Infection‐driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells

DATA AVAILABILITYThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD017117.International audienceTransglutaminase 2 (TG2) is a ubiquitously expressed enzyme with transamidating activity. We report here that both expression and activity of TG2 are enhanced in mammalian epithelial cells infected with the obligate intracellular bacteria Chlamydia trachomatis. Genetic or pharmacological inhibition of TG2 impairs bacterial development. We show that TG2 increases glucose import by up-regulating the transcription of the glucose transporter genes GLUT-1 and GLUT-3. Furthermore, TG2 activation drives one specific glucose-dependent pathway in the host, i.e., hexosamine biosynthesis. Mechanistically, we identify the glucosamine:fructose-6-phosphate amidotransferase (GFPT) among the substrates of TG2. GFPT modification by TG2 increases its enzymatic activity, resulting in higher levels of UDP-N-acetylglucosamine biosynthesis and protein O-GlcNAcylation. The correlation between TG2 transamidating activity and O-GlcNAcylation is disrupted in infected cells because host hexosamine biosynthesis is being exploited by the bacteria, in particular to assist their division. In conclusion, our work establishes TG2 as a key player in controlling glucose-derived metabolic pathways in mammalian cells, themselves hijacked by C. trachomatis to sustain their own metabolic needs

CBP-HSF2 structural and functional interplay in Rubinstein-Taybi neurodevelopmental disorder

Rubinstein-Taybi syndrome (RSTS) is a neurodevelopmental disorder with unclear underlying mechanisms. Here, the authors unravel the contribution of a stress-responsive pathway to RSTS where impaired HSF2 acetylation, due to RSTS-associated CBP/EP300 mutations, alters the expression of neurodevelopmental players, in keeping with hallmarks of cell-cell adhesion defects.Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology.</p

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Etude de la perturbation précoce des marques épigénétiques dans le cerveau fœtal exposé à l’alcool et de l’implication des voies de réponse au stress

Fetal brain is vulnerable to environmental stress such as prenatal alcohol exposure (PAE), the leading non-genetic cause of mental retardation. This stress induces a wide spectrum of neurodevelopmental defects that are often lately diagnosed. A better understanding of molecular mechanisms underlying these defects would help to develop reliable diagnostic tools for the early care of high-risk subjects.HSF2 transcription factor is a major actor of PAE response in the developing brain. Necessary for cortical physiological development, it also leads, in the context of chronic PAE stress, to abnormalities in brain development by changing its genomic targets. In addition, V. Mezger's team has demonstrated, in the embryonic cortex, that HSF2 binds DNMT3A - in a physiological context or following a PAE, DNMT3A being responsible for de novo DNA methylation.Since it has been shown that the methylome profile of adults that were exposed to a PAE stress, is often perturbed, it was conceivable that the DNMT3A/HSF2 interaction, in a stressful context, may, in part, be responsible for this methylome profile modifications. To test this hypothesis, three integrative high-throughput sequencing (NGS) studies were conducted, using cerebral cortices of mouse embryos exposed, or not, to PAE corresponding to a binge drinking alcoholization.ChIP-seq experiments, targeting HSF2 in alcohol-exposed fetal cortices, allowed us to map its binding sites in the genome, and identify 280 HSF2 targets. Most of these target sites are associated with genes involved in brain development or in stress response. Some of these genes are also linked, in the literature, with PAE effects.Few hours after PAE, 432 differentially methylated regions (DMRs) were identified between control (PBS treated) or alcohol-treated fetal cortices, using a methylome capture protocol. This analysis required the development of specific bioinformatics tools and approaches. These DMRs are mainly localized in active enhancers of the adult cortex. A high proportion of their associated genes correspond to imprinted genes or genes encoding clustered Protocadherins, both involved in neurodevelopment or brain function, and known to be impacted in adults prenatally exposed to an alcoholic stress. Because their deposition is linked to PAE per se and show some persistence in the postnatal/adut period, this strongly reinforces their potential as biomarkers of exposition. These results indicate that epigenetic ‘scars‘ are deposited very quickly after PAE and suggest, based on the literature, that some of them persist in adults.To estimate the functional consequences of PAE on the developing brain, a study of chromatin accessibility and gene expression over the stress period was conducted in a physiological context, analyzing public data (ENCODE) of ATAC-seq and RNA-seq from unstressed murine prefrontal cortices. This data mining study allowed us counting and identifying the chromatin regions that are differentially opened or closed, as well as the genes that are activated or repressed between the embryonic stages E13 and E16 in the developing cortex. Of note, a proportion of DMRs are significantly associated with chromatin regions whose accessibility varies - under physiological conditions - during the stress period, but also with genes whose expression increases during development, suggesting a particular vulnerability at these dynamic regions of the genome to stress.Our integrative analysis of the different NGS datasets did not reveal any correlation between HSF2 binding sites and the DMRs. However, since HSF2 target sequences contain often binding sites of methylome readers or chromatin remodellers, HSF2 might be involved in functional consequences of PAE-induced methylome disturbances, rather than in the establishment of these defects.Le cerveau fœtal est vulnérable aux stress environnementaux comme l’exposition prénatale à l’alcool (EPA), première cause non génétique de retard mental. Ce stress induit un large spectre de défauts neurodéveloppementaux souvent diagnostiqués tardivement. Mieux comprendre les mécanismes moléculaires à l’origine de ces défauts permettrait d’élaborer des outils diagnostics fiables, pour une prise en charge précoce des sujets à risques.Le facteur de transcription HSF2 est un acteur majeur de la réponse à l’EPA dans le cerveau en développement. Bien que nécessaire au développement physiologique du cortex, il conduit, en contexte de stress, à des anomalies du développement cérébral, en changeant de cibles génomiques. De plus, dans le cortex embryonnaire, dans un contexte physiologique ou suite à une EPA, HSF2 lie DNMT3A, protéine responsable de la méthylation de novo de l’ADN.Comme, il a été montré que des adultes ayant subi une EPA présentent une perturbation de leur méthylome, il était concevable que l’interaction DNMT3A/HSF2 dans un contexte de stress, puisse, en partie, être à l’origine de cette modification du méthylome. Pour tester cette hypothèse, une alcoolisation de type binge drinking dans des cortex embryonnaires murins a été réalisée et trois études de séquençage à haut débit (NGS) ont été menées en parallèle, puis intégrées.Un ChIP-seq ciblant HSF2 dans le cortex cérébral fœtal murin a permis de cartographier les sites de fixation de ce facteur et d’identifier 280 cibles de HSF2 après cette EPA. La plupart de ces cibles sont des gènes impliqués dans le développement cérébral, dans la réponse au stress et/ou associées dans la littérature à des effets d’une EPA.L’identification de 432 régions différentiellement méthylées (DMRs), immédiatement après EPA entre des cortex fœtaux témoins (traités au PBS) ou traités à l'alcool a été possible, par une capture du méthylome. Cette analyse a nécessité la mise au point d’un outil et d’une approche bio-informatiques spécifiques. Ces DMRs se situent majoritairement au niveau d’enhancers actifs du cortex adulte. Une forte proportion des gènes affectés correspond à des gènes soumis à l’empreinte ou des gènes codant des protocadhérines (impliquées dans le neurodéveloppement ou des fonctions cérébrales), connus comme étant perturbés chez l’adulte à la suite d’une EPA. Ces gènes sont donc porteurs de marques épigénétiques anormales, déposées dès la fin de l’EPA, et constituent de potentiels biomarqueurs d’exposition. Ainsi, des cicatrices épigénétiques sont mises en place rapidement après l’EPA et suggèrent, à la lumière de la littérature, que certaines persistent chez l’adulte.Pour estimer les conséquences fonctionnelles de l’EPA sur le cerveau en développement, une étude de l’accessibilité de la chromatine et de l’expression des gènes sur la période encadrant le stress a été réalisée en contexte physiologique, en analysant des données publiques (ENCODE) d’ATAC-seq et de RNA-seq provenant de cortex préfrontaux murins non stressés. Ce data mining, a permis de dénombrer et d’identifier les régions chromatiniennes différentiellement ouvertes ou fermées, ainsi que les gènes activés ou réprimés entre les stades embryonnaires E13 et E16 dans le cortex en développement. Les DMR sont associées à des régions chromatiniennes dont l’accessibilité varie au moment du stress, mais aussi au niveau de gènes dont l’expression augmente au cours du développement, suggérant une sensibilité particulière de ces régions dynamiques du génome.L’analyse intégrée des différents jeux de données NGS, n’a pas permis de mettre en évidence une corrélation entre les sites fixés par HSF2 et les DMR. En revanche, les sites de fixation de HSF2 étant souvent associés à des sites de liaison de readers du méthylome ou des remodellers de la chromatine, il est possible que HSF2 soit impliqué dans les conséquences fonctionnelles des perturbations du méthylome dues à l’EPA, plutôt que dans la mise en place de ces défauts

Analysis of early epigenetic mark disruptions caused by prenatal alcohol exposure in the mouse developing brain, and involvement of stress-response pathways

Le cerveau fœtal est vulnérable aux stress environnementaux comme l’exposition prénatale à l’alcool (EPA), première cause non génétique de retard mental. Ce stress induit un large spectre de défauts neurodéveloppementaux souvent diagnostiqués tardivement. Mieux comprendre les mécanismes moléculaires à l’origine de ces défauts permettrait d’élaborer des outils diagnostics fiables, pour une prise en charge précoce des sujets à risques.Le facteur de transcription HSF2 est un acteur majeur de la réponse à l’EPA dans le cerveau en développement. Bien que nécessaire au développement physiologique du cortex, il conduit, en contexte de stress, à des anomalies du développement cérébral, en changeant de cibles génomiques. De plus, dans le cortex embryonnaire, dans un contexte physiologique ou suite à une EPA, HSF2 lie DNMT3A, protéine responsable de la méthylation de novo de l’ADN.Comme, il a été montré que des adultes ayant subi une EPA présentent une perturbation de leur méthylome, il était concevable que l’interaction DNMT3A/HSF2 dans un contexte de stress, puisse, en partie, être à l’origine de cette modification du méthylome. Pour tester cette hypothèse, une alcoolisation de type binge drinking dans des cortex embryonnaires murins a été réalisée et trois études de séquençage à haut débit (NGS) ont été menées en parallèle, puis intégrées.Un ChIP-seq ciblant HSF2 dans le cortex cérébral fœtal murin a permis de cartographier les sites de fixation de ce facteur et d’identifier 280 cibles de HSF2 après cette EPA. La plupart de ces cibles sont des gènes impliqués dans le développement cérébral, dans la réponse au stress et/ou associées dans la littérature à des effets d’une EPA.L’identification de 432 régions différentiellement méthylées (DMRs), immédiatement après EPA entre des cortex fœtaux témoins (traités au PBS) ou traités à l'alcool a été possible, par une capture du méthylome. Cette analyse a nécessité la mise au point d’un outil et d’une approche bio-informatiques spécifiques. Ces DMRs se situent majoritairement au niveau d’enhancers actifs du cortex adulte. Une forte proportion des gènes affectés correspond à des gènes soumis à l’empreinte ou des gènes codant des protocadhérines (impliquées dans le neurodéveloppement ou des fonctions cérébrales), connus comme étant perturbés chez l’adulte à la suite d’une EPA. Ces gènes sont donc porteurs de marques épigénétiques anormales, déposées dès la fin de l’EPA, et constituent de potentiels biomarqueurs d’exposition. Ainsi, des cicatrices épigénétiques sont mises en place rapidement après l’EPA et suggèrent, à la lumière de la littérature, que certaines persistent chez l’adulte.Pour estimer les conséquences fonctionnelles de l’EPA sur le cerveau en développement, une étude de l’accessibilité de la chromatine et de l’expression des gènes sur la période encadrant le stress a été réalisée en contexte physiologique, en analysant des données publiques (ENCODE) d’ATAC-seq et de RNA-seq provenant de cortex préfrontaux murins non stressés. Ce data mining, a permis de dénombrer et d’identifier les régions chromatiniennes différentiellement ouvertes ou fermées, ainsi que les gènes activés ou réprimés entre les stades embryonnaires E13 et E16 dans le cortex en développement. Les DMR sont associées à des régions chromatiniennes dont l’accessibilité varie au moment du stress, mais aussi au niveau de gènes dont l’expression augmente au cours du développement, suggérant une sensibilité particulière de ces régions dynamiques du génome.L’analyse intégrée des différents jeux de données NGS, n’a pas permis de mettre en évidence une corrélation entre les sites fixés par HSF2 et les DMR. En revanche, les sites de fixation de HSF2 étant souvent associés à des sites de liaison de readers du méthylome ou des remodellers de la chromatine, il est possible que HSF2 soit impliqué dans les conséquences fonctionnelles des perturbations du méthylome dues à l’EPA, plutôt que dans la mise en place de ces défauts.Fetal brain is vulnerable to environmental stress such as prenatal alcohol exposure (PAE), the leading non-genetic cause of mental retardation. This stress induces a wide spectrum of neurodevelopmental defects that are often lately diagnosed. A better understanding of molecular mechanisms underlying these defects would help to develop reliable diagnostic tools for the early care of high-risk subjects.HSF2 transcription factor is a major actor of PAE response in the developing brain. Necessary for cortical physiological development, it also leads, in the context of chronic PAE stress, to abnormalities in brain development by changing its genomic targets. In addition, V. Mezger's team has demonstrated, in the embryonic cortex, that HSF2 binds DNMT3A - in a physiological context or following a PAE, DNMT3A being responsible for de novo DNA methylation.Since it has been shown that the methylome profile of adults that were exposed to a PAE stress, is often perturbed, it was conceivable that the DNMT3A/HSF2 interaction, in a stressful context, may, in part, be responsible for this methylome profile modifications. To test this hypothesis, three integrative high-throughput sequencing (NGS) studies were conducted, using cerebral cortices of mouse embryos exposed, or not, to PAE corresponding to a binge drinking alcoholization.ChIP-seq experiments, targeting HSF2 in alcohol-exposed fetal cortices, allowed us to map its binding sites in the genome, and identify 280 HSF2 targets. Most of these target sites are associated with genes involved in brain development or in stress response. Some of these genes are also linked, in the literature, with PAE effects.Few hours after PAE, 432 differentially methylated regions (DMRs) were identified between control (PBS treated) or alcohol-treated fetal cortices, using a methylome capture protocol. This analysis required the development of specific bioinformatics tools and approaches. These DMRs are mainly localized in active enhancers of the adult cortex. A high proportion of their associated genes correspond to imprinted genes or genes encoding clustered Protocadherins, both involved in neurodevelopment or brain function, and known to be impacted in adults prenatally exposed to an alcoholic stress. Because their deposition is linked to PAE per se and show some persistence in the postnatal/adut period, this strongly reinforces their potential as biomarkers of exposition. These results indicate that epigenetic ‘scars‘ are deposited very quickly after PAE and suggest, based on the literature, that some of them persist in adults.To estimate the functional consequences of PAE on the developing brain, a study of chromatin accessibility and gene expression over the stress period was conducted in a physiological context, analyzing public data (ENCODE) of ATAC-seq and RNA-seq from unstressed murine prefrontal cortices. This data mining study allowed us counting and identifying the chromatin regions that are differentially opened or closed, as well as the genes that are activated or repressed between the embryonic stages E13 and E16 in the developing cortex. Of note, a proportion of DMRs are significantly associated with chromatin regions whose accessibility varies - under physiological conditions - during the stress period, but also with genes whose expression increases during development, suggesting a particular vulnerability at these dynamic regions of the genome to stress.Our integrative analysis of the different NGS datasets did not reveal any correlation between HSF2 binding sites and the DMRs. However, since HSF2 target sequences contain often binding sites of methylome readers or chromatin remodellers, HSF2 might be involved in functional consequences of PAE-induced methylome disturbances, rather than in the establishment of these defects

: Analyse de troubles neurodéveloppementaux à la lumière de modifications de l’épigénome : exemple du syndrome d’alcoolisation fœtale

National audienc

The “HSF connection”: Pleiotropic regulation and activities of Heat Shock Factors shape pathophysiological brain development

International audienceThe Heat Shock Factors (HSFs) have been historically identified as a family of transcription factors that are activated and work in a stress-responsive manner, after exposure to a large variety of stimuli. However, they are also critical in normal conditions, in a life long manner, in a number of physiological processes that encompass gametogenesis, embryonic development and the integrity of adult organs and organisms. The importance of such roles is emphasized by the devastating impact of their deregulation on health, ranging from reproductive failure, neurodevelopmental disorders, cancer, and aging pathologies, including neurodegenerative disorders. Here, we provide an overview of the delicate choreography of the regulation of HSFs during neurodevelopment, at prenatal and postnatal stages. The regulation of HSFs acts at multiple layers and steps, and comprises the control of (i) HSF mRNA and protein levels, (ii) HSF activity in terms of DNA-binding and transcription, (iii) HSF homo- and hetero-oligomerization capacities, and (iv) HSF combinatory set of post-translational modifications. We also describe how these regulatory mechanisms operate in the normal developing brain and how their perturbation impact neurodevelopment under prenatal or perinatal stress conditions. In addition, we put into perspective the possible role of HSFs in the evolution of the vertebrate brains and the importance of the HSF pathway in a large variety of neurodevelopmental disorders

Immediate perturbation of DNA methylation upon acute prenatal alcohol exposure in the mouse developing brain cortex

AbstractThe reshaping of the DNA methylome landscape after prenatal alcohol exposure (PAE) has been well-documented in the adult brain, therefore a long time after the end of the exposure. However, the question of the immediate deposition or loss of DNA methylation marks in the prenatal neocortex, just after the end of PAE has not yet been directly addressed, genome widely.Using a binge-drinking-like model of PAE and capture of the DNA methylome, we have identified differentially methylated regions (DMRs) that are established immediately, within two hours after the end of PAE. Remarkably, these DMRs are prominently and statistically associated with: (i) enhancers that are active in the brain, associated with GO terms of importance for neurogenesis, neurodevelopment, and neuronal differentiation; (ii) genes that, in physiological conditions show dynamic gain in chromatin accessibility and/or upregulation of their expression in the time-window of exposure; (iii) imprinted genes and members of protocadherin genes clusters, two gene families playing key roles in neurodevelopment, whose mono-allelically expression is regulated by DNA methylation and impaired upon PAE. We observed that DMR-containing mono-allelically expressed genes, as well as other genes important for neurodevelopment, are also immediately upregulated upon PAE, suggesting that these early DNA methylation perturbations are thus highly susceptible to rapidly alter gene expression after PAE.DMRs in imprinted and protocadherin genes have been previously identified, both in the adult rodent brain prior-exposed to alcohol prenatally, and in cohorts of children diagnosed with fetal alcohol spectrum disorders (FASD). Our study thus strongly suggests that the DNA methylation profiles of key regulatory regions of these gene families are very quickly disturbed after the PAE and that these immediate altered regions could be persistently affected long after the stress. This strongly reinforces their potential as future biomarkers of PAE.Ethical issuesThe breeding and treatments of wild type C57BL/6N mice, used for the experimental protocols described in this study have been approved by the Institutional Animal Care and Use Ethical Committee of the Paris University (registration number CEEA-40). The project has been recorded under the following reference by the Ministère de l’Enseignement Supérieur et de la Recherche (#2016040414515579). All efforts were made to reduce stress and pain to animals.</jats:sec

Immediate perturbation of DNA methylation upon acute prenatal alcohol exposure in the mouse developing brain cortex

Funding information VM was funded by the Agence Nationale de la Recherche « HSF-EPISAME », SAMENTA ANR-13-SAMA-0008-01) and CNRS. AD was supported by a Doctoral Fellowship from the French Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation (MESRI) and FM from a Doctoral Fellowship from the CNRS. This study contributes to the Université de Paris IdEx #ANR-18-IDEX-0001 funded by the French Government through its "Investments for the Future" program. DSD benefited from a CNRS Délégation de Recherche (2018-2020). OTT benefited from travel grants from Université Paris Diderot