Clock Gene Network Research Articles

The emergence of circadian timekeeping in the intestine.

The circadian clock is a molecular timekeeper, present from cyanobacteria to mammals, that coordinates internal physiology with the external environment. The clock has a 24-h period however development proceeds with its own timing, raising the question of how these interact. Using the intestine of Drosophila melanogaster as a model for organ development, we track how and when the circadian clock emerges in specific cell types. We find that the circadian clock begins abruptly in the adult intestine and gradually synchronizes to the environment after intestinal development is complete. This delayed start occurs because individual cells at earlier stages lack the complete circadian clock gene network. As the intestine develops, the circadian clock is first consolidated in intestinal stem cells with changes in Ecdysone and Hnf4 signalling influencing the transcriptional activity of Clk/cyc to drive the expression of tim, Pdp1, and vri. In the mature intestine, stem cell lineage commitment transiently disrupts clock activity in differentiating progeny, mirroring early developmental clock-less transitions. Our data show that clock function and differentiation are incompatible and provide a paradigm for studying circadian clocks in development and stem cell lineages.

The Circadian Clock as a Potential Biomarker and Therapeutic Target in Gastrointestinal Cancers.

The circadian clock consists of a hierarchical multi-oscillator network of intracellular and intercellular mechanisms throughout the body that contributes to anticipating metabolic activity and maintaining system homeostasis in response to environmental cues and intrinsic stimuli. Over the past few years, genetic variations of core clock genes have been associated with cancer risk in several epidemiological studies. A growing number of epidemiological research studies have demonstrated a direct correlation between the disturbance of circadian rhythms and the growth of tumors, indicating that shift workers are more susceptible to malignancies of the colon, prostate, ovarian, breast, lung, and liver. One of the most related cancers with circadian rhythm is Gastrointestinal (GI) cancer, which is a leading cause of cancer-related mortality nowadays. The aim of this review was to demonstrate the effect of the clock gene network on the growth of GI cancer, providing molecular targets for GI cancer treatment, possible prognostic biomarkers, and guidance for treatment choices.

Exposure to unmethylated CpG oligonucleotides disrupts blood pressure circadian rhythms and placental clock gene network in pregnant rats.

Bacterial infections and impaired circulating mitochondrial DNA dynamics are associated with adverse pregnancy outcomes. Unmethylated cytosine-guanine dinucleotide (CpG) motifs are common in bacterial and mitochondrial DNA and act as potent immunostimulators. We tested the hypothesis that exposure to CpG oligonucleotides (ODN) during pregnancy would disrupt blood pressure circadian rhythms and placental molecular clock network, mediating aberrant fetoplacental growth dynamics. Rats were repeatedly treated with CpG ODN in the third trimester [gestational days (GD) 14, 16, and 18] and euthanized on GD20 (near term) or treated with a single dose of CpG ODN on GD14 and euthanized 4 h after treatment. Hemodynamic circadian rhythms were analyzed via Lomb-Scargle periodogram analysis on 24-h raw data collected continuously via radiotelemetry. A P value ≥ 0.05 indicates the absence of a circadian rhythm. Following the first treatment with CpG ODN, maternal systolic and diastolic blood pressure circadian rhythms were lost (P ≥ 0.05). Blood pressure circadian rhythm was restored by GD16 and remained unaffected after the second treatment with CpG ODN (P < 0.0001). Diastolic blood pressure circadian rhythm was again lost after the last treatment on GD18 (P ≥ 0.05). CpG ODN increased placental expression of Per2, Per3, and Tnfα (P ≤ 0.05) and affected fetoplacental growth dynamics. Reduced fetal and placental weights were disproportionately associated with increases in the number of resorptions in ODN-treated dams compared with controls. In conclusion, gestational exposure to unmethylated CpG ODN dysregulates the placental molecular clock network and fetoplacental growth dynamics and disrupts blood pressure circadian rhythms.NEW & NOTEWORTHY Gestational exposure to unmethylated CpG ODN dysregulates placental molecular clock network and fetoplacental growth dynamics and disrupts blood pressure circadian rhythms. These findings provide novel insights into the relationship between circadian rhythms and immune responses in pregnancy and propose new mechanisms by which maternal responses to immune triggers could dictate circadian rhythms of cardiovascular processes and placental clock machinery function to determine fetal growth trajectories.

Prediction of mammalian tissue-specific CLOCK–BMAL1 binding to E-box DNA motifs

The Brain and Muscle ARNTL-Like 1 protein (BMAL1) forms a heterodimer with either Circadian Locomotor Output Cycles Kaput (CLOCK) or Neuronal PAS domain protein 2 (NPAS2) to act as a master regulator of the mammalian circadian clock gene network. The dimer binds to E-box gene regulatory elements on DNA, activating downstream transcription of clock genes. Identification of transcription factor binding sites and genomic features that correlate to DNA binding by BMAL1 is a challenging problem, given that CLOCK–BMAL1 or NPAS2–BMAL1 bind to several distinct binding motifs (CANNTG) on DNA. Using three different types of tissue-specific machine learning models with features based on (1) DNA sequence, (2) DNA sequence plus DNA shape, and (3) DNA sequence and shape plus histone modifications, we developed an interpretable predictive model of genome-wide BMAL1 binding to E-box motifs and dissected the mechanisms underlying BMAL1–DNA binding. Our results indicated that histone modifications, the local shape of the DNA, and the flanking sequence of the E-box motif are sufficient predictive features for BMAL1–DNA binding. Our models also provide mechanistic insights into tissue specificity of DNA binding by BMAL1.

Clarifying the Temporal Dynamics of the Circadian Clock and Flowering Gene Network Using Overexpression and Targeted Mutagenesis of Soybean EARLY FLOWERING 3-1 ( GmELF3-1 ).

With progressing climate fluctuations, an understanding of the molecular mechanisms of crop plants that regulate their flowering responses to environments is crucial. To achieve this goal, we aimed at clarifying the gene regulatory networks among the circadian clock and flowering genes in soybean ( Glycine max ). Based on our network inference approach , we hypothesize that GmELF3-1 , one of the Evening Complex (EC) gene homologs in soybean's circadian clock, may have an integrative role in transcriptional regulation of the circadian clock and flowering gene network. In this study, we verify GmELF3-1 ' s regulatory roles in its potential downstream genes by modulating the activity of GmELF3-1 using overexpression and CRISPR-Cas9 in soybean protoplasts. Our results indicate that GmELF3-1 may control the expression of the PRR genes in the circadian clock and the flowering gene GmCOL1a .

Experimental Verification of Inferred Regulatory Interactions of EARLY FLOWERING 3 ( GmELF3-1 ) in Glycine max.

Understanding the roles of evening complex (EC) genes in the circadian clock of plants can inform how diurnal transcriptional loops in the clock gene network function to regulate key physiological and developmental events, including flowering transition. Gene regulatory interactions among soybean's circadian clock and flowering genes were inferred using time-series RNA-seq data and the network inference algorithmic package CausNet. In this study, we seek to clarify the inferred regulatory interactions of the EC gene GmELF3-1. A gene expression analysis using soybean protoplasts as a transient model indicated regulatory roles of GmELF3-1 in expression of selected flowering genes.

Testing the inferred transcription rates of a dynamic, gene network model in absolute units

AbstractThe circadian clock coordinates plant physiology and development. Mathematical clock models have provided a rigorous framework to understand how the observed rhythms emerge from disparate, molecular processes. However, models of the plant clock have largely been built and tested against RNA time-series data in arbitrary, relative units. This limits model transferability, refinement from biochemical data and applications in synthetic biology. Here, we incorporate absolute mass units into a detailed model of the clock gene network in Arabidopsis thaliana. We re-interpret the established P2011 model, highlighting a transcriptional activator that overlaps the function of REVEILLE 8/LHY-CCA1-LIKE 5. The new U2020 model incorporates the repressive regulation of PRR genes, a key feature of the most detailed clock model KF2014, without greatly increasing model complexity. We tested the experimental error distributions of qRT–PCR data calibrated for units of RNA transcripts per cell and of circadian period estimates, in order to link the models to data more appropriately. U2019 and U2020 models were constrained using these data types, recreating previously described circadian behaviours with RNA metabolic processes in absolute units. To test their inferred rates, we estimated a distribution of observed, transcriptome-wide transcription rates (Plant Empirical Transcription Rates, PETR) in units of transcripts per cell per hour. The PETR distribution and the equivalent degradation rates indicated that the models’ predicted rates are biologically plausible, with individual exceptions. In addition to updated clock models, FAIR (Findable, Accessible, Interoperable, Re-usable) data resources and a software environment in Docker, this validation process represents an advance in biochemical realism for models of plant gene regulation.

Statistical mechanics of clock gene networks underlying circadian rhythms

All multicellular organisms embed endogenous circadian oscillators or clocks that rhythmically regulate a wide variety of processes in response to daily environmental cycles. Previous molecular studies using rhythmic mutants for several model systems have identified a set of genes responsible for rhythmic activities and illustrated the molecular mechanisms underlying how disruptions in circadian rhythms are associated with the sort of aberrant cell cycling. However, the wide use of these forward genetic studies is impaired by a limited number of mutations that can be identified or induced only in a single genome, limiting the identification of many other conserved or non-conserved clock genes. Genetic linkage or association mapping provides an unprecedented glimpse into the genome-wide scanning and characterization of genes underlying circadian rhythms. The implementation of sophisticated statistical models into genetic mapping studies can not only identify key clock genes or clock quantitative trait loci (cQTL) but also, more importantly, reveal a complete atlas of the genetic control mechanisms constituted by gene interactomes. Here, we introduce and review an advanced statistical mechanics framework for coalescing all possible clock genes into intricate but well-organized interaction networks that regulate rhythmic cycles. The application of this framework to widely available mapping populations will reshape and further our understanding of the genetic signatures behind circadian rhythms for an enlarged range of species including microbes, plants, and humans.

Mathematical modeling of mammalian circadian clocks affecting drug and disease responses.

To align with daily environmental changes, most physiological processes in mammals exhibit a time-of-day rhythmicity. This circadian control of physiology is intrinsically driven by a cell-autonomous clock gene network present in almost all cells of the body that drives rhythmic expression of genes that regulate numerous molecular and cellular processes. Accordingly, many aspects of pharmacology and toxicology also oscillate in a time-of-day manner giving rise to diverse effects on pharmacokinetics and pharmacodynamics. Genome-wide studies and mathematical modeling are available tools that have significantly improved our understanding of these nonlinear aspects of physiology and therapeutics. In this manuscript current literature and our prior work on the model-based approaches that have been used to explore circadian genomic systems of mammals are reviewed. Such basic understanding and having an integrative approach may provide new strategies for chronotherapeutic drug treatments and yield new insights for the restoration of the circadian system when altered by diseases.

Mediators of Host-Microbe Circadian Rhythms in Immunity and Metabolism.

Simple SummaryCircadian rhythms serve as the body’s internal metronome, driving responses to environmental cues over a 24-h period. Essential to nearly all life forms, the core circadian clock gene network drives physiological outputs associated with metabolic and immune responses. Modern-day disruptions to host circadian rhythms, such as shift work and jet lag, result in aberrant metabolic responses and development of complex diseases, including obesity and Type 2 Diabetes. These complex diseases are also impacted by interactions between gut microbes and the host immune system, driving a chronic low-grade inflammatory response. Gut microbes exhibit circadian dynamics that are closely tied to host circadian networks and disrupting microbial rhythmicity contributes to metabolic diseases. The underlying mediators that drive communication between host metabolism, the immune system, gut microbes, and circadian networks remain unknown, particularly in humans. Here, we explore the current state of knowledge regarding the transkingdom control of circadian networks and discuss gaps and challenges to overcome to push the field forward from the preclinical to clinical setting.Circadian rhythms are essential for nearly all life forms, mediated by a core molecular gene network that drives downstream molecular processes involved in immune function and metabolic regulation. These biological rhythms serve as the body’s metronome in response to the 24-h light:dark cycle and other timed stimuli. Disrupted circadian rhythms due to drastic lifestyle and environmental shifts appear to contribute to the pathogenesis of metabolic diseases, although the mechanisms remain elusive. Gut microbiota membership and function are also key mediators of metabolism and are highly sensitive to environmental perturbations. Recent evidence suggests rhythmicity of gut microbes is essential for host metabolic health. The key molecular mediators that transmit rhythmic signals between microbes and host metabolic networks remain unclear, but studies suggest the host immune system may serve as a conduit between these two systems, providing homeostatic signals to maintain overall metabolic health. Despite this knowledge, the precise mechanism and communication modalities that drive these rhythms remain unclear, especially in humans. Here, we review the current literature examining circadian dynamics of gut microbes, the immune system, and metabolism in the context of metabolic dysregulation and provide insights into gaps and challenges that remain.

Diversified regulation of circadian clock gene expression following whole genome duplication.

Across taxa, circadian control of physiology and behavior arises from cell-autonomous oscillations in gene expression, governed by a networks of so-called 'clock genes', collectively forming transcription-translation feedback loops. In modern vertebrates, these networks contain multiple copies of clock gene family members, which arose through whole genome duplication (WGD) events during evolutionary history. It remains unclear to what extent multiple copies of clock gene family members are functionally redundant or have allowed for functional diversification. We addressed this problem through an analysis of clock gene expression in the Atlantic salmon, a representative of the salmonids, a group which has undergone at least 4 rounds of WGD since the base of the vertebrate lineage, giving an unusually large complement of clock genes. By comparing expression patterns across multiple tissues, and during development, we present evidence for gene- and tissue-specific divergence in expression patterns, consistent with functional diversification of clock gene duplicates. In contrast to mammals, we found no evidence for coupling between cortisol and circadian gene expression, but cortisol mediated non-circadian regulated expression of a subset of clock genes in the salmon gill was evident. This regulation is linked to changes in gill function necessary for the transition from fresh- to sea-water in anadromous fish. Overall, this analysis emphasises the potential for a richly diversified clock gene network to serve a mixture of circadian and non-circadian functions in vertebrate groups with complex genomes.

Ammonia exposure causes lung injuries and disturbs pulmonary circadian clock gene network in a pig study

Ammonia toxicity to respiratory system in pig faming is of particular concern, but the molecular mechanism remains still unclear. The present study was devoted to assess the impacts of the ammonia exposure on the lung tissues based on a pig study using 80 ppm ammonia exposing to piglets for different days. The histology analysis revealed ammonia exposure induced lung injury and inflammatory response, as indicated by epithelial-mesenchymal transition (EMT), significant thickening of alveolar septa, infiltration of inflammatory cells and excessive mucus production. The transcriptome analysis revealed many more up-regulated genes in exposure groups when compared with the control group, and these genes were significantly enriched in the GO term of extracellular exosome, proteolysis, and regulation of circadian rhythm. The study discovered the induction of seven genes (CRY2, CIART, CREM, NR1D1, NR1D2, PER1 and PER3) that encode repressors of circadian clock. One gene (ARNTL) that encodes activator of circadian clock was down-regulated after ammonia exposure. The results of this study suggest that ammonia exposure disturbed the pulmonary circadian clock gene expression, which may establish new evidence for further understanding the toxicity of ammonia to lungs.

LUX ARRHYTHMO mediates crosstalk between the circadian clock and defense in Arabidopsis

The circadian clock is known to regulate plant innate immunity but the underlying mechanism of this regulation remains largely unclear. We show here that mutations in the core clock component LUX ARRHYTHMO (LUX) disrupt circadian regulation of stomata under free running and Pseudomonassyringae challenge conditions as well as defense signaling mediated by SA and JA, leading to compromised disease resistance. RNA-seq analysis reveals that both clock- and defense-related genes are regulated by LUX. LUX binds to clock gene promoters that have not been shown before, expanding the clock gene networks that require LUX function. LUX also binds to the promoters of EDS1 and JAZ5, likely acting through these genes to affect SA- and JA-signaling. We further show that JA signaling reciprocally affects clock activity. Thus, our data support crosstalk between the circadian clock and plant innate immunity and imply an important role of LUX in this process.

Circadian Clock Gene Expression in Regions of Interest to Respiratory Control

Circadian rhythms are endogenous, ~24‐hour cycles that synchronize biological functions to external cues known as zeitgebers. Circadian rhythms emerge from self‐sustaining oscillations in a transcription‐translation feedback loop of core clock genes (Bmal1, Clock, Per1 and Per2). The central clock (suprachiasmatic nucleus) and peripheral tissues throughout the body utilize this same circadian machinery. Core clock components also exert tissue‐specific effects, including tissue‐appropriate gene regulation. Although clock gene networks have been established throughout the body, little is known concerning clock genes in the neuromuscular system regulating breathing. We tested the hypothesis that clock genes oscillate in key regions of the respiratory circuit by characterizing mRNA expression patterns of core clock genes in the medulla, ventral spinal cord and diaphragm throughout the day via qPCR. Samples were collected from Sprague Dawley rats entrained to a 12‐hour light/dark cycle at 4 zeitgeber times (ZT), including (n = 8 per group): ZT5 (11am), ZT11 (5pm), ZT17 (11pm) and ZT23 (5am); ZT0 corresponds to beginning of light cycle. Cosinor analysis revealed that Bmal1, Clock, Per1 and Per2 clock genes were rhythmic in all 3 regions (all p&lt;0.01). The expression pattern of Per1 and Per2 was antiphase to Bmal1 and Clock in all regions. We hypothesize that clock genes may influence the potential to elicit serotonin‐dependent respiratory motor plasticity, such as intermittent‐hypoxia‐induced phrenic long‐term facilitation. Thus, we tested the specific hypotheses that BMAL1 protein is expressed within phrenic motor neurons and molecules necessary for phrenic long‐term facilitation (5‐HT2A receptors, BDNF and TrkB) exhibit rhythmic gene expression in brainstem and spinal regions involved in this form of plasticity. Using immunofluorescence, we demonstrate that the clock protein BMAL1 is expressed within cholera toxin B labelled phrenic motor neurons. The plasticity‐related gene Bdnf was expressed rhythmically in medulla and in ventral spinal (C3–C5) homogenates (p&lt;0.001), peaking in the active phase. TrkB mRNA (Ntrk2) was expressed rhythmically within the medulla (p&lt;0.01), but not the ventral spinal cord. 5‐HT2A receptor mRNA (Htr2a) did not exhibit rhythmic expression in either region. In conclusion, daily rhythms are observed in transcripts from clock genes and molecules necessary and sufficient for phrenic long‐term facilitation (e.g. BDNF). Although we did not uncouple cellular clocks from zeitgebers (light/dark) in this initial study, we confirm the potential for local circadian regulation in regions of interest to respiratory motor control.Support or Funding InformationUniversity of Florida Graduate School Preeminence Award (MNK), NIH T32 HD043730 (MDS) and the McKnight Brain Institute (GSM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Smad3 Suppresses Epithelial Cell Migration and Proliferation via the Clock Gene Dec1, Which Negatively Regulates the Expression of Clock Genes Dec2 and Per1

Smad3 has circadian expression; however, whether Smad3 affects the expression of clock genes is poorly understood. Here, we investigated the regulatory mechanisms between Smad3 and the clock genes Dec1, Dec2, and Per1. In Smad3 knockout mice, the amplitude of locomotor activity was decreased, and Dec1 expression was decreased in the suprachiasmatic nucleus, liver, kidney, and tongue compared with control mice. Conversely, Dec2 and Per1 expression was increased compared with that of control mice. In Smad3 knockout mice, immunohistochemical staining revealed that Dec1 expression decreased, whereas Dec2 and Per1 expression increased in the endothelial cells of the kidney and liver. In NIH3T3 cells, Smad3 overexpression increased Dec1 expression, but decreased Dec2 and Per1 expression. In a wound-healing experiment that used Smad3 knockout mice, Dec1 expression decreased in the basal cells of squamous epithelium, promoting wound healing of the mucosa. Finally, the migration and proliferation of Smad3 knockdown squamous carcinoma cells was suppressed by Dec1 overexpression but was promoted by Dec2 overexpression. Dec1 overexpression decreased E-cadherin and proliferating cell nuclear antigen expression, whereas these expression levels were increased by Dec2 overexpression. These results suggest Smad3 is relevant to circadian rhythm and regulates cell migration and proliferation through Dec1, Dec2, and Per1 expression.

Research of cryptochrome gene 1 in human osteosarcoma

Objective Aim at investigating the function and mechanism of cryptochrome gene 1 in human osteosarcoma. Methods The lentivirus vector with short hairpin RNA (shRNA) targeting CRY1 was transfected into human osteosarcoma cells (HOS). The knockdown of CRY1 in HOS was confirmed by Western blotting; the proliferation of HOS cells was detected by cell counting kit-8 (CCK-8) assay; the downstream molecules was confirmed by Western blotting; the mRNA expression level of clock gene cryptochrome gene (CRY1, CRY2), period gene (PER1, PER2), BMAL1, CLOCK was tested by quantitative real-time polymerase chain reaction (RT-qPCR). Results After the knockdown of CRY1, the proliferation of HOS was enhanced (0.839±0.020 vs. 0.553±0.019, P<0.01) and the canonical Wnt signaling was found activated (0.729±0.067 vs. 1.119±0.018, P<0.01), as well as the mRNA levels of all clock network key genes but CRY1 were increased. What’s more, proliferation of HOS cell was inhibited in rescue experiment (1.012±0.151 vs. 1.483±0.095 vs. 0.541±0.129, P<0.01). Conclusion CRY1 controls proliferation of human osteosarcoma cells via Wnt signaling pathway and regulates the expression levels of other clock network key genes, including CRY2, PER1, PER2, BMAL1 and CLOCK, which indicates that CRY1 plays an important role in human osteosarcoma progression. Key words: Cryptochrome gene 1; Wnt signaling pathway; Clock gene networks; Osteosarcoma

ELONGATED HYPOCOTYL 5 mediates blue light signalling to the Arabidopsis circadian clock.

Circadian clocks are gene networks producing 24-h oscillations at the level of clock gene expression that are synchronized to environmental cycles via light signals. The ELONGATED HYPOCOTYL 5 (HY5) transcription factor is a signalling hub acting downstream of several photoreceptors and is a key mediator of photomorphogenesis. Here we describe a mechanism by which light quality could modulate the pace of the circadian clock through governing abundance of HY5. We show that hy5 mutants display remarkably shorter period rhythms in blue but not in red light or darkness, and blue light is more efficient than red to induce accumulation of HY5 at transcriptional and post-transcriptional levels. We demonstrate that the pattern and level of HY5 accumulation modulates its binding to specific promoter elements of the majority of clock genes, but only a few of these show altered transcription in the hy5 mutant. Mathematical modelling suggests that the direct effect of HY5 on the apparently non-responsive clock genes could be masked by feedback from the clock gene network. We conclude that the information on the ratio of blue and red components of the white light spectrum is decoded and relayed to the circadian oscillator, at least partially, by HY5.

Cryptochrome 2 (CRY2) Suppresses Proliferation and Migration and Regulates Clock Gene Network in Osteosarcoma Cells.

BackgroundCircadian disruption is a potential cancer risk factor in humans. However, the role of the clock gene, cryptochrome 2 (CRY2), in osteosarcoma (OS) is still not clear.Material/MethodsTo evaluate the potential role of CRY2 in HOS osteosarcoma cells, CRY2-silenced cell lines were established. Furthermore, we investigated the effect of CRY2 knockdown on HOS cells by CCK-8, colony formation, migration assay, and flow cytometry, in vitro.ResultsCRY2 knockdown promoted HOS OS cell proliferation and migration. We used a cell cycle assay to show that CRY2 knockdown increased the S phase cell population and reduced the G1 phase cell population. Western blot analyses showed that CRY2 knockdown decreased P53 expression and increased expression of c-myc and cyclin D1. Simultaneously, CRY2 knockdown increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, but did not change the phosphorylation of c-Jun N terminal kinase (JNK) and P38. CRY2 knockdown also increased the expression of matrix metalloproteinase (MMP)-2 and β-catenin, and increased OS cell proliferation and migration by inducing cell cycle progression and promoting mitogen-activated protein kinase (MAPK) and Wnt/β-catenin signaling pathways. Although it has previously been unclear whether the expression of CRY2 affects the expression of other clock genes in the clock gene network, our results show that knockdown of CRY2 significantly increased the mRNA expression of CRY1, Period (PER) 1, PER2, BMAL1, and CLOCK.ConclusionsOur results suggest that CRY2 may be an anti-oncogene in OS, whose functions involve both downstream genes and other circadian genes.

Obesity Disrupts Rhythmic Clock Gene Expression in Maternal Adipose Tissue during Rat Pregnancy.

Obesity during pregnancy causes numerous maternal and fetal health complications, but the underlying mechanisms remain unclear. Adipose tissue dysfunction in obesity has previously been linked to disruption of the intrinsic adipose clock gene network that is crucial for normal metabolic function. This adipose clock also undergoes major change as part of the maternal metabolic adaptation to pregnancy, but whether this is affected by maternal obesity is unknown. Consequently, in this study we tested the hypothesis that obesity disturbs rhythmic gene expression in maternal adipose tissue across pregnancy. A rat model of maternal obesity was established by cafeteria (CAF) feeding, and adipose expression of clock genes and associated nuclear receptors ( Ppars and Pgc1α) was measured across days 15-16 and 21-22 of gestation (term = 23 days). CAF feeding suppressed the mesor and/or amplitude of adipose tissue clock genes (most notably Bmal1, Per2, and Rev-erbα) relative to chow-fed controls (CON) across both days of gestation. On day 15, the CAF diet also induced adipose Pparα, Pparδ, and Pgc1α rhythmicity but repressed that of Pparγ, while expression of Pparα, Pparδ, and Pgc1α was reduced at select time points. CAF mothers were hyperleptinemic at both stages of gestation, and at day 21 this effect was time-of-day dependent. Fetal plasma leptin exhibited clear rhythmicity, albeit with low amplitude, but interestingly these levels were unaffected by CAF feeding. Our data show that maternal obesity disrupts rhythmic expression of clock and metabolic genes in maternal adipose tissue and leads to maternal but not fetal hyperleptinemia.

Diel pattern of circadian clock and storage protein gene expression in leaves and during seed filling in cowpea (Vigna unguiculata)

BackgroundCowpea (Vigna unguiculata) is an important source of protein supply for animal and human nutrition. The major storage globulins VICILIN and LEGUMIN (LEG) are synthesized from several genes including LEGA, LEGB, LEGJ and CVC (CONVICILIN). The current hypothesis is that the plant circadian core clock genes are conserved in a wide array of species and that primary metabolism is to a large extent controlled by the plant circadian clock. Our aim was to investigate a possible link between gene expression of storage proteins and the circadian clock.ResultsWe identified cowpea orthologues of the core clock genes VunLHY, VunTOC1, VunGI and VunELF3, the protein storage genes VunLEG, VunLEGJ, and VunCVC as well as nine candidate reference genes used in RT-PCR. ELONGATION FACTOR 1-A (ELF1A) resulted the most suitable reference gene. The clock genes VunELF3, VunGI, VunTOC1 and VunLHY showed a rhythmic expression profile in leaves with a typical evening/night and morning/midday phased expression. The diel patterns were not completely robust and only VungGI and VungELF3 retained a rhythmic pattern under free running conditions of darkness. Under field conditions, rhythmicity and phasing apparently faded during early pod and seed development and was regained in ripening pods for VunTOC1 and VunLHY. Mature seeds showed a rhythmic expression of VunGI resembling leaf tissue under controlled growth chamber conditions. Comparing time windows during developmental stages we found that VunCVC and VunLEG were significantly down regulated during the night in mature pods as compared to intermediate ripe pods, while changes in seeds were non-significant due to high variance. The rhythmic expression under field conditions was lost under growth chamber conditions.ConclusionsThe core clock gene network is conserved in cowpea leaves showing a robust diel expression pattern except VunELF3 under growth chamber conditions. There appears to be a clock transcriptional reprogramming in pods and seeds compared to leaves. Storage protein deposition may be circadian regulated under field conditions but the strong environmental signals are not met under artificial growth conditions. Diel expression pattern in field conditions may result in better usage of energy for protein storage.