Projet de thèse en Biologie
Sous la direction de Leila Tirichine delacour.
Thèses en préparation à Paris Saclay , dans le cadre de Sciences du Végétal : du gène à l'écosystème , en partenariat avec Institut de Biologie de l'École Normale Supérieure (laboratoire) et de Université Paris-Sud (établissement de préparation de la thèse) depuis le 12-09-2016 .
Les profils de chromatine ont été édifiés chez plusieurs espèces ce qui a permis une meilleure compréhension des mécanismes fondamentaux de la régulation des gènes. Cependant, plusieurs groupes eucaryotes majeurs connaissent de nombreuses lacunes dans ce domaine. Un tel exemple est les Stramenopiles (également connu sous le nom Heterokonta), que nous représentons ici avec le modèle de diatomée marine Phaeodactylum tricornutum. Les diatomées sont des espèces de phytoplancton très diverses et omniprésentes, qui seraient responsables de plus de 20% de la production primaire mondiale, jouant ainsi un rôle clé dans les cycles biogéochimiques mondiaux. L'étude de la régulation des gènes médiée par la chromatine chez les diatomées va aider à comprendre le succès écologique de ces organismes dans les océans contemporains et compte tenu de la conservation de la chromatine chez les eucaryotes, ce travail apportera sans aucun doute des éléments supplémentaires sur la compréhension de la régulation épigénétique chez les animaux et les plantes en réponse aux signaux environnementaux. Dans ce travail, nous avons grâce à la spéctrométrie de masse à haute résolution identifié un répertoire complet des modifications post-traductionnelles (PTM) sur les histones de P. tricornutum, y compris onze nouvelles modifications. Nous utilisons actuellement des approches biochimiques, génétiques et génomiques afin de caractériser un nouveau type de résidu modifié qui va sans doute révéler de nouveaux mécanismes et aidera à comprendre le succès écologique de cet organisme modèle émergent.
The marine diatom Phaeodactylum tricornutum an emerging model for revealing novel histone modifications and molecular mechanisms in Eukaryotic cells
Diatoms (Bacillariophyta) are one of the major groups of chromalveolates and represent one of the most abundant, diverse and ecologically significant groups of algae in both freshwater and marine ecosystems. They are essential for life on Earth, providing one fifth of the oxygen we breathe, and sustain life as primary producers at the base of the food chain. Although chronically understudied from a molecular perspective, they are a fundamental component of phytoplankton in most aquatic ecosystems, and are believed to contribute around 40% of primary production in marine ecosystems 1. Diatoms also represent a potentially important source of innovation for nanotechnology, biofuel and pharmaceutical industries. Complete sequencing of the Phaeodactylum tricornutum genome 2 showed that it has an unusual genetic composition, which arose through successive endosymbioses and horizontal gene transfers from bacteria. The combination of genes from different origins has attributed them with novel and unusual metabolic capacities for photosynthetic organisms, such as fatty acid oxidation pathways and a urea cycle centred in their mitochondria 3. These pathways are central hubs of diatom primary metabolism and are also used for diatom-specific processes such as for the construction of their silicified cell walls known as frustules 3,4. Diatoms are remarkably successful organisms with a broad distribution in contemporary oceans and with a well-known capacity to adapt rapidly and outcompete other phytoplankton when favorable conditions arise 5. It is possible that chromatin-based processes of gene regulation underlie their ecological success. Recent work published by us and others has demonstrated that Phaeodactylum is an ideal species to investigate the role of epigenetic processes in stramenopile biology and diatom ecological success in the oceans because it possesses all the major components of the epigenetic machinery, including DNA methylation 6, histone modifications 7, Veluchamy et al (submitted) and small RNAs. We have also evidence for the existence of a histone code in Phaeodactylum and its implication in regulating genes in response to ecologically relevant environmental changes (Veluchamy et al., submitted). P. tricornutum with its chimeric genome, metabolic innovations and ecological robustness and flexibility represents a huge potential for exploring new molecules, metabolic pathways and adaptive responses and mechanisms. Therefore, we used this emerging model to explore the role of epigenetic mechanisms, in particular histone modifications in the ecological success of diatoms in contemporary oceans. This work represents an important initial step for understanding the evolutionary history of chromatin and how epigenetic modifications affect gene expression in response to environmental cues in marine environments. In collaboration with the Curie institute in Paris, we have recently used a high accuracy mass spectrometer (MS) combined with different enzymes digest, synthetic peptides, manual inspection and validation of MS data (allow a high level of confidence in discriminating between modified sites with the same nominal masses) and identified a broad range of histone modifications in Phaeodactylum 8. Eleven of the 65 histone modifications detected are novel 8 (Tirichine, unpublished). Most of the novel modifications are on the core domain, namely acetylation of lysine 59 of histone H4, which was instead reported to be methylated in bovine calf thymus histones 9, acetylation of lysine 31 of histone H4 (reported only in Toxoplasma10 although not confirmed in an independent study11) and acetylation of lysines 2 of H2B. Interestingly, P. tricornutum combines histone PTMs found in both mammals and plants, such as acetylation and mono- di-methylation of lysine 79 of histone H3 found only in human and yeast 12 but not in Arabidopsis 13, underlying the mosaic nature of the P. tricornutum genome. Another interesting example is the acetylation of lysine 20 of histone H4 which is shared with Arabidopsis but different from human where this residue is only methylated 13. Novel histone core domain modifications identified in P. tricornutum are probably ancient modifications that likely were lost from the divergent lineages (or have not yet been detected). We are currently investigating the putative function of one of the novel histone modifications in Phaeodactylum and human cells. The successful PhD candidate will pursue the project using molecular biology as well as biochemical techniques in order to further characterize this novel chromatin mark. The PhD student will also participate to the characterization of a candidate gene (enzyme reader) using CRISPR cas9 editing which has been recently set up in the laboratory. Details of the thesis tasks: In collaboration with Active Motif, we raised an antibody against two of the novel modifications. Antibodies have been validated on whole cell extracts and chromatin. The successful PhD student we will perform chromatin IP followed by deep sequencing in order to investigate the distribution of these PTMs genome wide on genes, transposons and intergenic regions. He will perform in parallel a quantification of transcript levels using RNA seq in order to investigate the gene expression patterns associated with the histone modification (Bioinformatic analysis will be performed by the in house bioinformatics platform). Because of the nature of the histone modification, we performed in silico search in Phaeodactylum for proteins that might be involved in the deposition of the mark and found one candidate gene that we are investigating by reverse genetics. We generated knockdown (KD) and overexpressed (OX) lines, with decreased and increased levels of gene expression respectively monitored by Quantitative real time PCR (QPCR). In collaboration with partner Curie Institute, we used mass spectrometry to quantify the levels of this histone modification in transgenic lines compared to the wild type control. Our guess of the candidate gene is obviously correct and we could detect a significant reduction of the level of the histone mark in KD lines but increased levels in OX lines while wild type levels remain intermediate. The PhD student will validate these important results and examine additional KD and OX lines in Phaeodactylum. These results will be validated by another quantification method using the antibodies against this histone modification (western blot). He or she will characterize the mutants by monitoring their growth (flow cytometry), phenotype (light microscopy) and physiological studies related to the nature of the candidate gene. The distribution of this histone modification genome wide and its impact on transcriptional regulation of genes and TEs will be investigated using Chip Seq coupled to RNA seq. Recently, we succeeded in establishing CRISPR cas9 editing in Phaeodactylum which will be used to generate knockdown lines of the candidate gene which will be further characterized. If time allows, the PhD student will extend the analysis to human cells by knocking down using Si RNA the candidate gene homologue in Hela cells which we have identified in silico. Transgenic cell lines will be screened using QPCR, WB and Mass spectrometry. This modification seems to be cell cycle dependent as its increases at mid mitosis and our preliminary results show affected cell division and growth in KD cell lines. To better understand its function, the PhD student will investigate its dynamic by QPCR and WB at different stages of the cell cycle in wild type as well as transgenic synchronized cells of Phaeodactylum and human cells.