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Role of miR-142-3p in the Post-Transcriptional Regulation of the Clock Gene Bmal1 in the Mouse SCN
Shende, Vikram R.
Neuendorff, Nichole
Earnest, David J.
PLoS ONE
MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional modulators by regulating stability or translation of target mRNAs. Recent studies have implicated miRNAs in the regulation of mammalian circadian rhythms. To explore the role of miRNAs in the post-transcriptional modulation of core clock genes in the master circadian pacemaker, we examined miR-142-3p for evidence of circadian expression in the suprachiasmatic nuclei (SCN), regulation of its putative clock gene target Bmal1 via specific binding sites in the 3′ UTR and overexpression-induced changes in the circadian rhythm of BMAL1 protein levels in SCN cells. In mice exposed to constant darkness (DD), miR-142-3p levels in the SCN were characterized by circadian rhythmicity with peak expression during early subjective day at CT 3. Mutagenesis studies indicate that two independent miRNA recognition elements located at nucleotides 1–7 and 335–357 contribute equally to miR-142-3p-induced repression of luciferase-reported Bmal1 3′ UTR activity. Importantly, overexpression of miR-142-3p in immortalized SCN cells abolished circadian variation in endogenous BMAL1 protein levels in vitro. Collectively, our results suggest that miR-142-3p may play a role in the post-transcriptional modulation of Bmal1 and its oscillatory regulation in molecular feedback loops mediating SCN circadian function.
Related Articles
Genome-Wide and Phase-Specific DNA-Binding Rhythms of BMAL1 Control Circadian Output Functions in Mouse Liver
Rey, Guillaume
Cesbron, François
Rougemont, Jacques
Reinke, Hans
Brunner, Michael
Naef, Felix
PLoS Biology
Temporal mapping during a circadian day of binding sites for the BMAL1 transcription factor in mouse liver reveals genome-wide daily rhythms in DNA binding and uncovers output functions that are controlled by the circadian oscillator.
The mammalian circadian clock uses interlocked negative feedback loops in which the heterodimeric basic helix-loop-helix transcription factor BMAL1/CLOCK is a master regulator. While there is prominent control of liver functions by the circadian clock, the detailed links between circadian regulators and downstream targets are poorly known. Using chromatin immunoprecipitation combined with deep sequencing we obtained a time-resolved and genome-wide map of BMAL1 binding in mouse liver, which allowed us to identify over 2,000 binding sites, with peak binding narrowly centered around Zeitgeber time 6. Annotation of BMAL1 targets confirms carbohydrate and lipid metabolism as the major output of the circadian clock in mouse liver. Moreover, transcription regulators are largely overrepresented, several of which also exhibit circadian activity. Genes of the core circadian oscillator stand out as strongly bound, often at promoter and distal sites. Genomic sequence analysis of the sites identified E-boxes and tandem E1-E2 consensus elements. Electromobility shift assays showed that E1-E2 sites are bound by a dimer of BMAL1/CLOCK heterodimers with a spacing-dependent cooperative interaction, a finding that was further validated in transactivation assays. BMAL1 target genes showed cyclic mRNA expression profiles with a phase distribution centered at Zeitgeber time 10. Importantly, sites with E1-E2 elements showed tighter phases both in binding and mRNA accumulation. Finally, analyzing the temporal profiles of BMAL1 binding, precursor mRNA and mature mRNA levels showed how transcriptional and post-transcriptional regulation contribute differentially to circadian expression phase. Together, our analysis of a dynamic protein-DNA interactome uncovered how genes of the core circadian oscillator crosstalk and drive phase-specific circadian output programs in a complex tissue.
Author Summary
The circadian clock is a timing system that allows organisms to keep behavioral, physiological, and cellular rhythms in resonance with daily environmental cycles. In mammals, such clocks use transcriptional regulatory loops in which the heterodimeric transcription factor BMAL1/CLOCK plays a central role. While defects in circadian clock function have been associated with diabetes, obesity, and cancer, the molecular links between the circadian clock and such output pathways are poorly characterized. Here, we mapped DNA-binding sites of BMAL1 in mouse liver during one circadian cycle. Our temporal analysis revealed widespread daily rhythms in DNA binding, with maximum levels peaking at midday. In the list of candidates, core circadian genes stood out as the most strongly bound, often showing multiple binding sites. Interestingly, BMAL1 targets were highly enriched in genes involved in carbohydrate and lipid metabolism, and also in transcription factors, in particular nuclear receptors. Our results suggest that the mammalian clock uses BMAL1 to control transcriptional output programs both directly and indirectly. Additionally, the DNA specificity of BMAL1 binding revealed the importance of tandem E-box elements, which may favor strong binding and precise timing of daily gene expression. Taken together, our work confirms BMAL1's primary function as a master regulator of the core circadian oscillator, while demonstrating that it also contributes in a more distributed fashion to a variety of output programs.
Related Articles
Expression and Rhythmic Modulation of Circulating MicroRNAs Targeting the Clock Gene Bmal1 in Mice
Shende, Vikram R.
Goldrick, Marianna M.
Ramani, Suchitra
Earnest, David J.
PLoS ONE
MicroRNAs (miRNAs) interact with 3′ untranslated region (UTR) elements of target genes to regulate mRNA stability or translation and thus play a role in regulating many different biological processes, including circadian rhythms. However, specific miRNAs mediating the regulation of essential clock genes remain largely unknown. Because vesicles containing membrane-bound miRNAs are present in the circulatory system, we examined miRNAs predicted to target the clock gene, Bmal1, for evidence of rhythmic fluctuations in circulating levels and modulatory effects on the 3′ UTR activity of Bmal1. A number of miRNAs with Bmal1 as a predicted target were expressed in the serum of mice exposed to LD 12∶12 and of these miRNAs, miR-152 and miR-494 but not miR-142-3p were marked by diurnal oscillations with bimodal peaks in expression occurring near the middle of the day and 8 or 12 hr later during the night. Co-transfection of pre-miR over-expression constructs for miR-494 and miR-142-3p in HEK293 cells had significant effects in repressing luciferase-reported Bmal1 3′ UTR activity by as much as 60%, suggesting that these miRNAs may function as post-transcriptional modulators of Bmal1. In conjunction with previous studies implicating miRNAs as extracellular regulatory signals, our results suggest that circulating miRNAs may play a role in the regulation of the molecular clockworks in peripheral circadian oscillators.
Related Articles
Redundant Function of REV-ERBα and β and Non-Essential Role for Bmal1 Cycling in Transcriptional Regulation of Intracellular Circadian Rhythms
Liu, Andrew C.
Tran, Hien G.
Zhang, Eric E.
Priest, Aaron A.
Welsh, David K.
Kay, Steve A.
PLoS Genetics
The mammalian circadian clockwork is composed of a core PER/CRY feedback loop and additional interlocking loops. In particular, the ROR/REV/Bmal1 loop, consisting of ROR activators and REV-ERB repressors that regulate Bmal1 expression, is thought to "stabilize" core clock function. However, due to functional redundancy and pleiotropic effects of gene deletions, the role of the ROR/REV/Bmal1 loop has not been accurately defined. In this study, we examined cell-autonomous circadian oscillations using combined gene knockout and RNA interference and demonstrated that REV-ERBα and β are functionally redundant and are required for rhythmic Bmal1 expression. In contrast, the RORs contribute to Bmal1 amplitude but are dispensable for Bmal1 rhythm. We provide direct in vivo genetic evidence that the REV-ERBs also participate in combinatorial regulation of Cry1 and Rorc expression, leading to their phase-delay relative to Rev-erbα. Thus, the REV-ERBs play a more prominent role than the RORs in the basic clock mechanism. The cellular genetic approach permitted testing of the robustness of the intracellular core clock function. We showed that cells deficient in both REV-ERBα and β function, or those expressing constitutive BMAL1, were still able to generate and maintain normal Per2 rhythmicity. Our findings thus underscore the resilience of the intracellular clock mechanism and provide important insights into the transcriptional topologies underlying the circadian clock. Since REV-ERB function and Bmal1 mRNA/protein cycling are not necessary for basic clock function, we propose that the major role of the ROR/REV/Bmal1 loop and its constituents is to control rhythmic transcription of clock output genes.
Author Summary
Circadian clocks in plants, fungi, insects, and mammals all share a common transcriptional network architecture. At the cellular level, the mammalian clockwork consists of a core Per/Cry negative feedback loop and additional interlocking loops. We wished to address experimentally the contribution of the interlocking Bmal1 loop to clock function in mammals. Because behavioral rhythms do not always reflect cell-autonomous phenotypes and are subject to pleiotropic effects, we employed cell-based genetic approaches and monitored rhythms longitudinally using bioluminescent reporters of clock gene expression. We showed that REV-ERB repressors play a more prominent role than ROR activators in regulating the Bmal1 rhythm. However, significant rhythmicity remains even with constitutive expression of Bmal1, pointing to the resilience of the core loop to perturbations of the Bmal1 loop. We conclude that while the interlocking loop contributes to fine-tuning of the core loop, its primary function is to provide discrete waveforms of clock gene expression for control of local physiology. This study has important general implications not only for circadian biology across species, but also for the emerging field of systems biology that seeks to understand complex interactions in genetic networks.
Related Articles
New role of zCRY and zPER2 as regulators of sub-cellular distributions of zCLOCK and zBMAL proteins
Hirayama, Jun
Fukuda, Itsuki
Ishikawa, Tomoko
Kobayashi, Yuri
Todo, Takeshi
Nucleic Acids Research
The core oscillator that generates circadian rhythm in eukaryotes consists of transcription/translation-based autoregulatory feedback loops by which clock gene products negatively regulate their own expression. Control of the accumulation and nuclear entry of the negative regulators PER and CRY is believed to be a key step in these loops. We clarified the mutual interaction between zebrafish clock-related proteins and their sub-cellular localizations in NIH3T3 cells. Six CRYs exist in zebrafish, of which zCRY1a strongly represses zCLOCK1: zBMAL3-mediated transcription, but zCRY3 does not. We show that zCRY1a interacts with zCLOCK1 and zBMAL3, facilitating nuclear accumulation, whereas zCRY3 associates with neither one and does not influence their sub-cellular distributions. We cloned zPer2 cDNA and showed that the protein product encoded by the cDNA acts as a moderate transcriptional repressor. In our sub-cellular localization studies we also found that zPER2 interacts with the zCLOCK1:zBMAL3 heterodimer, causing its cytoplasmic retention. zCRY1a and zPER2 apparently have opposite effects on the sub-cellular distribution of zCLOCK:zBMAL heterodimer. We speculate that the opposite regulation of the sub-cellular distribution of this is associated with the different transcriptional repression abilities of zCRY1a and zPER2. zCRY1a acts as a potent transcriptional inhibitor by interacting directly with the zCLOCK:zBMAL heterodimer in the nucleus, whereas zPER2 maintains the zCLOCK:zBMAL heterodimer in the cytoplasm, resulting in transactivation repression.
Related Articles
CLOCK and NPAS2 have overlapping roles in the circadian oscillation of arylalkylamine N-acetyltransferase (Aanat) mRNA in chicken cone photoreceptors
Haque, Rashidul
Ali, Fatima G.
Biscoglia, Rebecca
Abey, Jane
Weller, Joan
Klein, David
Iuvone, P. Michael
Journal of neurochemistry
Circadian clocks in vertebrates are thought to be composed of transcriptional-translational feedback loops involving a highly conversed set of "clock genes": namely, period (Per1–3) and cryptochrome (Cry1–2), which encode negative transcriptional regulators; and Bmal1, Clock, and Npas2, which encode positive regulators. Aanat, which encodes arylalkylamine N-acetyltransferase (AANAT), the key regulatory enzyme that drives the circadian rhythm of melatonin synthesis, contains a circadian E-box element (CACGTG) in its proximal promoter that is potentially capable of binding CLOCK: BMAL1 and NPAS2: BMAL1 heterodimers. The present study was conducted to investigate whether CLOCK and/or NPAS2 regulates Aanat expression in photoreceptor cells. Npas2 and Clock are both expressed in photoreceptor cells in vivo and in vitro. To assess the roles of CLOCK and NPAS2 in Aanat expression, gene specific microRNA (miR) vectors were used to knock down expression of these clock genes in photoreceptor-enriched cell cultures. The knockdown of CLOCK protein significantly reduced the circadian expression of Npas2, Per2, and Aanat transcripts but had no effect on the circadian rhythm of Bmal1 transcript level. The knockdown of NPAS2 significantly damped the circadian rhythm of Aanat mRNAs but had no effect on circadian expression of any of clock genes examined, except Npas2 itself. Chromatin immunoprecipitation studies indicated that both CLOCK and NPAS2 bound to the Aanat promoter in situ. Thus, CLOCK and NPAS2 have overlapping roles in the clock output pathway that regulates the rhythmic expression of Aanat in photoreceptors. However, CLOCK plays the predominant role in the chicken photoreceptor circadian clockwork mechanism, including the regulation of NPAS2 expression.
circadian clock genes; circadian rhythms; retina; melatonin; transcription factors; RNA interference
Related Articles
CBP/p300 is a cell type-specific modulator of CLOCK/BMAL1-mediated transcription
Hosoda, Hiroshi
kato, Kenichi
Asano, Hidenori
Ito, Motonori
Kato, Haruno
Iwamoto, Taku
Suzuki, Akinobu
Masushige, Shoichi
Kida, Satoshi
Molecular Brain
Previous studies have demonstrated tissue-specific regulation of the rhythm of circadian transcription, suggesting that transcription factor complex CLOCK/BMAL1, essential for maintaining circadian rhythm, regulates transcription in a tissue-specific manner. To further elucidate the mechanism of the cell type-specific regulation of transcription by CLOCK/BMAL1 at the molecular level, we investigated roles of CBP/p300 and tissue-specific cofactors in CLOCK/BMAL1-mediated transcription.
As shown previously, CBP/p300 stimulates CLOCK/BMAL1-mediated transcription in COS-1 cells. However, CBP/p300 repressed CLOCK/BMAL1-mediated transcription in NIH3T3 cells and knockdown of CBP or p300 expression by siRNA enhanced this transcription. Studies using GAL4-fusion proteins suggested that CBP represses CLOCK/BMAL1-mediated transcription by targeting CLOCK. We further investigated mechanisms of this cell type-specific modulation of CLOCK/BMAL1-mediated transcription by CBP by examining roles of co-repressor HDAC3 and co-activator pCAF, which are highly expressed in NIH3T3 and COS cells, respectively. CBP repressed CLOCK/BMAL1-mediated transcription in COS-1 cells when HDAC3 was overexpressed, but activated it in NIH3T3 cells when pCAF was overexpressed. CBP forms a complex with CLOCK by interacting with HDAC3 or pCAF; however, direct interaction of CBP with CLOCK was not observed.
Our findings indicate possible mechanisms by which CBP/p300 tissue-specifically acts cooperatively with pCAF and HDAC3 either as a co-activator or co-repressor, respectively, for CLOCK/BMAL1.
Related Articles
Intermolecular recognition revealed by the complex structure of human CLOCK-BMAL1 basic helix-loop-helix domains with E-box DNA
Wang, Zixi
Wu, Yaling
Li, Lanfen
Su, Xiao-Dong
Cell Research
CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1) are both transcription factors of the circadian core loop in mammals. Recently published mouse CLOCK-BMAL1 bHLH (basic helix-loop-helix)-PAS (period-ARNT-single-minded) complex structure sheds light on the mechanism for heterodimer formation, but the structural details of the protein-DNA recognition mechanisms remain elusive. Here we have elucidated the crystal structure of human CLOCK-BMAL1 bHLH domains bound to a canonical E-box DNA. We demonstrate that CLOCK and BMAL1 bHLH domains can be mutually selected, and that hydrogen-bonding networks mediate their E-box recognition. We identified a hydrophobic contact between BMAL1 Ile80 and a flanking thymine nucleotide, suggesting that CLOCK-BMAL1 actually reads 7-bp DNA and not the previously believed 6-bp DNA. To find potential non-canonical E-boxes that could be recognized by CLOCK-BMAL1, we constructed systematic single-nucleotide mutations on the E-box and measured their relevant affinities. We defined two non-canonical E-box patterns with high affinities, AACGTGA and CATGTGA, in which the flanking A7-T7′ base pair is indispensable for recognition. These results will help us to identify functional CLOCK-BMAL1-binding sites in vivo and to search for clock-controlled genes. Furthermore, we assessed the inhibitory role of potential phosphorylation sites in bHLH regions. We found that the phospho-mimicking mutation on BMAL1 Ser78 could efficiently block DNA binding as well as abolish normal circadian oscillation in cells. We propose that BMAL1 Ser78 should be a key residue mediating input signal-regulated transcriptional inhibition for external cues to entrain the circadian clock by kinase cascade.
crystal structure; circadian; CLOCK; BMAL1; non-canonical E-box; phosphorylation
Related Articles
Direct Regulation of CLOCK Expression by REV-ERB
Crumbley, Christine
Burris, Thomas P.
PLoS ONE
Circadian rhythms are regulated at the cellular level by transcriptional feedback loops leading to oscillations in expression of key proteins including CLOCK, BMAL1, PERIOD (PER), and CRYPTOCHROME (CRY). The CLOCK and BMAL1 proteins are members of the bHLH class of transcription factors and form a heterodimer that regulates the expression of the PER and CRY genes. The nuclear receptor REV-ERBα plays a key role in regulation of oscillations in BMAL1 expression by directly binding to the BMAL1 promoter and suppressing its expression at certain times of day when REV-ERBα expression levels are elevated. We recently demonstrated that REV-ERBα also regulates the expression of NPAS2, a heterodimer partner of BMAL1. Here, we show that REV-ERBα also regulates the expression another heterodimer partner of BMAL1, CLOCK. We identified a REV-ERBα binding site within the 1st intron of the CLOCK gene using a chromatin immunoprecipitation – microarray screen. Suppression of REV-ERBα expression resulted in elevated CLOCK mRNA expression consistent with REV-ERBα's role as a transcriptional repressor. A REV-ERB response element (RevRE) was identified within this region of the CLOCK gene and was conserved between humans and mice. Additionally, the CLOCK RevRE conferred REV-ERB responsiveness to a heterologous reporter gene. Our data suggests that REV-ERBα plays a dual role in regulation of the activity of the BMAL1/CLOCK heterodimer by regulation of expression of both the BMAL1 and CLOCK genes.
Related Articles
Sleep Loss Reduces the DNA-Binding of BMAL1, CLOCK, and NPAS2 to Specific Clock Genes in the Mouse Cerebral Cortex
Mongrain, Valérie
La Spada, Francesco
Curie, Thomas
Franken, Paul
PLoS ONE
We have previously demonstrated that clock genes contribute to the homeostatic aspect of sleep regulation. Indeed, mutations in some clock genes modify the markers of sleep homeostasis and an increase in homeostatic sleep drive alters clock gene expression in the forebrain. Here, we investigate a possible mechanism by which sleep deprivation (SD) could alter clock gene expression by quantifying DNA-binding of the core-clock transcription factors CLOCK, NPAS2, and BMAL1 to the cis-regulatory sequences of target clock genes in mice. Using chromatin immunoprecipitation (ChIP), we first showed that, as reported for the liver, DNA-binding of CLOCK and BMAL1 to target clock genes changes in function of time-of-day in the cerebral cortex. Tissue extracts were collected at ZT0 (light onset), −6, −12, and −18, and DNA enrichment of E-box or E'-box containing sequences was measured by qPCR. CLOCK and BMAL1 binding to Cry1, Dbp, Per1, and Per2 depended on time-of-day, with maximum values reached at around ZT6. We then observed that SD, performed between ZT0 and −6, significantly decreased DNA-binding of CLOCK and BMAL1 to Dbp, consistent with the observed decrease in Dbp mRNA levels after SD. The DNA-binding of NPAS2 and BMAL1 to Per2 was also decreased by SD, although SD is known to increase Per2 expression in the cortex. DNA-binding to Per1 and Cry1 was not affected by SD. Our results show that the sleep-wake history can affect the clock molecular machinery directly at the level of chromatin binding thereby altering the cortical expression of Dbp and Per2 and likely other targets. Although the precise dynamics of the relationship between DNA-binding and mRNA expression, especially for Per2, remains elusive, the results also suggest that part of the reported circadian changes in DNA-binding of core clock components in tissues peripheral to the suprachiasmatic nuclei could, in fact, be sleep-wake driven.
Related Articles
Genomic Convergence among ERRα, PROX1, and BMAL1 in the Control of Metabolic Clock Outputs
Dufour, Catherine R.
Levasseur, Marie-Pier
Pham, Nguyen Hoai Huong
Eichner, Lillian J.
Wilson, Brian J.
Charest-Marcotte, Alexis
Duguay, David
Poirier-Héon, Jean-François
Cermakian, Nicolas
Giguère, Vincent
PLoS Genetics
Metabolic homeostasis and circadian rhythms are closely intertwined biological processes. Nuclear receptors, as sensors of hormonal and nutrient status, are actively implicated in maintaining this physiological relationship. Although the orphan nuclear receptor estrogen-related receptor α (ERRα, NR3B1) plays a central role in the control of energy metabolism and its expression is known to be cyclic in the liver, its role in temporal control of metabolic networks is unknown. Here we report that ERRα directly regulates all major components of the molecular clock. ERRα-null mice also display deregulated locomotor activity rhythms and circadian period lengths under free-running conditions, as well as altered circulating diurnal bile acid and lipid profiles. In addition, the ERRα-null mice exhibit time-dependent hypoglycemia and hypoinsulinemia, suggesting a role for ERRα in modulating insulin sensitivity and glucose handling during the 24-hour light/dark cycle. We also provide evidence that the newly identified ERRα corepressor PROX1 is implicated in rhythmic control of metabolic outputs. To help uncover the molecular basis of these phenotypes, we performed genome-wide location analyses of binding events by ERRα, PROX1, and BMAL1, an integral component of the molecular clock. These studies revealed the existence of transcriptional regulatory loops among ERRα, PROX1, and BMAL1, as well as extensive overlaps in their target genes, implicating these three factors in the control of clock and metabolic gene networks in the liver. Genomic convergence of ERRα, PROX1, and BMAL1 transcriptional activity thus identified a novel node in the molecular circuitry controlling the daily timing of metabolic processes.
Author Summary
The molecular basis for coordinated control of circadian rhythms and metabolism is not well understood. Although integral components of the molecular clock such as the transcription factor BMAL1 can directly regulate some metabolic genes, the output from the circadian oscillator is believed to be in large part mediated through the action of transcription factors whose patterns of expression are rhythmic in metabolic tissues. The estrogen-related receptor α (ERRα, NR3B1) and its corepressor PROX1, two major metabolic regulators, could be well suited for this function. Indeed, we show that proper maintenance of daily glucose, insulin, bile acid, lipid, and locomotor rhythms in mice are dependent on the presence of ERRα. Ablation of PROX1 in synchronized HepG2 cells revealed the importance of PROX1 in regulating the rhythmic expression of clock and metabolic genes. Using genome-wide analysis of promoter occupancy and gene expression analyses, we identify ERRα and PROX1 as novel transcriptional regulators of the molecular clock and show that the direct participation of BMAL1 in the clock output pathway related to metabolic control is highly extensive. ERRα and BMAL1 thus serve as key bidirectional regulators connecting the peripheral clock and cellular energy metabolism.
Related Articles
Molecular assembly of the period-cryptochrome circadian transcriptional repressor complex
Nangle, Shannon N
Rosensweig, Clark
Koike, Nobuya
Tei, Hajime
Takahashi, Joseph S
Green, Carla B
Zheng, Ning
The mammalian circadian clock is driven by a transcriptional–translational feedback loop, which produces robust 24-hr rhythms. Proper oscillation of the clock depends on the complex formation and periodic turnover of the Period and Cryptochrome proteins, which together inhibit their own transcriptional activator complex, CLOCK-BMAL1. We determined the crystal structure of the CRY-binding domain (CBD) of PER2 in complex with CRY2 at 2.8 Å resolution. PER2-CBD adopts a highly extended conformation, embracing CRY2 with a sinuous binding mode. Its N-terminal end tucks into CRY adjacent to a large pocket critical for CLOCK-BMAL1 binding, while its C-terminal half flanks the CRY2 C-terminal helix and sterically hinders the recognition of CRY2 by the FBXL3 ubiquitin ligase. Unexpectedly, a strictly conserved intermolecular zinc finger, whose integrity is important for clock rhythmicity, further stabilizes the complex. Our structure-guided analyses show that these interspersed CRY-interacting regions represent multiple functional modules of PERs at the CRY-binding interface.
DOI: http://dx.doi.org/10.7554/eLife.03674.001
eLife digest
Since the very simplest organisms emerged on earth, the rhythms of life have been synchronized with the rising and setting of the sun. Even the most basic life forms have internal clocks that help them to maintain daily routines and adapt to shifting seasons. In animals, these internal clocks regulate processes such as the release of hormones that wake an animal up and the expression of genes necessary to carry out the activities of daily life. Later on, the clocks then trigger the release of hormones that cause drowsiness and the expression of the genes that are active during rest.
In mammals, these internal circadian rhythms are maintained by a feedback loop governed by four key proteins. Two of these proteins—CLOCK and BMAL1—work together to begin a process called transcription, whereby sections of DNA are used as a template to copy the information needed to make a protein. The two activating proteins CLOCK and BMAL1 recognize the sections of DNA where the genes that are controlled by the circadian clock are located and selectively turn on the expression of those genes.
Expression of the two other key circadian proteins—Period and Cryptochrome—is switched on by CLOCK and BMAL1. As Period and Cryptochrome proteins accumulate, they begin to inhibit the activity of CLOCK and BMAL1, helping to reduce the rate at which the circadian genes are transcribed as the day progresses.
Nangle et al. provide new insights into how the Period and Cryptochrome proteins interact with each other, using X-ray crystallography to reveal the molecular level details of the bond between the two proteins. Period stretches out as it 'embraces' Cryptochrome. One end of the Period protein then tucks into part of the Cryptochrome structure that is next to a large pocket. This pocket is where the Cryptochrome protein binds to CLOCK and BMAL1, suggesting that Period can influence whether this binding occurs.
The other end of the Period protein covers one end of the Cryptochrome protein. By doing so, enzymes cannot bind there, and so cannot break down Cryptochrome. Nangle et al. also discovered that a finger-like projection that includes a zinc ion acts as a clasp, strengthening the bond between Period and Cryptochrome.
These findings help to demonstrate how Period proteins act as a timekeeper that regulates how long Cryptochrome can turn down the activity of CLOCK and BMAL1. A deeper understanding of the molecular choreography among the four clock proteins holds promise for developing medications to treat the sleep disorders and circadian clock disruptions associated with a modern lifestyle.
DOI: http://dx.doi.org/10.7554/eLife.03674.002
circadian rhythm; cryptochrome; period; mouse
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Regulation of Retinal Inflammation by Rhythmic Expression of MiR-146a in Diabetic Retina
Wang, Qi
Bozack, Svetlana N.
Yan, Yuanqing
Boulton, Michael E.
Grant, Maria B.
Busik, Julia V.
Investigative Ophthalmology & Visual Science
Chronic inflammation and dysregulation of circadian rhythmicity are involved in the pathogenesis of diabetic retinopathy. MicroRNAs (miRNAs) can regulate inflammation and circadian clock machinery. We tested the hypothesis that altered daily rhythm of miR-146a expression in diabetes contributes to retinal inflammation.
Nondiabetic and STZ-induced diabetic rats kept in 12/12 light/dark cycle were killed every 2 hours over a 72-hour period. Human retinal endothelial cells (HRECs) were synchronized with dexamethasone. Expression of miR-146a, IL-1 receptor-associated kinase 1 (IRAK1), IL-1β, VEGF and ICAM-1, as well as clock genes was examined by real-time PCR and Western blot. To modulate expression levels of miR-146a, mimics and inhibitors were used.
Diabetes inhibited amplitude of negative arm (per1) and enhanced amplitude of the positive arm (bmal1) of clock machinery in retina. In addition to clock genes, miR-146a and its target gene IRAK1 also exhibited daily oscillations in antiphase; however, these patterns were lost in diabetic retina. This loss of rhythmic pattern was associated with an increase in ICAM-1, IL-β, and VEGF expression. Human retinal endothelial cells had robust miR-146a expression that followed circadian oscillation pattern; however, HRECs isolated from diabetic donors had reduced miR-146a amplitude but increased amplitude of IRAK1 and ICAM-1. In HRECs, miR-146a mimic or inhibitor caused 1.6- and 1.7-fold decrease or 1.5- and 1.6-fold increase, respectively, in mRNA and protein expression levels of ICAM-1 after 48 hours.
Diabetes-induced dysregulation of daily rhythms of miR-146a and inflammatory pathways under miR-146a control have potential implications for the development of diabetic retinopathy.
Diabetes-induced dysregulation of daily rhythms of miR-146a and inflammatory pathways under miR-146a control have potential implications for the development of diabetic retinopathy.
diabetic retinopathy; miRNA; circadian rhythm; intercellular adhesion molecule 1; inflammatory response; endothelial cells
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Circadian Clock Genes as Modulators of Sensitivity to Genotoxic Stress
Antoch, Marina P.
Kondratov, Roman V.
Takahashi, Joseph S.
Cell cycle (Georgetown, Tex.)
A broad variety of organisms display circadian rhythms (i.e., oscillations with 24-hr periodicities) in many aspects of their behavior, physiology and metabolism. These rhythms are under genetic control and are generated endogenously at the cellular level. In mammals, the core molecular mechanism of the oscillator consists of two transcriptional activators, CLOCK and BMAL1, and their transcriptional targets, CRYPTOCHROMES (CRYS) and PERIODS (PERS). The CRY and PER proteins function as negative regulators of CLOCK/BMAL1 activity, thus forming the major circadian autoregulatory feedback loop. It is believed that the circadian clock system regulates daily variations in output physiology and metabolism through periodic activation/repression of the set of clock-controlled genes that are involved in various metabolic pathways. Importantly, circadian-controlled pathways include those that determine in vivo responses to genotoxic stress. By using circadian mutant mice deficient in different components of the molecular clock system, we have established genetic models that correlate with the two opposite extremes of circadian cycle as reflected by the activity of the CLOCK/BMAL1 transactivation complex. Comparison of the in vivo responses of these mutants to the chemotherapeutic drug, cyclophosphamide (CY), has established a direct correlation between drug toxicity and the functional status of the CLOCK/BMAL1 transcriptional complex. We have also demonstrated that CLOCK/BMAL1 modulates sensitivity to drug-induced toxicity by controlling B cell responses to active CY metabolites. These results suggest that the sensitivity of cells to genotoxic stress induced by anticancer therapy may be modulated by CLOCK/BMAL1 transcriptional activity. Further elucidation of the molecular mechanisms of circadian control as well as identification of specific pharmacological modulators of CLOCK/BMAL1 activity are likely to lead to the development of new anti-cancer treatment schedules with increased therapeutic index and reduced morbidity.
Circadian; CLOCK; BMAL1; transcription; anticancer therapy
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Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans
Watthanasurorot, Apiruck
Saelee, Netnapa
Phongdara, Amornrat
Roytrakul, Sittiruk
Jiravanichpaisal, Pikul
Söderhäll, Kenneth
Söderhäll, Irene
PLoS Genetics
Daily, circadian rhythms influence essentially all living organisms and affect many physiological processes from sleep and nutrition to immunity. This ability to respond to environmental daily rhythms has been conserved along evolution, and it is found among species from bacteria to mammals. The hematopoietic process of the crayfish Pacifastacus leniusculus is under circadian control and is tightly regulated by astakines, a new family of cytokines sharing a prokineticin (PROK) domain. The expression of AST1 and AST2 are light-dependent, and this suggests an evolutionarily conserved function for PROK domain proteins in mediating circadian rhythms. Vertebrate PROKs are transmitters of circadian rhythms of the suprachiasmatic nucleus (SCN) in the brain of mammals, but the mechanism by which they function is unknown. Here we demonstrate that high AST2 expression is induced by melatonin in the brain. We identify RACK1 as a binding protein of AST2 and further provide evidence that a complex between AST2 and RACK1 functions as a negative-feedback regulator of the circadian clock. By DNA mobility shift assay, we showed that the AST2-RACK1 complex will interfere with the binding between BMAL1 and CLK and inhibit the E-box binding activity of the complex BMAL1-CLK. Finally, we demonstrate by gene knockdown that AST2 is necessary for melatonin-induced inhibition of the complex formation between BMAL1 and CLK during the dark period. In summary, we provide evidence that melatonin regulates AST2 expression and thereby affects the core clock of the crustacean brain. This process may be very important in all animals that have AST2 molecules, i.e. spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, and Blattodea, but not Diptera and Coleoptera. Our findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops.
Author Summary
Most living organisms are able to sense the time and in particular time of day by their internal clocks. So-called clock proteins control these internal clockworks. BMAL1 and CLK are two important clock proteins, which together form a complex that serves as a transcription factor and controls the production of diurnal proteins. These diurnal proteins, in turn, inhibit the formation of clock proteins so that the concentration of the different proteins in the cell oscillates back and forth throughout the day. External factors may affect the balance of clock proteins, and one such important factor is light. Melatonin is a darkness hormone produced in the brain of most animals during the night, and here we show that melatonin controls the formation of a protein named AST2 in crayfish. AST2 belongs to a group of proteins found in many arthropods, such as spiders, scorpions, crustaceans, and some insects, whose function has been unknown until now. Now we demonstrate that AST2 is induced by melatonin at night and then functions in the internal biological clock by preventing BMAL1 and CLK to form a complex. In this way, AST2 acts as a link between melatonin and the internal biological clock.
Related Articles
Tuning the Mammalian Circadian Clock: Robust Synergy of Two Loops
Relógio, Angela
Westermark, Pal O.
Wallach, Thomas
Schellenberg, Katja
Kramer, Achim
Herzel, Hanspeter
PLoS Computational Biology
The circadian clock is accountable for the regulation of internal rhythms in most living organisms. It allows the anticipation of environmental changes during the day and a better adaptation of physiological processes. In mammals the main clock is located in the suprachiasmatic nucleus (SCN) and synchronizes secondary clocks throughout the body. Its molecular constituents form an intracellular network which dictates circadian time and regulates clock-controlled genes. These clock-controlled genes are involved in crucial biological processes including metabolism and cell cycle regulation. Its malfunction can lead to disruption of biological rhythms and cause severe damage to the organism. The detailed mechanisms that govern the circadian system are not yet completely understood. Mathematical models can be of great help to exploit the mechanism of the circadian circuitry. We built a mathematical model for the core clock system using available data on phases and amplitudes of clock components obtained from an extensive literature search. This model was used to answer complex questions for example: how does the degradation rate of Per affect the period of the system and what is the role of the ROR/Bmal/REV-ERB (RBR) loop? Our findings indicate that an increase in the RNA degradation rate of the clock gene Period (Per) can contribute to increase or decrease of the period - a consequence of a non-monotonic effect of Per transcript stability on the circadian period identified by our model. Furthermore, we provide theoretical evidence for a potential role of the RBR loop as an independent oscillator. We carried out overexpression experiments on members of the RBR loop which lead to loss of oscillations consistent with our predictions. These findings challenge the role of the RBR loop as a merely auxiliary loop and might change our view of the clock molecular circuitry and of the function of the nuclear receptors (REV-ERB and ROR) as a putative driving force of molecular oscillations.
Author Summary
Most organisms have evolved an internal clock which allows them to anticipate and react to the light/dark daily rhythm and is able to generate oscillation with a circa 24 hour rhythm. A molecular network involving feedback loops is responsible for the rhythm generation. A large number of clock-controlled genes pass on time messages and control several biological processes. In spite of its medical importance (role in cancer, sleep disorders, diabetes and others) the mechanism of action of the circadian clock and the role of its constituent's feedback loops remains partially unknown. Using a mathematical model, we were able to bring insight in open circadian biology questions. Firstly, increasing the mRNA degradation rate of Per can contribute to increase or decrease of the period which might explain contradictory experimental findings. Secondly, our data points to a more relevant role of the ROR/Bmal/REV-ERB loop. In particular, that this loop can be an oscillator on its own. We provide experimental evidence that overexpression of members of the ROR/Bmal/REV-ERB lead to loss of Bmal reporter mRNA oscillations. The fact that REV-ERB and ROR are nuclear receptors and therefore important regulators in many cellular processes might have important implications for molecular biology and medicine.
Related Articles
Nascent-Seq reveals novel features of mouse circadian transcriptional regulation
Menet, Jerome S
Rodriguez, Joseph
Abruzzi, Katharine C
Rosbash, Michael
A substantial fraction of the metazoan transcriptome undergoes circadian oscillations in many cells and tissues. Based on the transcription feedback loops important for circadian timekeeping, it is commonly assumed that this mRNA cycling reflects widespread transcriptional regulation. To address this issue, we directly measured the circadian dynamics of mouse liver transcription using Nascent-Seq (genome-wide sequencing of nascent RNA). Although many genes are rhythmically transcribed, many rhythmic mRNAs manifest poor transcriptional rhythms, indicating a prominent contribution of post-transcriptional regulation to circadian mRNA expression. This analysis of rhythmic transcription also showed that the rhythmic DNA binding profile of the transcription factors CLOCK and BMAL1 does not determine the transcriptional phase of most target genes. This likely reflects gene-specific collaborations of CLK:BMAL1 with other transcription factors. These insights from Nascent-Seq indicate that it should have broad applicability to many other gene expression regulatory issues.
DOI: http://dx.doi.org/10.7554/eLife.00011.001
eLife digest
Many biological processes oscillate with a period of roughly 24 hr, and the ability of organisms as diverse as bacteria and humans to maintain such circadian rhythms, even under conditions of continuous darkness, influences a range of phenomena, including sleep, migration and reproduction. One characteristic of circadian rhythms is that they can adjust to local time (with humans suffering from jet lag as they wait for this to happen).
Experiments have shown that the circadian system in mammals relies on feedback loops that operate at the level of individual cells. These loops are controlled by two particular proteins, which comprise the transcription factor complex called BMAL1:CLK. Transcription factors cause particular sequences of bases in the DNA of cells to be transcribed into messenger RNA, thus starting the process by which target genes are expressed as proteins. In the case of BMAL1:CLK, these proteins are then modified, which inhibits any further transcription of the target genes. A reversal of these modifications is then followed by the synthesis of new proteins, which allows a new cycle of the transcription process to begin.
The amounts of many messenger RNAs (mRNAs) in a cell also increases and decreases with a period of 24 hr, and it was generally assumed that this was due to the changes in the level of transcription. More recently, however, it was suggested that other processes, such as splicing and translation, might also contribute to rhythmic changes in the amount of mRNA associated with particular genes. Such post-transcriptional processes are known to have a role in other areas of cell biology, including aspects of the circadian system, but until very recently this had not been studied in detail for all genes.
Now Menet et al. have directly assayed rhythmic transcription by measuring the amount of nascent mRNA being produced at a given time, six times a day, across all the genes in mouse liver cells using a high-throughput sequencing approach called Nascent-Seq. They compared this with the amount of liver mRNA expressed at six time points of the day. Although the authors found that many genes exhibit rhythmic mRNA expression in the mouse liver, about 70% of them did not show comparable transcriptional rhythms. Post-transcriptional regulation must, therefore, have a major role in the circadian system of mice and, presumably, other mammals.
Menet et al. also found that the influence of CLK:BMAL1 differed from what was expected, which suggests that it collaborates with a number of other transcription factors to effect transcription of most target genes. In addition to showing that circadian systems of mammals are more complex than previously believed, the results also illustrate the potential of Nascent-Seq as a genome-wide assay technique for exploring a range of questions related to gene expression and gene regulation.
DOI: http://dx.doi.org/10.7554/eLife.00011.002
Circadian rhythms; transcription; nascent RNA; high-throughput sequencing; RNA processing; post-transcriptional regulation; Mouse
Related Articles
Modeling an Evolutionary Conserved Circadian Cis-Element
Paquet, Eric R
Rey, Guillaume
Naef, Felix
PLoS Computational Biology
Circadian oscillator networks rely on a transcriptional activator called CLOCK/CYCLE (CLK/CYC) in insects and CLOCK/BMAL1 or NPAS2/BMAL1 in mammals. Identifying the targets of this heterodimeric basic-helix-loop-helix (bHLH) transcription factor poses challenges and it has been difficult to decipher its specific sequence affinity beyond a canonical E-box motif, except perhaps for some flanking bases contributing weakly to the binding energy. Thus, no good computational model presently exists for predicting CLK/CYC, CLOCK/BMAL1, or NPAS2/BMAL1 targets. Here, we use a comparative genomics approach and first study the conservation properties of the best-known circadian enhancer: a 69-bp element upstream of the Drosophila melanogaster period gene. This fragment shows a signal involving the presence of two closely spaced E-box–like motifs, a configuration that we can also detect in the other four prominent CLK/CYC target genes in flies: timeless, vrille, Pdp1, and cwo. This allows for the training of a probabilistic sequence model that we test using functional genomics datasets. We find that the predicted sequences are overrepresented in promoters of genes induced in a recent study by a glucocorticoid receptor-CLK fusion protein. We then scanned the mouse genome with the fly model and found that many known CLOCK/BMAL1 targets harbor sequences matching our consensus. Moreover, the phase of predicted cyclers in liver agreed with known CLOCK/BMAL1 regulation. Taken together, we built a predictive model for CLK/CYC or CLOCK/BMAL1-bound cis-enhancers through the integration of comparative and functional genomics data. Finally, a deeper phylogenetic analysis reveals that the link between the CLOCK/BMAL1 complex and the circadian cis-element dates back to before insects and vertebrates diverged.
Author Summary
Life on earth is subject to daily light/dark and temperature cycles that reflect the earth rotation about its own axis. Under such conditions, organisms ranging from bacteria to human have evolved molecularly geared circadian clocks that resonate with the environmental cycles. These clocks serve as internal timing devices to coordinate physiological and behavioral processes as diverse as detoxification, activity and rest cycles, or blood pressure. In insects and vertebrates, the clock circuitry uses interlocked negative feedback loops which are implemented by transcription factors, among which the heterodimeric activators CLOCK and CYCLE play a key role. The specific DNA elements recognized by this factor are known to involve E-box motifs, but the low information content of this sequence makes it a poor predictor of the targets of CLOCK/CYCLE on a genome-wide scale. Here, we use comparative genomics to build a more specific model for a CLOCK-controlled cis-element that extends the canonical E-boxes to a more complex dimeric element. We use functional data from Drosophila and mouse circadian experiments to test the validity and assess the performance of the model. Finally, we provide a phylogenetic analysis of the cis-elements across insect and vertebrates that emphasizes the ancient link between CLOCK/CYCLE and the modeled enhancer. These results indicate that comparative genomics provides powerful means to decipher the complexity of the circadian cis-regulatory code.
Related Articles
Cell Type-Specific Functions of Period Genes Revealed by Novel Adipocyte and Hepatocyte Circadian Clock Models
Ramanathan, Chidambaram
Xu, Haiyan
Khan, Sanjoy K.
Shen, Yang
Gitis, Paula J.
Welsh, David K.
Hogenesch, John B.
Liu, Andrew C.
PLoS Genetics
In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.
Author Summary
Various aspects of our daily rhythms in physiology and behavior such as the sleep-wake cycle are regulated by endogenous circadian clocks that are present in nearly every cell. It is generally accepted that these oscillators share a similar biochemical negative feedback mechanism, consisting of transcriptional activators and repressors. In this study, we developed cell-autonomous, metabolically relevant clock models in mouse hepatocytes and adipocytes. Each clock model has an integrated luciferase reporter that allows for kinetic luminescence recording with an inexpensive microplate reader and thus is feasible for most laboratories. These models are amenable to high throughput screening of small molecules or genomic entities for impacts on cell-autonomous clocks relevant to metabolism. We validated these new models by RNA interference via lentivirus-mediated knockdown of known clock genes. As expected, we found that many core clock components have similar functions across cell types. To our surprise, however, we also uncovered previously under-appreciated cell type-specific functions of core clock genes, particularly Per1, Per2, and Per3. Because the circadian system is integrated with, and influenced by, the local physiology that is under its control, our studies provide important implications for future studies into cell type-specific mechanisms of various circadian systems.
Related Articles
Comprehensive analysis of microRNA-mRNA co-expression in circadian rhythm
Na, Young-Ji
Sung, Jung Hwan
Lee, Suk Chan
Lee, Young-Ju
Choi, Yeun Joo
Park, Woong-Yang
Shin, Hee Sup
Kim, Ju Han
Experimental & Molecular Medicine
To investigate the potential role of microRNA (miRNA) in the regulation of circadian rhythm, we performed microarray-based expression profiling study of both miRNA and mRNA in mouse liver for 48 h at 4-hour intervals. Circadian miRNA-mRNA target pair is defined as the pair both elements of which show circadian expression patterns and the sequence-based target relationship of which can be predicted. Circadian initiators, Clock and Bmal1, showed inversely correlated circadian expression patterns against their corresponding miRNAs, miR-181d and miR-191, targeting them. In contrast, circadian suppressors, Per, Cry, CKIe and Rev-erba, exhibited positively correlated circadian expression patterns to their corresponding miRNAs. Genomic location analysis revealed that intronic region showed higher abundance of cyclic than non-cyclic miRNAs targeting circadian genes while other (i.e., 3'-UTR, exon and intergenic) regions showed no difference. It is suggested that miRNAs are involved in the regulation of peripheral circadian rhythm in mouse liver by modulating Clock:Bmal1 complex. Identifying specific miRNAs and their targets that are critically involved in circadian rhythm will provide a better understanding of the regulation of circadian-clock system.
circadian rhythm; gene expression profiling; gene expression regulation; liver; mice; micro-RNA
Related Articles
Effect of Resveratrol, a SIRT1 Activator, on the Interactions of the CLOCK/BMAL1 Complex
Park, Insung
Lee, Yool
Kim, Hee-Dae
Kim, Kyungjin
Endocrinology and Metabolism
In mammals, the CLOCK/BMAL1 heterodimer is a key transcription factor complex that drives the cyclic expression of clock-controlled genes involved in various physiological functions and behavioral consequences. Recently, a growing number of studies have reported a molecular link between the circadian clock and metabolism. In the present study, we explored the regulatory effects of SIRTUIN1 (SIRT1), an NAD+-dependent deacetylase, on CLOCK/BMAL1-mediated clock gene expression.
To investigate the interaction between SIRT1 and CLOCK/BMAL1, we conducted bimolecular fluorescence complementation (BiFC) analyses supplemented with immunocytochemistry assays. BiFC experiments employing deletion-specific mutants of BMAL1 were used to elucidate the specific domains that are necessary for the SIRT1-BMAL1 interaction. Additionally, luciferase reporter assays were used to delineate the effects of SIRT1 on circadian gene expression.
BiFC analysis revealed that SIRT1 interacted with both CLOCK and BMAL1 in most cell nuclei. As revealed by BiFC assays using various BMAL1 deletion mutants, the PAS-B domain of BMAL1 was essential for interaction with SIRT1. Activation of SIRT1 with resveratrol did not exert any significant change on the interaction with the CLOCK/BMAL1 complex. However, promoter analysis using Per1-Luc and Ebox-Luc reporters showed that SIRT1 significantly downregulated both promoter activities. This inhibitory effect was intensified by treatment with resveratrol, indicating a role for SIRT1 and its activator in CLOCK/BMAL1-mediated transcription of clock genes.
These results suggest that SIRT1 may form a regulatory complex with CLOCK/BMAL1 that represses clock gene expression, probably via deacetylase activity.
Circadian clocks; CLOCK/BMAL1 heterodimer; SIRT1; BiFC analysis; Resveratrol
Related Articles
SRC-2 Is an Essential Coactivator for Orchestrating Metabolism and Circadian Rhythm
Stashi, Erin
Lanz, Rainer B.
Mao, Jianqiang
Michailidis, George
Zhu, Bokai
Kettner, Nicole M.
Putluri, Nagireddy
Reineke, Erin L.
Reineke, Lucas C.
Dasgupta, Subhamoy
Dean, Adam
Stevenson, Connor R.
Sivasubramanian, Natarajan
Sreekumar, Arun
DeMayo, Francesco
York, Brian
Fu, Loning
O'Malley, Bert W.
Cell reports
Synchrony of the mammalian circadian clock is achieved by complex transcriptional and translational feedback loops centered on the BMAL1: CLOCK heterodimer. Modulation of circadian feedback loops is essential for maintaining rhythmicity, yet the role of transcriptional coactivators in driving BMAL1:CLOCK transcriptional networks is largely unexplored. Here, we show diurnal hepatic steroid receptor coactivator 2 (SRC-2) recruitment to the genome that extensively overlaps with the BMAL1 cistrome during the light phase, targeting genes that enrich for circadian and metabolic processes. Notably, SRC-2 ablation impairs wheel-running behavior, alters circadian gene expression in several peripheral tissues, alters the rhythmicity of the hepatic metabolome, and deregulates the synchronization of cell-autonomous metabolites. We identify SRC-2 as a potent coregulator of BMAL1:CLOCK and find that SRC-2 targets itself with BMAL1:CLOCK in a feedforward loop. Collectively, our data suggest that SRC-2 is a transcriptional coactivator of the BMAL1:CLOCK oscillators and establish SRC-2 as a critical positive regulator of the mammalian circa-dian clock.
Related Articles
Machine Learning Helps Identify CHRONO as a Circadian Clock Component
Anafi, Ron C.
Lee, Yool
Sato, Trey K.
Venkataraman, Anand
Ramanathan, Chidambaram
Kavakli, Ibrahim H.
Hughes, Michael E.
Baggs, Julie E.
Growe, Jacqueline
Liu, Andrew C.
Kim, Junhyong
Hogenesch, John B.
PLoS Biology
Two independent studies, one of them using a computational approach, identified CHRONO, a gene shown to modulate the activity of circadian transcription factors and alter circadian behavior in mice.
Over the last decades, researchers have characterized a set of "clock genes" that drive daily rhythms in physiology and behavior. This arduous work has yielded results with far-reaching consequences in metabolic, psychiatric, and neoplastic disorders. Recent attempts to expand our understanding of circadian regulation have moved beyond the mutagenesis screens that identified the first clock components, employing higher throughput genomic and proteomic techniques. In order to further accelerate clock gene discovery, we utilized a computer-assisted approach to identify and prioritize candidate clock components. We used a simple form of probabilistic machine learning to integrate biologically relevant, genome-scale data and ranked genes on their similarity to known clock components. We then used a secondary experimental screen to characterize the top candidates. We found that several physically interact with known clock components in a mammalian two-hybrid screen and modulate in vitro cellular rhythms in an immortalized mouse fibroblast line (NIH 3T3). One candidate, Gene Model 129, interacts with BMAL1 and functionally represses the key driver of molecular rhythms, the BMAL1/CLOCK transcriptional complex. Given these results, we have renamed the gene CHRONO (computationally highlighted repressor of the network oscillator). Bi-molecular fluorescence complementation and co-immunoprecipitation demonstrate that CHRONO represses by abrogating the binding of BMAL1 to its transcriptional co-activator CBP. Most importantly, CHRONO knockout mice display a prolonged free-running circadian period similar to, or more drastic than, six other clock components. We conclude that CHRONO is a functional clock component providing a new layer of control on circadian molecular dynamics.
Author Summary
Daily rhythms are ever-present in the living world, driving the sleep–wake cycle and many other physiological changes. In the last two decades, several labs have identified "clock genes" that interact to generate underlying molecular oscillations. However, many aspects of circadian molecular physiology remain unexplained. Here, we used a simple "machine learning" approach to identify new clock genes by searching the genome for candidate genes that share clock-like features such as cycling, broad-based tissue RNA expression, in vitro circadian activity, genetic interactions, and homology across species. Genes were ranked by their similarity to known clock components and the candidates were screened and validated for evidence of clock function in vitro. One candidate, which we renamed CHRONO (Gm129), interacted with the master regulator of the clock, BMAL1, disrupting its transcriptional activity. We found that Chrono knockout mice had prolonged locomotor activity rhythms, getting up progressively later each day. Our experiments demonstrated that CHRONO interferes with the ability of BMAL1 to recruit CBP, a bona fide histone acetylase and key transcriptional coactivator of the circadian clock.
Related Articles
Profiling of Circadian Genes Expressed in the Uterus Endometrial Stromal Cells of Pregnant Rats as Revealed by DNA Microarray Coupled with RNA Interference
Tasaki, Hirotaka
Zhao, Lijia
Isayama, Keishiro
Chen, Huatao
Nobuhiko Yamauchi,
Yasufumi Shigeyoshi,
Hashimoto, Seiichi
Hattori, Masa-aki
Frontiers in Endocrinology
The peripheral circadian oscillator plays an essential role in synchronizing local physiology to operate in a circadian manner via regulation of the expression of clock-controlled genes. The present study aimed to evaluate the circadian rhythms of clock genes and clock-controlled genes expressed in the rat uterus endometrial stromal cells (UESCs) during the stage of implantation by a DNA microarray. Of 12,252 genes showing significantly expression, 7,235 genes displayed significant alterations. As revealed by the biological pathway analysis using the database for annotation, visualization, and integrated discovery online annotation software, genes were involved in cell cycle, glutathione metabolism, MAPK signaling pathway, fatty acid metabolism, ubiquitin mediated proteolysis, focal adhesion, and PPAR signaling pathway. The clustering of clock genes were mainly divided into four groups: the first group was Rorα, Timeless, Npas2, Bmal1, Id2, and Cry2; the second group Per1, Per2, Per3, Dec1, Tef, and Dbp; the third group Bmal2, Cry1, E4bp4, Rorβ, and Clock; the fourth group Rev-erbα. Eleven implantation-related genes and 24 placenta formation-related genes displayed significant alterations, suggesting that these genes involved in implantation and placenta formation are controlled under circadian clock. Some candidates as clock-controlled genes were evaluated by using RNA interference to Bmal1 mRNA. Down-regulation of Igf1 gene expression was observed by Bmal1 silencing, whereas the expression of Inhβa was significantly increased. During active oscillation of circadian clock, the apoptosis-related genes Fas and Caspase3 remained no significant changes, but they were significantly increased by knockdown of Bmal1 mRNA. These results indicate that clock-controlled genes are up- or down-regulated in rat UESCs during the stage of decidualization. DNA microarray analysis coupled with RNA interference will be helpful to understand the physiological roles of some oscillating genes in blastocyst implantation and placenta formation.
uterus endometrial stromal cells; clock genes; clock-controlled genes; DNA microarray; implantation; decidualization; Bmal1 siRNA; Per2-dLuc reporter gene
Related Articles
Loss of Bmal1 leads to uncoupling and impaired glucose-stimulated insulin secretion in β-cells
Lee, Jeongkyung
Kim, Mi-Sun
Li, Rongying
Liu, Victoria Y.
Fu, Loning
Moore, David D.
Ma, Ke
Yechoor, Vijay K.
The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia suggesting β-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of β-cell function by Bmal1. We studied β-cell function in global Bmal1-/- mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1-/- mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1-/- islets and in Bmal1 knockdown cells, as compared with controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1-/- mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1-/- islets. This defect in GSIS occurs as a result of increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2 in isolated islets leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1-/- islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.
Bmal1; circadian clock; diabetes; insulin secretion; mitochondria; β-cells
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Welcome to the National Student Television Association: The one true home of Student Television. We have been at the heart of student media for over 40 years and we are still going strong. So come in, sit down and let us tell you all about NaSTA ...
The last Pick of the Week for September is the first from our London Regional Officer, Catriona O'Sullivan. She has chosen rubarbTV's Society Challenge Episode 3: "Rhubarb has been working... More
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Miss Peregrine's Home for Peculiar Children
The latest adventure from Tim Burton would seem tailor-made for his tastes but it's a convoluted slog, dense in mythology and explanatory dialogue but woefully…
The latest adventure from Tim Burton would seem tailor-made for his tastes but it's a convoluted slog, dense in mythology and explanatory dialogue but woefully…
Everything here feels routine—more like an inevitability than a work of art or even a piece of entertainment.
Roger Ebert on James Ivory's "Howards End".
"The Ballad of Narayama" is a Japanese film of great beauty and elegant artifice, telling a story of startling cruelty. What a space it opens…
Ken Loach's "I, Daniel Blake" wins the Palme d'Or at the 69th Cannes Film Festival.
Barbara Scharres on the cats of Cannes 2016.
An article about Hillary Clinton's historic nomination as the first female presidential candidate and the most qualified
An article about the upcoming premiere of Issa Rae's HBO series, "Insecure," in Chicago.
A comparison between the recent version of "Ben-Hur" and the classic 1959 version by William Wyler.
A table of contents for Omer M. Mozaffar's four-part essay series on Islam in Western cinema.
A preview of the 54th New York Film Festival, including "Son of Joseph," "The Rehearsal," "Graduation," "Sieranevada" and much more.
Matt Zoller Seitz celebrates "Beyond the Valley of the Dolls," now available on Criterion Blu-ray and DVD.
There's a small tomb in Southwark Cathedral that I like to visit when I am in London. It contains the bones of a teenage girl who died three centuries ago. I know the inscription by heart:
This world to her
Was but a tragic play.
She came, saw, dislik'd,
And passed away.
I thought of those words while I was watching "Ghost World," the story of an 18-year-old girl from Los Angeles who drifts forlorn through her loneliness, cheering herself up with an ironic running commentary. The girl is named Enid, she has just graduated from high school, and she has no plans for college, marriage, a career or even next week. She's stuck in a world of stupid, shallow phonies, and she makes her personal style into a rebuke.
Unfortunately, Enid is so smart, so advanced, and so ironically doubled back upon herself, that most of the people she meets don't get the message. She is second-level satire in a one-level world, and so instead of realizing, for example, that she is mocking the 1970s punk look, stupid video store clerks merely think she's 25 years out of style.
Enid is played by Thora Birch, from "American Beauty," and in a sense this character is a continuation of that one--she certainly looks at her father the same way, with disbelief and muted horror. Her running-mate is Rebecca (Scarlett Johansson). There's a couple like this in every high school: The smart outsider girls who are best friends for the purpose of standing back to back and fighting off the world. At high school graduation, they listen to a speech from a classmate in a wheelchair, and Enid whispers, "I liked her so much better when she was an alcoholic and drug addict. She gets in one stupid car crash and suddenly she's Little Miss Perfect." But now Rebecca is showing alarming signs of wanting to get on with her life, and Enid is abandoned to her world of thrift shops, strip malls, video stores and 1950s retro diners. One day, in idle mischief, she answers a personal ad in a local paper, and draws into her net a pathetic loner named Seymour (Steve Buscemi). At first she strings him along. Then, unexpectedly, she starts to like him--this collector who lives hermetically sealed in a world of precious 78 rpm records and old advertising art.
By day, Seymour is an insignificant fried chicken executive. By night, he catalogs his records and wonders how to meet a woman. Why does Enid like him? "He's the exact opposite of all the things I hate." Why does he like her? Don't get ahead of the story. "Ghost World" isn't a formula romance where opposites attract and march toward the happy ending.
Seymour and Enid are too similar to fall in love; they both specialize in complex personal lifestyles that send messages no one is receiving. Enid even offers to try to fix up Seymour, but he sees himself as a bad candidate for a woman: "I don't want to meet someone who shares my interests. I hate my interests." Seymour resembles someone I know, and that person is Terry Zwigoff, who directed this movie. It's his first fiction film. Zwigoff earlier made two documentaries, the masterpiece "Crumb" (1995), about the comic artist R. Crumb, and "Louie Bluie," about the old-timey Chicago string band Martin, Bogan and the Armstrongs. He looks a little like Buscemi, and acts like a Buscemi character: worn down, dubious, ironic, resigned. Zwigoff was plagued by agonizing back pain all during the period when he was making "Crumb," and slept with a gun under his pillow, he told me, in case he had to end his misery in the middle of the night. When Crumb didn't want to cooperate with the documentary, Zwigoff threatened to shoot himself. Crumb does not often meet his match, but did with Zwigoff.
Both Zwigoff and his character Seymour collect old records that are far from the mainstream. Both are morose and yet have a bracing black humor that sees them through. Seymour and Enid connect because they are kindred spirits, and it's hard to find someone like that when you've cut yourself off from mankind.
The movie is based on an underground comic book by Daniel Clowes, who co-wrote the screenplay with Zwigoff. It listens carefully to how people talk. Illeana Douglas, for example, has a perfectly observed role as the art teacher of Enid's summer make-up class, who has fallen for political correctness hook, line and sinker, and praises art not for what it looks like but for what it "represents." There are also some nice moments from Teri Garr, who plays the take-charge girlfriend of Enid's father (Bob Balaban).
One scene I especially like involves a party of Seymour's fellow record collectors. They meet to exchange arcane information, and their conversations are like encryptions of the way most people talk. This event must seem strange to Enid, but see how she handles it. It's Seymour's oddness, his tactless honesty, his unapologetic aloneness, that Enid responds to. He works like the homeopathic remedy for angst: His loneliness drives out her own.
I wanted to hug this movie. It takes such a risky journey and never steps wrong. It creates specific, original, believable, lovable characters, and meanders with them through their inconsolable days, never losing its sense of humor. The Buscemi role is one he's been pointing toward during his entire career; it's like the flip side of his alcoholic barfly in "Trees Lounge," who also becomes entangled with a younger girl, not so fortunately.
The movie sidesteps the happy ending Hollywood executives think lobotomized audiences need as an all-clear to leave the theater. Clowes and Zwigoff find an ending that is more poetic, more true to the tradition of the classic short story, in which a minor character finds closure that symbolizes the next step for everyone. "Ghost World" is smart enough to know that Enid and Seymour can't solve their lives in a week or two. But their meeting has blasted them out of lethargy, and now movement is possible. Who says that isn't a happy ending?
This message came to me from a reader named Peter Svensland. He and a fr...
An article about Hillary Clinton's historic nomination as the first female presidential candidate and the most qualif...


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Benjamin's Modern Glam Bachelor Pad
- • The perfect color for any home, palace white on the main walls.
• Accents walls in truffle brown and matte black.
• Bedroom walls are custom faux finished in a metallic plaster in charcoal gray. I wanted the walls to look like worn leather.
• The bathroom is Ralph Lauren beep blue.
- • Marble top table: Create and Barrel
• Hide skin and horn bench: Art by God
• Mirror: Z Gallery
• Fabric: Fishman's
- • Sofa: Boston Store Furniture Gallery
• High gloss white coffee table: West Elm
• Sisel rug with black leather trim: Bombay Company
• Crystal chandeliers: Swarovski
• Lucite lamps with black shades: Lamps plus
• 2 armed chrome side lamp: Macy's
• Black wood side table: vintage
• Iron glass top writing desk: Bombay Company
• Black lucite ghost chair- Design within Reach
• Zebra framed beveled mirror: vintage
• Gold Italian rope and tassel beverage cart: vintage (along with the barware on them)
• Black wood cabinet: vintage
• Telescope: Bombay Company
• Leather slipper chairs: Elements
- • Brown leather tufted bed: Z Gallery
• Bedside tables: Design within Reach
• Lamps: vintage (grandma's basement)
• Side chair: vintage
• Wood stool with leopard top: vintage
• Faux finished walls to look like warn leather: Kath's Creations
Jason Loper • HOUSE TOUR ARCHIVE Check out past house tours here • Interested in sharing your home with Apartment Therapy? Contact the editors through our House Tour Submission Form. • Are you a designer/architect/decorator interested in sharing a residential project with Apartment Therapy readers? Contact the editors through our Professional Submission Form.


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According to hiphollywood.com Tami Roman has suddenly gotten a whole lot lighter. Now while you can immediately tell how much weight she's lost…we mean lighter in another way! The internets are swirling with activity claiming that Miss Tami has indeed gone the way of Sammy Sosa and bleached her skin in order to be lighter complected.
RELATED::Algebra Blessett Shows Growth In R&B [EXCLUSIVE VIDEO]
Tami took to social media and said,
"I see the little bootleg bloggers jumped on a bullsh*t rumor for clicks LMAO. GM world…dont believe the hype – stay blessed."
But even she must admit the pics that she recently posted on her instagram are startling! Especially since we thought she was absolutely gorgeous the way she was!
Here is another pic that she posted that is prompting whispers below!
The problem is that this appears to be a totally different day and totally different clothes. You know how the rumor mill does it…and once it's out there…it's sort of out there. The word is that Tami and or her camp are saying that it was simply the flash from the camera. Before you screw your face all the way up at that notion…this could actually be a real thing as some of us have had it happen in pictures before. But we digress.
If it's not true Miss Roman should be thrilled to know that while the rumor part is no doubt annoying people really feel she is a beauty as she is. If it is true…then everyone is upset with her for doing something that never needed to be done. Tami you're a knockout…we hope that it's not true because your coffee and cream complexion was just stunning. Don't be mad…cause that's love…even if it might "get us all popped" for saying it!
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50 Brown-Skinned Beauties In Hollywood
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incinerator Companies serving Equatorial Guinea
Current filtering criteriaYou can remove a filter by clicking on the x
based in Novate Milanese, ITALY
Testori is an international group that has been operating for over 100 years in industrial filtration. The core business is in designing, producing and marketing textile products (fabrics, felts, filter bags) for gas filtration and fabrics (cloths, bags) for liquid filtration. The main areas of application are: cement and aluminium industries, power generation, and the ...
Environmental Source Samplers, Inc.
based in Wilmington, NORTH CAROLINA (USA)
Environmental Source Samplers, Inc. (ESS) has a long and proven track record. We have been providing air quality related services since 1979. Headquartered in Wilmington, North Carolina, ESS has provided stack testing services in most of the fifty states and internationally. ESS serves clients in many industries including: Aluminum, Asphalt, Automobile, Carbon, ...
Catalytic Products International (CPI)
based in Lake Zurich, ILLINOIS (USA)
Catalytic Products International is the preferred resource for innovative and efficient industrial air pollution control solutions. Customers from across the globe rely on our expertise to solve their most complex VOC, NOx, and Odor pollution challenges. The custom engineered systems include, Thermal Oxidizers, Catalytic Oxidizers, Regenerative Thermal Oxidizers, Heat ...
Procal - Parker Hannifin Corp
based in Peterborough, UNITED KINGDOM
Procal design and manufacture continuous emissions monitors suitable for applications that come under the European Large Combustion Plant and Waste Incineration Directives. These monitors are also used on US EPA approved sites. Certified under MCERTS for the measurement of NO, SO2, CO, CO2, H2O, and providing information as required for compliance with EN14181, Procal ...
Babcock & Wilcox MEGTEC
based in De Pere, WISCONSIN (USA)
Babcock & Wilcox MEGTEC provides environmental compliance solutions that make economic sense. B&W MEGTEC offers a portfolio of proven, high-efficiency technologies including wet electrostatic precipitators, scrubbers and liquid atomization systems for particulate and acid gas control, DeNOx and evaporative gas cooling, regenerative thermal oxidizers (RTOs) for VOC ...
Anguil Environmental Systems, Inc.
based in Milwaukee, WISCONSIN (USA)
Anguil Environmental Systems is a global provider of industrial air pollution control, emission abatement and energy recovery systems. We design, manufacturer, service and install thermal and catalytic oxidation technologies as well as heat recovery systems for a variety of applications and industries. Anguil Aqua was developed to help our industrial clients with water ...
NEO Monitors AS
based in Skedsmokorset, NORWAY
NEO Monitors is one of the leading suppliers of gas analysers and dust monitors based on tuneable diode laser (TDL) technology. Our instruments are the preferred choice for a broad range of applications within process and emission control. Laser based Gas Monitors for Environmental Monitoring or Process Control. NEO Monitors supply state of the art LaserGas gas monitors ...
Macpresse Europa S.R.L
based in Vernate, ITALY
Macpresse International is an Italian company headquartered in Vernate (Milan). Macpresse has been designing and installing recovery facilities for recycling secondary scrap materials for over 40 years. Macpresse provides scrap-handling solutions in the following sectors: recovery of first/secondary materials, processing of municipal solid waste (MSW) and industrial ...
Bionomic Industries Inc.
based in Mahwah, NEW JERSEY (USA)
Bionomic Industries' recently redesigned website. We appreciate your interest in our company and hope you find our new site user-friendly, most informative and helpful in solving your present or future air pollution, emission control, product or waste heat recovery application. As the dominant worldwide leader in providing conventional and advanced gas absorption, ...
Macrotek Inc.
based in Markham, ONTARIO (CANADA)
With more than 80 years of experience, Macrotek Engineering offers a variety of air pollution control systems to suite a broad range of environmental requirements. Using some of the most innovative and effective technologies available in the industry, Macrotek provides cost-efficient applications for industrial air purification. Our mission is to produce customized ...
SatrindTech Srl
based in Arluno, ITALY
SatrindTech is a mechanical and electrical engineering company based in Arluno (Milano) with well consolidated experience and leadership in engineering, manufacturing and installation of industrial shredders and complex plants to handle many different types of waste.
Advanced Cyclone Systems, S. A.
based in Porto, PORTUGAL
Advanced Cyclone Systems, S.A. (ACS) is a company exclusively dedicated to the development and commercialization of high efficiency cyclone systems, worldwide. The company works in a very close cooperation with its clients in order to design custom made cyclone systems that really solve their unmet needs.
based in Noerresundby, DENMARK
DESMI is a global company specialised in the development and manufacture of pump solutions for marine, industry, oil spill combating, defence & fuel and utility both locally and globally. Our product range supplemented with agency products from other leading, world class manufacturers – is completed by related services such as the design and installation of pump plants, ...
Granutech-Saturn Systems
based in Grand Prairie, TEXAS (USA)
Granutech Saturn Systems, offers a diversified line of recycling equipment systems that can be easily integrated into a single processing facility. From shredders to granulators to powederizers and refiner mills -- to auto crushers and balers/loggers, our engineers continuously develop products that meet the needs of your industry and application. But more importantly, ...
Franke-Filter GmbH
based in Bad Salzdetfurth, GERMANY
Franke-Filter manufactures oil mist separators for generating adjustable vacuum in the lube oil system and filtering the oil mist with an efficiency of more than 99.9%. Turbine engineers, maintenance managers and plant managers turn to Franke-Filter to solve the following problems:- lack of vacuum in the lube oil tank- leakages at the turbine bearings- oil mist ...
Midwesco Filter Resources, Inc.
based in Winchester, VIRGINIA (USA)
Midwesco Filter Resources designs and manufactures value-added industrial air filtration solutions for dust collectors, baghouses and air inlet houses on a worldwide basis. These systems are box-like structures where particulates, usually from industrial and utility sources, are removed from exhaust gases while passing through filter elements. Midwesco Filter makes filter ...
High Temperature Technologies Corp. Canada
based in Chateauguay, QUEBEC (CANADA)
Waste to Energy Conversion Systems. High Temperature Technologies Corp. is a leader in design, development and manufacturing of the plasma based waste treatment solutions. High Temperature Technologies Company is a privately-owned firm based in Montreal, Canada. It offers environmentally-friendly solutions for waste treatment and disposal. Its highly innovative technology ...
EnGlobe Corp.
based in Quebec, QUEBEC (CANADA)
EnGlobe Corp. is a worldwide leader when it comes to integrated environmental services. We have developed a unique expertise in the management of organic waste materials and in reusing or recovering decontaminated soils as well as in the precision testing and calibration of bulk tanks and storage systems. Our clients are in public administration, in business and industry ...
Begg Cousland Envirotec Limited
based in Glasgow, UNITED KINGDOM
Begg Cousland has specialised in the design & manufacture of Mist Eliminators, a broad range of wire & fibre form products used for industrial air pollution control and in-process filtration.In recent years we are proud to have developed innovative designs and equipment solutions for gas scrubbing and odour control problems. The Company's Technical Sales Team use our ...
ANDRITZ Separation - a division of the Andritz Group
based in Graz, AUSTRIA
ANDRITZ Separation, trusted partner for process solutions for waste water treatment. There is a wide range of challenges in municipal drinking water and waste water treatment: whereas in developing countries the focus is often on setting up a base structure for drinking water and wastewater, in industrialized countries water quality, efficiency and recycling of residual ...
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Day 145 Birds of a Feather
- Posted Aug. 29, 2010 by Marjorie Mcdonald in Planes Trains And Automobiles. Viewed 3680 times
- This is a migrated legacy post. Image resolution is low. Info
Proud Honda Goldwing owners, out for a late summer ride - wish I was doing the same :(
2 There are 2 comments, add yours!
Well spotted! Great capture :-)
Sandra Vermeulen
Fantastic machines,
wonderful shot. You captured the colors and shine very well. ;)