leaf movements of leguminous plants belong to the physiological processes controlled by the clock. However, their function and the way they are synchronized by light constitute a controversial subject of plant physiology. Thus, the aim of our study is to explore the role of circadian clock genes in the synchronization of the daily rhythmic leaf movements of the leguminous plant Phaseolus vulgaris. By applying a prototype experimental approach that differentiates the effect of light according to the circadian time, we were able to show that the synchronization of rhythmic leaf movements and of expression of the clock genes PvLHY and PvTOC1 proceeds according to different patterns. By our detailed gene expression analysis we demonstrate that these genes are strongly coupled regarding their adaption to the new phase which suggests that they are interlocked elements of the bean oscillator. Under the same conditions, the leaves' synchronization evolved independently from that of the genes mimicking a rhythm not related to the imposed photoperiod. This novel finding reveals for the first time that the crosstalk between rhythmic transcription and leaf movements is not direct. Our observations establish that the control of the endogenous clock underlying bean leaf movements is exerted on at a level beyond transcription, suggesting many new directions for study.

An adipokine feedback regulating diurnal food intake rhythms in mice.

Endogenous circadian clocks have evolved to anticipate 24 hr rhythms in environmental demands. Recent studies suggest that circadian rhythm disruption is a major risk factor for the development of metabolic disorders in humans. Conversely, alterations in energy state can disrupt circadian rhythms of behavior and physiology, creating a vicious circle of metabolic dysfunction. How peripheral energy state affects diurnal food intake, however, is still poorly understood. We here show that the adipokine adiponectin (ADIPOQ) regulates diurnal feeding rhythms through clocks in energy regulatory centers of the mediobasal hypothalamus (MBH). Adipoq-deficient mice show increased rest phase food intake associated with disrupted transcript rhythms of clock and appetite-regulating genes in the MBH. ADIPOQ regulates MBH clocks via AdipoR1-mediated upregulation of the core clock gene Bmal1. BMAL1, in turn, controls expression of orexigenic neuropeptide expression in the MBH. Together, these data reveal a systemic metabolic circuit to regulate central circadian clocks and energy intake.

Parthenolide reduces the freezing tolerance of hydrated lettuce seeds by inhibiting the proteolysis of seed storage globulins

Moisture content is a key factor affecting success of both field seed survival and seed cryobanks. Studying the freezing tolerance of hydrated seeds can provide information for above issues. Our previous study indicated that the ubiquitin-mediated proteolysis pathway was involved in the freezing tolerance of hydrated lettuce seeds. In this study, parthenolide, a repressor of ring-finger-type ubiquitin E3 ligase, was found to significantly reduce the freezing tolerance of lettuce seeds in slow cooling treatment. Differential scanning calorimeter (DSC) proved that parthenolide led to earlier and more ice crystal formation in the embryo of hydrated seeds in slow cooling, compared with control treatment. Real time PCR analysis showed that parthenolide changed the transcription rhythm of the RING-type E3 ligase COP1 and cold-induced gene ICE1 (Inducer of CBF Expression 1). Results of RNA-seq and real time PCR indicated that parthenolide affected the function of endoplasmic reticulum associated degradation (ERAD) pathway, in which Hsp90 was significantly up-regulated and Hsp20 down-regulated after slow cooling. Two-dimensional electrophoresis of total proteins and SDS-PAGE of soluble proteins found that parthenolide inhibited the proteolysis of seed storage globulins in slow cooling treatment. The inhibition of proteolysis for seed storage globulins and the reduction of small molecular chaperons that can prevented aggregation of misfolded proteins in parthenolide-treated seeds may be responsible for the reduction of freezing tolerance of the hydrated seeds. Parthenolide can disrupt the expression pattern of COP1 and cold responsive genes, reduce the level of small molecular chaperons and the proteolysis of seed globulins, which makes hydrated seeds freezing-sensitive.

Damped circadian oscillation in the absence of KaiA in Synechococcus

Proteins KaiA, KaiB and KaiC constitute a biochemical circadian oscillator in the cyanobacterium Synechococcus elongatus. It has been reported kaiA inactivation completely abolishes circadian oscillations. However, we show here that kaiBC promoter activity exhibits a damped, low-amplitude oscillation with a period of approximately 24 h in kaiA-inactivated strains. The damped rhythm resonates with external cycles with a period of 24–26 h, indicating that its natural frequency is similar to that of the circadian clock. Double-mutation experiments reveal that kaiC, kaiB, and sasA (encoding a KaiC-binding histidine kinase) are all required for the damped oscillation. Further analysis suggests that the kaiA-less damped transcriptional rhythm requires KaiB-KaiC complex formation and the transcription-translation feedback loop, but not the KaiC phosphorylation cycle. Our results provide insights into mechanisms that could potentially underlie the diurnal/circadian behaviors observed in other bacterial species that possess kaiB and kaiC homologues but lack a kaiA homologue.

ATP and spontaneous calcium oscillations control neural stem cell fate determination in Huntington's disease: a novel approach for cell clock research.

Calcium, the most versatile second messenger, regulates essential biology including crucial cellular events in embryogenesis. We investigated impacts of calcium channels and purinoceptors on neuronal differentiation of normal mouse embryonic stem cells (ESCs), with outcomes being compared to those of in vitro models of Huntington's disease (HD). Intracellular calcium oscillations tracked via real-time fluorescence and luminescence microscopy revealed a significant correlation between calcium transient activity and rhythmic proneuronal transcription factor expression in ESCs stably expressing ASCL-1 or neurogenin-2 promoters fused to luciferase reporter genes. We uncovered that pharmacological manipulation of L-type voltage-gated calcium channels (VGCCs) and purinoceptors induced a two-step process of neuronal differentiation. Specifically, L-type calciumchannel-mediated augmentation of spike-like calcium oscillations first promoted stable expression of ASCL-1 in differentiating ESCs, which following P2Y2 purinoceptor activation matured into GABAergic neurons. By contrast, there was neither spike-like calcium oscillations nor responsive P2Y2 receptors in HD-modelingstem cells in vitro. The data shed new light on mechanisms underlying neurogenesis of inhibitory neurons. Moreover, our approach may be tailored to identify pathogenic triggers of other developmental neurological disorders for devising targeted therapies.

Rhythms of Transcription in Field-Grown Sugarcane Are Highly Organ Specific

Circadian clocks improve plant fitness in a rhythmic environment. As each cell has its own circadian clock, we hypothesized that sets of cells with different functions would have distinct rhythmic behaviour. To test this, we investigated whether different organs in field-grown sugarcane follow the same rhythms in transcription. We assayed the transcriptomes of three organs during a day: leaf, a source organ; internodes 1 and 2, sink organs focused on cell division and elongation; and internode 5, a sink organ focused on sucrose storage. The leaf had twice as many rhythmic transcripts (>68%) as internodes, and the rhythmic transcriptomes of the internodes were more like each other than to those of the leaves. Among the transcripts expressed in all organs, only 7.4% showed the same rhythmic pattern. Surprisingly, the central oscillators of these organs — the networks that generate circadian rhythms — had similar dynamics, albeit with different amplitudes. The differences in rhythmic transcriptomes probably arise from amplitude differences in tissue-specific circadian clocks and different sensitivities to environmental cues, highlighted by the sampling under field conditions. The vast differences suggest that we must study tissue-specific circadian clocks in order to understand how the circadian clock increases the fitness of the whole plant.

How will farmed populations of freshwater fish deal with the extreme climate scenario in 2100? Transcriptional responses of Colossoma macropomum from two Brazilian climate regions.

Tambaqui (Colossoma macropomum Cuvier, 1818) is an endemic fish of the Amazon and Orinoco basins, and it is the most economically important native species in Brazil being raised in five climatically distinct regions. In the face of current global warming, environmental variations in farm ponds represent additional challenges that may drive new adaptive regional genetic variations among broodstocks of tambaqui. In an experimental context based on the high-emission scenario of the 5th Intergovernmental Panel on Climate Change (IPCC) report, we used two farmed tambaqui populations to test this hypothesis. RNA-seq transcriptome analysis was performed in the liver of juvenile tambaqui from northern (Balbina Experimental Station, Balbina, AM) and southeastern (Brumado Fish Farming, Mogi Mirim, SP) Brazilian regions kept for 30 days in artificial environmental rooms mimicking the current and extreme climate scenarios. Three Illumina MiSeq runs produced close to 120 million 500 bp paired-end reads; 191,139 contigs were assembled with N50=1595. 355 genes were differentially expressed for both populations in response to the extreme scenario. After enrichment analysis, each population presented a core set of genes to cope with climate change. Northern fish induced genes related to the cellular response to stress, activation of MAPK activity, response to unfolded protein, protein metabolism and cellular response to DNA damage stimuli. Genes biologically involved in regulating cell proliferation, protein stabilisation and protein ubiquitination for degradation through the ubiquitin-proteasome system were downregulated. Genes associated with biological processes, including the cellular response to stress, MAPK cascade activation, homeostatic processes and positive regulation of immune responses were upregulated in southeastern fish. The downregulated genes were related to cytoskeleton organisation, energy metabolism, and the regulation of transcription and biological rhythms. Our findings reveal the signatures of promising candidate genes involved in the regional plasticity of each population of tambaqui in dealing with upcoming climate changes.

Circadian Regulation of Immunity Through Epigenetic Mechanisms.

The circadian clock orchestrates daily rhythms in many physiological, behavioral and molecular processes, providing means to anticipate, and adapt to environmental changes. A specific role of the circadian clock is to coordinate functions of the immune system both at steady-state and in response to infectious threats. Hence, time-of-day dependent variables are found in the physiology of immune cells, host-parasite interactions, inflammatory processes, or adaptive immune responses. Interestingly, the molecular clock coordinates transcriptional-translational feedback loops which orchestrate daily oscillations in expression of many genes involved in cellular functions. This clock function is assisted by tightly controlled transitions in the chromatin fiber involving epigenetic mechanisms which determine how a when transcriptional oscillations occur. Immune cells are no exception, as they also present a functional clock dictating transcriptional rhythms. Hereby, the molecular clock and the chromatin regulators controlling rhythmicity represent a unique scaffold mediating the crosstalk between the circadian and the immune systems. Certain epigenetic regulators are shared between both systems and uncovering them and characterizing their dynamics can provide clues to design effective chronotherapeutic strategies for modulation of the immune system.

The Tissue Clock Network: Driver and Gatekeeper of Circadian Physiology: Circadian rhythms are integrated outputs of central and peripheral tissue clocks interacting in a complex manner - from drivers to gatekeepers.

In mammals, a network of cellular circadian clocks organizes physiology and behavior along the 24-h day cycle. The traditional hierarchical model of circadian clock organization with a central pacemaker and peripheral slave oscillators has recently been challenged by studies combining tissue-specific mouse mutants with transcriptome analyses. First, a surprisingly small number of tissue rhythms are lost when only local clocks are ablated and, second, transcriptional circadian rhythms appear to be regulated by a complex mix of local and systemic factors. As reviewed here, these findings suggest a more integrated model of clock network interaction with the central pacemaker as the main source of behavioral and systemic-physiological rhythms and peripheral clocks controlling some local rhythms while at the same time acting as gatekeepers that temporally adjust cellular responses to external stimuli.

Interaction between the Circadian Clock and Regulators of Heat Stress Responses in Plants.

The circadian clock is found ubiquitously in nature, and helps organisms coordinate internal biological processes with environmental cues that inform the time of the day or year. Both temperature stress and the clock affect many important biological processes in plants. Specifically, clock-controlled gene regulation and growth are impacted by a compromised clock or heat stress. The interactions linking these two regulatory pathways include several rhythmic transcription factors that are important for coordinating the appropriate response to temperature stress. Here we review the current understanding of clock control of the regulators involved in heat stress responses in plants.

12-h clock regulation of genetic information flow by XBP1s.

Our group recently characterized a cell-autonomous mammalian 12-h clock independent from the circadian clock, but its function and mechanism of regulation remain poorly understood. Here, we show that in mouse liver, transcriptional regulation significantly contributes to the establishment of 12-h rhythms of mRNA expression in a manner dependent on Spliced Form of X-box Binding Protein 1 (XBP1s). Mechanistically, the motif stringency of XBP1s promoter binding sites dictates XBP1s’s ability to drive 12-h rhythms of nascent mRNA transcription at dawn and dusk, which are enriched for basal transcription regulation, mRNA processing and export, ribosome biogenesis, translation initiation, and protein processing/sorting in the Endoplasmic Reticulum (ER)-Golgi in a temporal order consistent with the progressive molecular processing sequence described by the central dogma information flow (CEDIF). We further identified GA-binding proteins (GABPs) as putative novel transcriptional regulators driving 12-h rhythms of gene expression with more diverse phases. These 12-h rhythms of gene expression are cell autonomous and evolutionarily conserved in marine animals possessing a circatidal clock. Our results demonstrate an evolutionarily conserved, intricate network of transcriptional control of the mammalian 12-h clock that mediates diverse biological pathways. We speculate that the 12-h clock is coopted to accommodate elevated gene expression and processing in mammals at the two rush hours, with the particular genes processed at each rush hour regulated by the circadian and/or tissue-specific pathways.

Clock regulation of protein secretion.

The molecular clock regulates the rhythmic transcription of myriad genes, leading to a circadian pattern of expression of the encoded proteins. A study demonstrates circadian regulation of expression of components of the protein secretory pathway, providing a mechanism to generate circadian patterns of secreted protein expression.

A single factor dominates the behavior of rhythmic genes in mouse organs

BackgroundCircadian rhythm, regulated by both internal and external environment of the body, is a multi-scale biological oscillator of great complexity. On the molecular level, thousands of genes exhibit rhythmic transcription, which is both organ- and species-specific, but it remains a mystery whether some common factors could potentially explain their rhythmicity in different organs. In this study we address this question by analyzing the transcriptome data in 12 mouse organs to determine such major impacting factors.ResultsWe found a strong positive correlation between the transcriptional level and rhythmic amplitude of circadian rhythmic genes in mouse organs. Further, transcriptional level could explain over 70% of the variation in amplitude. In addition, the functionality and tissue specificity were not strong predictors of amplitude, and the expression level of rhythmic genes was linked to the energy consumption associated with transcription.ConclusionExpression level is a single major factor impacts the behavior of rhythmic genes in mouse organs. This single determinant implicates the importance of rhythmic expression itself on the design of the transcriptional system. So, rhythmic regulation of highly expressed genes can effectively reduce the energetic cost of transcription, facilitating the long-term adaptive evolution of the entire genetic system.

Circadian regulation of astrocyte function: implications for Alzheimer's disease.

The circadian clock regulates rhythms in gene transcription that have a profound impact on cellular function, behavior, and disease. Circadian dysfunction is a symptom of aging and neurodegenerative diseases, and recent studies suggest a bidirectional relationship between impaired clock function and neurodegeneration. Glial cells possess functional circadian clocks which may serve to control glial responses to daily oscillations in brain activity, cellular stress, and metabolism. Astrocytes directly support brain function through synaptic interactions, neuronal metabolic support, neuroinflammatory regulation, and control of neurovascular coupling at blood and CSF barriers. Emerging evidence suggests that the astrocyte circadian clock may be involved in many of these processes, and that clock disruption could influence neurodegeneration by disrupting several aspects of astrocyte function. Here we review the literature surrounding circadian control of astrocyte function in health and disease, and discuss the potential implications of astrocyte clocks for neurodegeneration.

Circadian sensitivity to the cardiac glycoside oleandrin is associated with diurnal intestinal P-glycoprotein expression

The cardiac glycoside oleandrin is a main active constituent of the botanical anti-cancer drug candidate PBI-05204, an extract of Nerium oleander. Here, we aimed to determine the circadian sensitivity of mice to oleandrin, and to investigate the role of intestinal P-gp in generating rhythmic drug toxicity. Toxicity and pharmacokinetic experiments were performed with wild-type, Bmal1iKO (intestine-specific Bmal1 knockout) and Bmal1fl/fl (control littermates of Bmal1iKO) mice. The cardiac toxicity (reflected by plasma CK-MB, LDH and cTn-I levels) varied significantly with the times of drug dosing in wild-type mice (a lower toxicity at ZT10 and more severe at ZT2/22). Dosing at ZT2 generated a higher drug exposure than ZT10, supporting a lower toxicity at ZT10. Intracellular accumulation of oleandrin (2.5–10 μM) was reduced in MDCKⅡ-MDR1 than in parental cells. MDR1 overexpression decreased the cell sensitivity to oleandrin toxicity. The net flux ratio (MDCKⅡ-MDR1 versus parental cells) was 2.9 for oleandrin. These data indicated oleandrin as a P-gp substrate. Both mdr1a mRNA and P-gp protein oscillated with the times of the day in small intestine of Bmal1fl/fl mice. Intestinal ablation of Bmal1 down-regulated mdr1a mRNA and P-gp protein, and abrogated their rhythms. Likewise, Bmal1 silencing led to down-regulated mdr1a mRNA and to a loss of its rhythmicity in serum-shocked CT26 cells. Based on luciferase reporter assays, Bmal1 regulated rhythmic mdr1a transcription through the clock output genes Hlf and E4bp4. Intestinal ablation of Bmal1 exacerbated oleandrin toxicity and enhanced drug exposure. Moreover, time dependency of toxicity and drug exposure were lost in Bmal1iKO mice. In conclusion, diurnal intestinal P-gp is a critical factor influencing daily oleandrin exposure and toxicity. Our findings have implications in minimizing oleandrin (and possibly Nerium oleander) toxicity and improving drug efficacy via dosing time optimization.

Diurnal expression of ABC and SLC transporters in jejunum is modulated by adrenalectomy

The circadian clock system drives many physiological processes, including plasma concentration of glucocorticoids and epithelial transport of some ions and nutrients. As glucocorticoids entrain the circadian rhythms in various peripheral organs, we examined whether adrenalectomy affects the expression and circadian rhythmicity of intestinal transporters of the solute carrier (SLC) and ATP-binding cassette (ABC) families, which participate in intestinal barriers for absorption of nutrients, nonnutrients and oral drugs. The rat jejunum showed rhythmic circadian profiles of Sglt1, Pept1, Nhe3, Mdr1 and Mrp2 but not Mct1, Oct1, Octn1, Oatp1, Cnt1 and Bcrp. With the exception of Pept1 and Mct1, adrenalectomy decreased the expression of all rhythmic and arrhythmic transporters including the amplitude of Sglt1 and Nhe3 rhythms but minimally affected the phases of rhythmic transporters except of Nhe3. Similarly, adrenalectomy downregulated the expression of rhythmic (Pparα, Hlf, Pgc1α) and arrhythmic (Hnf1β, Hnf4α) transcription factors, which are known to regulate the expression of transporters. We conclude that endogenous corticosteroids have a profound effect on the expression of intestinal SLC and ABC transporters and their nuclear transcription factors. The circulating corticosteroids are necessary for maintaining upregulated expression of Sglt1, Oct1, Octn1, Oatp1, Cnt1, Nhe3, Mdr1, Bcrp, Mrp2, Pparα, Pgc1α, Hnf1β, Hnf4α and Hlf and for maintaining the high amplitude of Sglt1, Nhe3, Pparα, Pgc1α and Hlf circadian rhythms. The study demonstrates that signals from the adrenal gland are necessary for maintaining the expression of arrhythmic and rhythmic intestinal transporters and that changes in the secretion of corticosteroids associated with stress might reorganize intestinal transport barriers.