Role of the antagonistic histone methylation marks H3K4me3 and H3K27me3 in the cold stress response of Arabidopsis thaliana

As sessile organisms, plants need to adapt to their changing environment, including temperature fluctuations. As low temperatures can have major noxious consequences on their development and survival, plants need to establish the proper defences in order to endure the stress. This requires a massive and very fast transcriptome reprogramming involving, among others, the induction of hundreds of cold-responsive (COR) genes. Following the immediate response to chilling stress, plants are also able to memorize cold spells, leading to an improved survival during a second stress episode. This process is associated with a revised transcriptomic response also called transcriptional memory. Overall, both the response to cold and the memory of this stress rely on the tight transcriptional regulation of the COR genes. While numerous transcription factors necessary for their induction were already identified, the role of chromatin modifications in this process remains largely undiscovered. As the combination of chromatin modifications (the “chromatin state”) is a key determinant of gene expression, this study aimed at uncovering the potential role of histone modifications in the transcriptional regulation of COR genes before, during and after a cold episode. First, a comprehensive in silico analysis of the chromatin state of COR genes prior to any cold occurrence revealed that a majority of those genes carry both the activating mark H3K4me3 and the silencing mark H3K27me3, forming a specific chromatin state called bivalency. The in vivo characterization of bivalent genes revealed that this chromatin state decorates not only cold-inducible genes but numerous reversibly silenced stress-responsive genes and might poise them for expression by maintaining them in an open chromatin conformation. Furthermore, the putative bivalency reader DEK2 was shown to prevent the over-induction of bivalent COR genes during a cold episode, suggesting that bivalency can also participate in transcriptional regulation in trans through the action of specific readers. In a second stage, the dynamics of H3K4me3 and H3K27me3 during a cold stress were analysed using genome-wide approaches, revealing that both marks underwent intensive redistribution already after three hours of low temperature. Those changes partially correlated with expression changes: in particular, the induction of COR genes was associated with a loss of the repressive mark H3K27me3 or a gain of the activating mark H3K4me3. However, each mark displayed different targets and dynamics, suggesting that they hold distinct roles in the cold response: H3K4me3 associated with immediate stress responses while H3K27me3 rather correlated with longer-term adaptation. Upon return to ambient temperature, the cold-induced variations reverted at a different pace depending on the gene and some changes were maintained for up to seven days. Both the maintenance of H3K4me3 and H3K27me3 changes were linked to transcriptional memory: higher levels of H3K4me3 were associated with sustained induction while lower levels of H3K27me3 were correlated with a faster re-induction during a second stress exposure. Finally, the H3K27me3 demethylase ELF6 was shown to be essential for cold stress memory. This led to the hypothesis that cold stress memory might rely on the maintained loss of H3K27me3 on specific COR genes, allowing a faster re-establishment of defences during a second stress episode. In conclusion, this study demonstrates that the antagonistic marks H3K4me3 and H3K27me3 jointly participate to the transcriptional regulation of COR genes and reveals a new role of bivalency in the plant cold stress response and memory.Da Pflanzen an ihren Standort gebunden sind, müssen sie sich ständig an veränderte Umweltbedingungen anpassen. Kälte kann schädliche Folgen für die Pflanzenentwicklung und sogar zum Absterben von Pflanzen führen. Daher müssen Pflanzen auf diesen Stress reagieren, indem sie eine geeignete Abwehr zum Überleben aufbauen. Dies erfordert eine erhebliche Umprogrammierung des Transkriptoms, welche die Induktion von zahlreichen kälteempfindlichen (COR) Genen enthält. Nach einer direkten Stressantwort sind Pflanzen in der Lage ein Kältegedächtnis aufzubauen, wodurch sie eine zweite Kälteepisode besser überstehen. Dieser Prozess ist mit massiven Änderungen auf Genexpressionsebene verbunden, die auch „transkriptionelles Gedächtnis“ genannt wird. Sowohl die unmittelbare Reaktion auf, als auch das Bilden eines längerfristigen Gedächtnisses an Kälte, sind auf eine präzise Transkriptionsregulation von COR Genen angewiesen. Obwohl der Chromatinzustand ein bestimmender Faktor für Genexpression ist, ist die Rolle von Chromatinmodifikationen in der Induktion von COR Genen noch weitgehend unbekannt. Deshalb war es das Ziel dieser Arbeit, die Rolle von Histonmodifikationen in der Transkriptionsregulation von COR Genen vor, während, und nach Kältestress zu analysieren. Zunächst offenbarte eine umfassende in silico Analyse des Chromatinzustands von COR Genen vor einem Kälteereignis, dass die Mehrheit dieser Gene sowohl die aktivierende Modifikation H3K4me3 als auch die repressive Modifikation H3K27me3 tragen. Dieser Chromatinzustand wird auch als bivalent bezeichnet. Die in vivo Charakterisierung bivalenter Gene zeigte, dass besonders stillgelegte, induzierbare Gene durch einen bivalenten Chromatinzustand markiert sind. Diese könnten dadurch für eine eventuelle Expression vorbereitet sein, indem diese Genbereiche in einer offenen Chromatin-Konformation verbleiben. Der vermeintliche Bivalenz-Leser DEK2 verhinderte die Überinduktion von bivalenten Genen während einer Kälteepisode. Dies weist darauf hin, dass Bivalenz auch an der Transkriptionsregulation in trans durch die Aktion von bestimmten Reader-Proteinen Anteil nehmen kann. Die Analyse der H3K4me3 und H3K27me3 Dynamik mittels genomweiter Methoden zeigte, dass bei niedrigen Temperaturen eine intensive Neuverteilung beider Modifikationen stattfindet, die teilweise mit Expressionsvariationen korrelierte. Insbesondere war die Induktion von COR Genen mit einem Verlust der repressiven Modifikation H3K27me3 oder einer Zunahme der aktivierenden Modifikation H3K4me3 assoziiert. Die Modifikationen haben jedoch distinkte Rollen in der Kälteantwort. H3K4me3 war mit der unmittelbaren Stressantwort assoziiert, während H3K27me3 eher mit Langzeitadaptation korreliert war. Nach der Rückkehr zu ambienter Umgebungstemperatur kehrte das Chromatin zu seinem Ausgangzustand in unterschiedlichem Tempo abhängig vom Gen zurück, wobei manche Veränderungen bis zu sieben Tage beibehalten wurden. Die Aufrechterhaltung von sowohl H3K4me3 als auch H3K27me3 Variationen waren mit transkriptionellem Gedächtnis assoziiert: höhere H3K4me3 Mengen korrelierten mit beständiger Induktion und niedrigere H3K27me3 Mengen waren mit einer schnelleren Re-Induktion während einer zweiten Kälteepisode assoziiert. Schließlich wurde gezeigt, dass die H3K27me3 Demethylase ELF6 unabdingbar für das Kältestressgedächtnis ist. Der fortbestehende Verlust von H3K27me3 auf spezifischen Genen könnte daher die molekulare Basis für das Kältestressgedächtnis sein, indem ein schnellerer Wiederaufbau der Abwehr während einer zweiten Stressepisode ermöglicht wird. Insgesamt zeigt diese Studie, dass die antagonistischen Modifikationen H3K4me3 und H3K27me3 gemeinsam an der Transkriptionsregulation von COR Genen teilhaben, und offenbart eine neue Rolle der Bivalenz bei der Kältestressreaktion von Pflanzen

Transcriptional and Post-Transcriptional Regulation and Transcriptional Memory of Chromatin Regulators in Response to Low Temperature

Chromatin regulation ensures stable repression of stress-inducible genes under non-stress conditions and transcriptional activation and memory of stress-related genes after stress exposure. However, there is only limited knowledge on how chromatin genes are regulated at the transcriptional and post-transcriptional level upon stress exposure and relief from stress. We reveal that the repressive modification histone H3 lysine 27 trimethylation (H3K27me3) targets genes which are quickly activated upon cold exposure, however, H3K27me3 is not necessarily lost during a longer time in the cold. In addition, we have set-up a quantitative reverse transcription polymerase chain reaction-based platform for high-throughput transcriptional profiling of a large set of chromatin genes. We find that the expression of many of these genes is regulated by cold. In addition, we reveal an induction of several DNA and histone demethylase genes and certain histone variants after plants have been shifted back to ambient temperature (deacclimation), suggesting a role in the memory of cold acclimation. We also re-analyze large scale transcriptomic datasets for transcriptional regulation and alternative splicing (AS) of chromatin genes, uncovering an unexpected level of regulation of these genes, particularly at the splicing level. This includes several vernalization regulating genes whose AS may result in cold-regulated protein diversity. Overall, we provide a profiling platform for the analysis of chromatin regulatory genes and integrative analyses of their regulation, suggesting a dynamic regulation of key chromatin genes in response to low temperature stress

Vuorovaikutteisen suunnittelun haasteet ja mahdollisuudet metsätalouden vesiensuojelussa

Rapport de l'expertise scientifique collectiveLes animaux peuvent-ils éprouver des émotions, peuvent-ils penser, ont-ils une histoire de vie ? Depuis l’Antiquité, les philosophes ont proposé des réponses contrastées à ces questions. Du XIXème siècle à nos jours, la réflexion sur ce que sont les animaux s’est enrichie d’apports scientifiques : théorie de l’évolution, éthologie, neurophysiologie, sciences cognitives. Mais la conscience animale reste toujours l’objet de débats importants dans la communauté scientifique. Ainsi en 2012 un groupe de scientifiques de premier plan a éprouvé la nécessité de publier un manifeste intitulé « Déclaration de Cambridge sur la Conscience », qui énonce qu’«…une convergence de preuves indique que les animaux non humains disposent des substrats neuro-anatomiques, neurochimiques et neurophysiologiques des états conscients ainsi que la capacité d’exprimer des comportements intentionnels...».Les connaissances actuelles, dont cette expertise collective propose une synthèse, montrent que les animaux possèdent un large éventail de capacités cognitives associées à des comportements plus ou moins complexes. Les formes de conscience étudiées chez les humains supposent des capacités cognitives distinctes que l’on retrouve chez certains animaux. Peut-on en postuler que ceux-ci ont des formes de consciences équivalentes à celles de l’homme, sans être forcément identiques ?L’étude des niveaux et des contenus de la conscience chez les animaux est en passe de devenir un enjeu scientifique important en raison de la complexité du sujet et des controverses qu’il ne manquera pas de susciter. Enfin, les acquis scientifiques dans ce domaine invitent à reprendre les réflexions morales concernant les relations que les hommes entretiennent avec les animaux (et particulièrement avec les animaux domestiques

The extruded non-template strand determines the architecture of R-loops

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The extruded non-template strand determines the architecture of R-loops.

Three-stranded R-loop structures have been associated with genomic instability phenotypes. What underlies their wide-ranging effects on genome stability remains poorly understood. Here we combined biochemical and atomic force microscopy approaches with single molecule R-loop footprinting to demonstrate that R-loops formed at the model Airn locus in vitro adopt a defined set of three-dimensional conformations characterized by distinct shapes and volumes, which we call R-loop objects. Interestingly, we show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks

La conscience animale. Résumé de l'expertise scientifique collective

Les animaux peuvent-ils éprouver des émotions ? Ont-ils une histoire de vie ? L’Inra (Délégation à l'Expertise scientifique collective, à la Prospective et aux Etudes) s’est saisi de ces questions en réalisant une expertise scientifique collective sur la conscience animale, à la demande de l’Autorité européenne de sécurité alimentaire (EFSA). Les résultats ont été présentés aux représentants des pays membres du réseau européen sur le Bien-Etre animal et de l’EFSA le 11 mai 2017 à Parme (Italie)

Animal consciousness. Summary of the multidisciplinary assesment: Summary

Summary of the multidisciplinary assesmentDo animals experience emotions? Think? Have a life story? Since antiquity, philosophers have contrasting answers to those questions. From the Nineteenth century onwards, thoughts on the nature of animals were enriched by scientific insights from evolutionary theory, ethology, neurophysiology and cognitive sciences. Meanwhile, animal consciousness remains controversial within the scientific community. In 2012, a group of leading scientists felt the need to publish a manifesto entitled the “Cambridge Declaration on Consciousness” it states that “convergent evidence indicates that nonhuman animals have the neuroanatomical, neurochemical, and neurophysiological substrates of conscious states along with the capacity to exhibit intentional behaviours”.Current knowledge, including the outcome of this multidisciplinary scientific assessment, shows that animals possess a wide range of cognitive abilities associated with behaviours of varying complexity. The forms of consciousness observed in humans presuppose distinct cognitive abilities which are found in certain animals. Could it be concluded that animals possess some sort of consciousness similar, albeit not identical, to that of humans?Research on levels and contents of consciousness in animals is emerging as a scientific topic of major interest due to its high level of complexity and associated controversies. Current scientific advances invite us to revisit ethical considerations regarding the relationships between humans and animals (domestic animals in particular)

Animal consciousness. Summary of the multidisciplinary assesment: Summary

Summary of the multidisciplinary assesmentDo animals experience emotions? Think? Have a life story? Since antiquity, philosophers have contrasting answers to those questions. From the Nineteenth century onwards, thoughts on the nature of animals were enriched by scientific insights from evolutionary theory, ethology, neurophysiology and cognitive sciences. Meanwhile, animal consciousness remains controversial within the scientific community. In 2012, a group of leading scientists felt the need to publish a manifesto entitled the “Cambridge Declaration on Consciousness” it states that “convergent evidence indicates that nonhuman animals have the neuroanatomical, neurochemical, and neurophysiological substrates of conscious states along with the capacity to exhibit intentional behaviours”.Current knowledge, including the outcome of this multidisciplinary scientific assessment, shows that animals possess a wide range of cognitive abilities associated with behaviours of varying complexity. The forms of consciousness observed in humans presuppose distinct cognitive abilities which are found in certain animals. Could it be concluded that animals possess some sort of consciousness similar, albeit not identical, to that of humans?Research on levels and contents of consciousness in animals is emerging as a scientific topic of major interest due to its high level of complexity and associated controversies. Current scientific advances invite us to revisit ethical considerations regarding the relationships between humans and animals (domestic animals in particular)

La conscience animale: Une expertise scientifique collective de l’Inra

Ce dossier a préalablement été publié par l’Inra sur son site : http://institut.Inra.fr/Missions/Eclairer-les-decisions/Expertises/Toutes-les-actualites/Conscience-animaleThis paper presents the outcome of a scientific multidisciplinary assessment on animal consciousness carried out by Inra from 2015 to 2017. The experts, produced in a 169 pages report in English, an exhaustive analysis of the scientific and philosophical knowledge available in the international literature. It first describes the knowledge about humans in which the concepts and tools describing consciousness have been developed. The available data on animals are much less numerous, but they make it possible to draw firm conclusions, even if they still require more data. Most of the reported results were obtained on laboratory and wild vertebrates and little on livestock species. Consciousness is defined as the subjective experience that the animal has of its environment and of its own knowledge. The overall view of the behavioral, cognitive and neurobiological studies analyzed in the report tends to show the existence of elaborate consciousness contents in the species studied. The consequences for animals of the presence of consciousness on pain and on ethical behavior towards them are presented. Additional researches should complement existing ones, particularly on farmed species.Ce dossier présente les résultats d’une expertise scientifique collective sur la conscience animale réalisée par l’Inra en 2015-2017. Les experts ont produit dans un rapport de 169 pages en anglais une analyse exhaustive des connaissances scientifiques et philosophiques disponibles dans la littérature internationale. Il décrit tout d’abord les connaissances sur les humains où ont été élaborés les concepts et les outils décrivant la conscience. Les données disponibles sur les animaux sont beaucoup moins nombreuses mais elles permettent cependant de tirer des conclusions solides, même si elles demandent à être confortées. La plupart des résultats rapportés ont été obtenus sur des vertébrés de laboratoire ou sauvages et peu sur les espèces de rente. La conscience est définie comme l’expérience subjective que l’animal a de son environnement et de ses propres connaissances. La vision d’ensemble des études comportementales, cognitives et neurobiologiques analysées dans le rapport tend à montrer l’existence de contenus élaborés de conscience chez les espèces étudiées. Les conséquences pour les animaux de la présence d’une conscience sur la douleur et sur les conduites éthiques vis-à-vis d’eux sont présentées. Des recherches supplémentaires devront compléter celles existantes, particulièrement sur les espèces d’élevage