ORCID Profile
0000-0002-4739-6773
Current Organisations
University of Oxford
,
University of Manchester
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Biochemistry and cell biology | Systems biology |
Publisher: eLife Sciences Publications, Ltd
Date: 05-08-2021
DOI: 10.7554/ELIFE.63324
Abstract: The circadian clock component NR1D1 (REVERBα) is considered a dominant regulator of lipid metabolism, with global Nr1d1 deletion driving dysregulation of white adipose tissue (WAT) lipogenesis and obesity. However, a similar phenotype is not observed under adipocyte-selective deletion ( Nr1d1 Flox2-6 :Adipoq Cre ), and transcriptional profiling demonstrates that, under basal conditions, direct targets of NR1D1 regulation are limited, and include the circadian clock and collagen dynamics. Under high-fat diet (HFD) feeding, Nr1d1 Flox2-6 :Adipoq Cre mice do manifest profound obesity, yet without the accompanying WAT inflammation and fibrosis exhibited by controls. Integration of the WAT NR1D1 cistrome with differential gene expression reveals broad control of metabolic processes by NR1D1 which is unmasked in the obese state. Adipocyte NR1D1 does not drive an anticipatory daily rhythm in WAT lipogenesis, but rather modulates WAT activity in response to alterations in metabolic state. Importantly, NR1D1 action in adipocytes is critical to the development of obesity-related WAT pathology and insulin resistance.
Publisher: Cold Spring Harbor Laboratory
Date: 19-04-2018
DOI: 10.1101/303941
Abstract: Using genome-wide data from 697,828 research participants from 23andMe and UK Biobank, we increase the number of identified loci associated with being a morning person, a behavioural indicator of a person’s underlying circadian rhythm, from 24 to 351. Using data from 85,760 in iduals with activity-monitor derived measures of sleep timing we show that the chronotype loci influence sleep timing: the mean sleep timing of the 5% of in iduals carrying the most “morningness” alleles was 25 minutes earlier than the 5% carrying the fewest. The loci were enriched for genes involved in circadian regulation, cAMP, glutamate and insulin signalling pathways, and those expressed in the retina, hindbrain, hypothalamus, and pituitary. We provide evidence that being a morning person is causally associated with better mental health but does not appear to affect BMI or Type 2 diabetes. This study offers new insights into the biology of circadian rhythms and links to disease in humans.
Publisher: Oxford University Press (OUP)
Date: 20-03-2007
DOI: 10.1093/HMG/DDM048
Abstract: MAF, one of a family of large Maf bZIP transcription factors, is mutated in human developmental ocular disorders that include congenital cataract, microcornea, coloboma and anterior segment dysgenesis. Expressed early in the developing lens vesicle, it is central to regulation of lens crystallin gene expression. We report a semi-dominant mouse c-Maf mutation recovered after ENU mutatgenesis which results in the substitution, D90V, at a highly conserved residue within the N-terminal 35 amino-acid minimal transactivation domain (MTD). Unlike null and loss-of-function c-Maf mutations, which cause severe runting and renal abnormalities, the phenotype caused by the D90V mutation is isolated cataract. In reporter assays, D90V results in increased promoter activation, a situation similar to MTD mutations of NRL that also cause human disease. In contrast to wild-type protein, the c-Maf D90V mutant protein is not inhibited by protein kinase A-dependent pathways. The MTD of large Maf proteins has been shown to interact with the transcriptional co-activator p300 and we demonstrate that c-Maf D90V enhances p300 recruitment in a cell-type dependent manner. We observed the same for the pathogenic human NRL MTD mutation S50T, which suggests a common mechanism of action.
Publisher: Proceedings of the National Academy of Sciences
Date: 28-09-2020
Abstract: The circadian clock protein REVERBα is proposed to be a key regulator of liver metabolism. We now show that REVERBα action is critically dependent on metabolic state. Using transgenic mouse models, we show that the true role of REVERBα is to buffer against aberrant responses to metabolic perturbation, rather than confer rhythmic regulation to programs of lipid synthesis and storage, as has been thought previously. Thus, in the case of liver metabolism, the clock does not so much drive rhythmic processes, as provide protection against mistimed feeding cues. Understanding how the clock is coupled to metabolism is critical for understanding metabolic disease and the impacts of circadian disruptors such as shift work and 24-h lifestyles.
Publisher: Wiley
Date: 30-07-2009
DOI: 10.1002/ART.24659
Publisher: Wiley
Date: 15-06-2022
Publisher: Wiley
Date: 05-1998
Publisher: American Diabetes Association
Date: 04-09-2014
DOI: 10.2337/DB13-1835
Abstract: Obesity is a major risk factor for metabolic disease, with white adipose tissue (WAT) inflammation emerging as a key underlying pathology. We detail that mice lacking Reverbα exhibit enhanced fat storage without the predicted increased WAT inflammation or loss of insulin sensitivity. In contrast to most animal models of obesity and obese human patients, Reverbα−/− mice exhibit elevated serum adiponectin levels and increased adiponectin secretion from WAT explants in vitro, highlighting a potential anti-inflammatory role of this adipokine in hypertrophic WAT. Indeed, adiponectin was found to suppress primary macrophage responses to lipopolysaccharide and proinflammatory fatty acids, and this suppression depended on glycogen synthase kinase 3β activation and induction of A20. Attenuated inflammatory responses in Reverbα−/− WAT depots were associated with tonic elevation of A20 protein and ex vivo shown to depend on A20. We also demonstrate that adipose A20 expression in obese human subjects exhibits a negative correlation with measures of insulin sensitivity. Furthermore, bariatric surgery–induced weight loss was accompanied by enhanced WAT A20 expression, which is positively correlated with increased serum adiponectin and improved metabolic and inflammatory markers, including C-reactive protein. The findings identify A20 as a mediator of adiponectin anti-inflammatory action in WAT and a potential target for mitigating obesity-related pathology.
Publisher: Proceedings of the National Academy of Sciences
Date: 03-01-2020
Abstract: The circadian clock regulates many aspects of immunity. Bacterial infections are affected by time of day, but the mechanisms involved remain undefined. Here we show that loss of the core clock protein BMAL1 in macrophages confers protection against pneumococcal pneumonia. Infected mice show both reduced weight loss and lower bacterial burden in circulating blood. In vivo studies of macrophage phagocytosis reveal increased bacterial ingestion following Bmal1 deletion, which was also seen in vitro. BMAL1 −/− macrophages exhibited marked differences in actin cytoskeletal organization, a phosphoproteome enriched for cytoskeletal changes, with reduced phosphocofilin and increased active RhoA. Further analysis of the BMAL1 −/− macrophages identified altered cell morphology and increased motility. Mechanistically, BMAL1 regulated a network of cell movement genes, 148 of which were within 100 kb of high-confidence BMAL1 binding sites. Links to RhoA function were identified, with 29 genes impacting RhoA expression or activation. RhoA inhibition restored the phagocytic phenotype to that seen in control macrophages. In summary, we identify a surprising gain of antibacterial function due to loss of BMAL1 in macrophages, associated with a RhoA-dependent cytoskeletal change, an increase in cell motility, and gain of phagocytic function.
Publisher: Bioscientifica
Date: 04-2019
DOI: 10.1530/JOE-18-0584
Abstract: We previously identified a critical pathogenic role for mineralocorticoid receptor (MR) activation in cardiomyocytes that included a potential interaction between the MR and the molecular circadian clock. While glucocorticoid regulation of the circadian clock is undisputed, studies on MR interactions with circadian clock signalling are limited. We hypothesised that the MR influences cardiac circadian clock signalling, and vice versa. Aldosterone or corticosterone (10 nM) regulated Cry1 , Per1 , Per2 and ReverbA ( Nr1d1 ) gene expression patterns in H9c2 cells over 24 h. MR-dependent regulation of circadian gene promoters containing GREs and E-box sequences was established for CLOCK, Bmal, CRY1 and CRY2, PER1 and PER2 and transcriptional activators CLOCK and Bmal modulated MR-dependent transcription of a subset of these promoters. We also demonstrated differential regulation of MR target gene expression in hearts of mice 4 h after administration of aldosterone at 08:00 h vs 20:00 h. Our data support MR regulation of a subset of circadian genes, with endogenous circadian transcription factors CLOCK and BMAL modulating the response. This unsuspected relationship links MR in the heart to circadian rhythmicity at the molecular level and has important implications for the biology of MR signalling in response to aldosterone as well as cortisol. These data are consistent with MR signalling in the brain where, like the heart, it preferentially responds to cortisol. Given the undisputed requirement for diurnal cortisol release in the entrainment of peripheral clocks, the present study highlights the MR as an important mechanism for transducing the circadian actions of cortisol in addition to glucocorticoid receptor (GR) in the heart.
Publisher: Springer Science and Business Media LLC
Date: 21-09-2017
DOI: 10.1038/S41598-017-09722-Z
Abstract: The glucocorticoid receptor (GR) is essential for the stress response in mammals. We investigated potential non-transcriptional roles of GR in cellular stress response using fission yeast as a model.We surprisingly discovered marked heat stress resistance in yeast ectopically expressing human GR, which required expression of both the N-terminal transactivation domain, and the C-terminal ligand binding domain, but not the DNA-binding domain of the GR. This effect was not affected by GR ligand exposure, and occurred without significant GR nuclear accumulation. Mechanistically, the GR survival effect required Hsp104, and, indeed, GR expression increased Hsp104 expression. Proteomic analysis revealed GR binding to translasome components, including eIF3, a known partner for Sty1, a pattern of protein interaction which we confirmed using yeast two-hybrid studies.Taken together, we find evidence for a novel pathway conferring stress resistance in yeast that can be activated by the human GR, acting by protein-protein mechanisms in the cytoplasm. This suggests that in organisms where GR is natively expressed, GR likely contributes to stress responses through non-transcriptional mechanisms in addition to its well-established transcriptional responses.
Publisher: Springer Science and Business Media LLC
Date: 25-02-2019
Publisher: Oxford University Press (OUP)
Date: 19-11-2010
DOI: 10.1111/J.1365-2249.2010.04289.X
Abstract: The aim of this study was to determine the genetic regulation of macrophage migration inhibitory factor (MIF). DNase I hypersensitivity was used to identify potential hypersensitive sites (HS) across the MIF gene locus. Reporter gene assays were performed in different human cell lines with constructs containing the native or mutated HS element. Following phylogenetic and transcription factor binding profiling, electrophoretic mobility shift assay (EMSA) and RNA interference were performed and the effects of incubation with mithramycin, an antibiotic that binds GC boxes, were also studied. An HS centred on the first intron of MIF was identified. The HS acted as an enhancer in human T lymphoblasts (CEMC7A), human embryonic kidney cells (HEK293T) and human monocytic cells (THP-1), but not in a fibroblast-like synoviocyte (FLS) cell line (SW982) or cultured FLS derived from rheumatoid arthritis (RA) patients. Two cis-elements within the first intron were found to be responsible for the enhancer activity. Mutation of the consensus Sp1 GC box on each cis-element abrogated enhancer activity and EMSA indicated Sp1 binding to one of the cis-elements contained in the intron. SiRNA knock-down of Sp1 alone or Sp1 and Sp3 together was incomplete and did not alter the enhancer activity. Mithramycin inhibited expression of MIF in CEMC7A cells. This effect was specific to the intronic enhancer and was not seen on the MIF promoter. These results identify a novel, cell type-specific enhancer of MIF. The enhancer appears to be driven by Sp1 or related Sp family members and is highly sensitive to inhibition via mithramycin.
Publisher: Cold Spring Harbor Laboratory
Date: 25-09-2020
DOI: 10.1101/2020.09.23.308155
Abstract: The circadian clock component REVERBα is considered a dominant regulator of lipid metabolism, with global Reverbα deletion driving dysregulation of white adipose tissue (WAT) lipogenesis and obesity. However, a similar phenotype is not observed under adipocyte-selective deletion ( Reverbα Flox2-6 Adipo Cre ), and transcriptional profiling demonstrates that, under basal conditions, direct targets of REVERBα regulation are limited, and include the circadian clock and collagen dynamics. Under high-fat diet (HFD) feeding, Reverbα Flox2-6 Adipo Cre mice do manifest profound obesity, yet without the accompanying WAT inflammation and fibrosis exhibited by controls. Integration of the WAT REVERBα cistrome with differential gene expression reveals broad control of metabolic processes by REVERBα which is unmasked in the obese state. Adipocyte REVERBα does not drive an anticipatory daily rhythm in WAT lipogenesis, but rather modulates WAT activity in response to alterations in metabolic state. Importantly, REVERBα action in adipocytes is critical to the development of obesity-related WAT pathology and insulin resistance.
Publisher: Proceedings of the National Academy of Sciences
Date: 06-04-2015
Abstract: We have discovered a role for the glucocorticoid receptor (GR) in coordinating cell ision. We find enrichment of GR to mitotic spindles and demonstrate that GR knockdown causes accumulation of mitotic defects, including delayed anaphase, ternary chromosome segregation, and death in mitosis. Mitotic GR function requires the ligand-binding domain but not ligand binding, revealing a nontranscriptional and ligand-independent mechanism of action. Analysis of GR haploinsufficient cells and tissues reveals increased aneuploidy and DNA damage, and mice show an increased incidence of tumors in vivo, with further GR loss within those incident tumors. We also identify reduced GR expression in several common human cancers, thereby implicating GR as a novel tumor suppressor gene.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 06-2021
DOI: 10.1161/STROKEAHA.120.031742
Abstract: Circadian biology modulates almost all aspects of mammalian physiology, disease, and response to therapies. Emerging data suggest that circadian biology may significantly affect the mechanisms of susceptibility, injury, recovery, and the response to therapy in stroke. In this review erspective, we survey the accumulating literature and attempt to connect molecular, cellular, and physiological pathways in circadian biology to clinical consequences in stroke. Accounting for the complex and multifactorial effects of circadian rhythm may improve translational opportunities for stroke diagnostics and therapeutics.
Publisher: BMJ
Date: 09-10-2018
DOI: 10.1136/THORAXJNL-2018-212021
Abstract: The importance of circadian factors in managing patients is poorly understood. We present two retrospective cohort studies showing that lungs reperfused between 4 and 8 AM have a higher incidence (OR 1.12 95% CI 1.03 to 1.21 p=0.01) of primary graft dysfunction (PGD) in the first 72 hours after transplantation. Cooling of the donor lung, occurring during organ preservation, shifts the donor circadian clock causing desynchrony with the recipient. The clock protein REV-ERBα directly regulates PGD biomarkers explaining this circadian regulation while also allowing them to be manipulated with synthetic REV-ERB ligands.
Publisher: Wiley
Date: 22-02-2019
Publisher: European Respiratory Society (ERS)
Date: 18-05-2023
DOI: 10.1183/23120541.00161-2023
Abstract: Asthma is a rhythmic inflammatory disease of the airway, regulated by the circadian clock. “Spill-over” of airway inflammation into the systemic circulation occurs in asthma and is reflected in circulating immune cell repertoire. The objective of the present study was to determine how asthma impacts peripheral blood diurnal rhythmicity. 10 healthy and 10 mild/moderate asthma participants were recruited to an overnight study. Blood was drawn every 6 h for 24 h. The molecular clock in blood cells in asthma is altered PER3 is significantly more rhythmic in asthma compared to healthy controls. Blood immune cell numbers oscillate throughout the day, in health and asthma. Peripheral blood mononucleocytes from asthma patients show significantly enhanced responses to immune stimulation and steroid suppression at 16:00 h, compared to at 04:00 h. Serum ceramides show complex changes in asthma: some losing and others gaining rhythmicity. This is the first report showing that asthma is associated with a gain in peripheral blood molecular clock rhythmicity. Whether the blood clock is responding to rhythmic signals received from the lung or driving rhythmic pathology within the lung itself is not clear. Dynamic changes occur in serum ceramides in asthma, probably reflecting systemic inflammatory action. The enhanced responses of asthma blood immune cells to glucocorticoid at 16:00 h may explain why steroid administration is more effective at this time.
Publisher: Cold Spring Harbor Laboratory
Date: 30-08-2018
DOI: 10.1101/393231
Abstract: CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the method’s success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles ( %), and a very small number of desired alleles ( %) requiring, on average 1,192 zygotes.
Publisher: Cold Spring Harbor Laboratory
Date: 25-09-2019
DOI: 10.1101/781666
Abstract: Pulmonary inflammatory responses lie under circadian control however the importance of circadian mechanisms in fibrosis is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of litude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the poorly described transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinβ1 focal adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of two human cohorts. In the UK Biobank circadian strain markers (sleep length, chronotype and shift work) are associated with pulmonary fibrosis making them novel risk factors. In a separate cohort REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, suggesting targeting REVERBα could be a viable therapeutic approach. The circadian clock plays an essential role in energy metabolism, and inflammation. In contrast the importance of the clock in the pathogenesis of fibrosis remains poorly explored. This study describes a striking alteration in circadian biology during pulmonary fibrosis where the relatively arrhythmic alveolar structures gain circadian but desynchronous rhythmicity due to infiltration by fibroblasts. Disruption of the clock in these cells, which are not widely implicated in circadian pathophysiology, results in a pro-fibrotic phenotype. Translation of these findings in humans revealed previously unrecognised important circadian risk factors for pulmonary fibrosis (sleep length, chronotype and shift work). In addition, targeting REVERBα repressed collagen secretion from human fibrotic lung tissue making this protein a promising therapeutic target.
Publisher: Springer Science and Business Media LLC
Date: 29-01-2019
DOI: 10.1038/S41467-018-08259-7
Abstract: Being a morning person is a behavioural indicator of a person’s underlying circadian rhythm. Using genome-wide data from 697,828 UK Biobank and 23andMe participants we increase the number of genetic loci associated with being a morning person from 24 to 351. Using data from 85,760 in iduals with activity-monitor derived measures of sleep timing we find that the chronotype loci associate with sleep timing: the mean sleep timing of the 5% of in iduals carrying the most morningness alleles is 25 min earlier than the 5% carrying the fewest. The loci are enriched for genes involved in circadian regulation, cAMP, glutamate and insulin signalling pathways, and those expressed in the retina, hindbrain, hypothalamus, and pituitary. Using Mendelian Randomisation, we show that being a morning person is causally associated with better mental health but does not affect BMI or risk of Type 2 diabetes. This study offers insights into circadian biology and its links to disease in humans.
Publisher: Proceedings of the National Academy of Sciences
Date: 26-12-2019
Abstract: Pulmonary inflammatory responses lie under circadian control however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of litude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinβ1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach.
Publisher: Springer Science and Business Media LLC
Date: 26-08-2019
DOI: 10.1186/S13059-019-1776-2
Abstract: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis , an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2023
End Date: 12-2025
Amount: $433,078.00
Funder: Australian Research Council
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