Une approche tridimensionnelle de génomique fonctionnelle pour identifier les cibles contrôlant la réponse au stress thermique chez le blé – 3DWheat

To understand the epigenome reprogramming of wheat in response to heat stress, we will analyze the transcriptome (Task1), the epigenome (Task2), chromatin accessibility (Task3), and chromatin architecture (Task 4) of 1-week old wheat seedling leaves subjected to heat stress. For the transcriptome, we will focus on coding RNA to get the indispensable information regarding the genes involved in heat stress tolerance, and on long non-coding RNA because they could control the formation of chromatin loops5. In Task 2, we will analyse selected chromatin features: RNAPlII (RNA Polymerase II, a mark of active promoters), anti-H3K9ac (euchromatin marks), anti-H3K4me3 (euchromatin marks) and anti-H3K27me3 (mark of inactive genes). To monitor chromatin accessibility (Task 3), we will use ATAC-Seq (assay for transposase-accessible chromatin using sequencing) which is a high-throughput procedure to isolate and map nucleosome-depleted chromatin regions. We have already established this technology for wheat. Finally in Task 4, we will combine two complementary approaches relying on the proximity-based ligation of sequences brought together by chromatin loops. The Hi-C technique is designed to sequence all chromatin contacts, giving a global picture of genome organization, whereas in the ChIA-PET technique, an immuno-precipitation step allows the targeted sequencing of chromatin loops associated with a protein of interest. Here we will focus on regions brought together by RNA Polymerase II. This approach will allow us to identify changes in chromatin architecture induced by heat, as well as distant regulatory elements such as enhancers. Selected chromatin loops will be validated using FISH (Task 6). The key goal of our project will be the integration of all data sets (Task 7) in order to understand the logic of transcriptome and epigenome reprogramming during heat stress.

We performed different chromatin immuno-staining experiments to analyse at the microscopic level the wheat nuclear organization and we observed a number of unique features. Notably, the wheat nucleus appears highly polarized, with constitutive (representing transposable elements and repeated sequences) and facultative (corresponding to repressed genes) heterochromatin occupying opposite poles. Wheat chromatin organization is thus different from what we know in Arabidopsis, and extrapolating knowledge acquired in models will not allow a full understanding of how wheat chromatin dynamics controls gene expression.

In parallel, we performed different ChIP-seq experiments to analyse the distribution of activating and repressive chromatin marks. This data was generated in the context of the wheat genome sequencing project. We are thus author of the submitted paper entitled “Shifting the limits in wheat research and breeding through a fully annotated and anchored reference genome sequence” to Science. First, we found that a significant difference exists in the H3K9ac level between homeologs suggesting that chromatin modifications play a crucial role in the differential regulation of wheat homeolog transcription. In addition, we observed that a large cohort of genes simultaneously display both activating H3K4me3 and repressive H3K27me3 marks, suggesting the existence of a bivalent chromatin state in wheat. Most of these genes are associated with stress response and a significant proportion of those corresponds to heat inducible genes. By displaying both active and repressive marks, bivalent genes are proposed to be in a poised state, enabling them to be rapidly activated upon suitable developmental cues and/or environmental stimuli, which is particularly relevant in the context of heat stress.
To gain insight into the spatial organization of the wheat genome inside the nucleus, we conducted a Hi-C experiment.

lots of questions are still open: what will be the consequence of a heat stress? What is the role of this reorganization? What are the molecular mechanisms behind it? Knowing that rice is diploid, what will be the impact of stress on chromatin architecture in a polyploid? This is why we need to study in deep this phenomenon. Exploring the chromatin-based regulation of gene expression in polyploid wheat represents a challenge, due to the complexity of its genome, however it may be particularly relevant to unravel new mechanisms promoting epigenome adaptation to environmental constraints.
In this project, we wish to monitor changes in the transcriptome, epigenome and 3D-chromatin structure in Triticum aestivum after a heat stress in order to obtain a global vision of how changes at the chromatin level are associated with changes in gene expression in a polyploid plant during heat stress response, and how this information can be used to improve wheat adaptation to environmental constraints.

ongoing

Du fait du changement climatique, le stress thermique est en passe de devenir une source majeure de perte de rendement en Europe. Il est donc urgent de décrypter les mécanismes impliqués dans la réponse à la chaleur afin de produire de nouvelles variétés mieux adaptées. Plusieurs études se sont intéressées à la réponse à la chaleur mais peu ont porté sur le rôle de la dynamique chromatinienne dans ces processus chez les plantes cultivées. Dans ce contexte, le projet 3DWheat vise à caractériser les modifications du transcriptome, de l'épigénome et de la structure 3D de la chromatine chez le blé afin de caractériser les modifications de l’architecture chromatinienne associées aux changements de l'expression des gènes au cours de la réponse au stress thermique. Pour aller au-delà de la simple description de la réponse au stress thermique, nous proposons d'étudier les mécanismes moléculaires contrôlant l'état bivalent de la chromatine observé sur les gènes répondant à la chaleur.