Project

Discipline

DNA methylation; DNA mismatch repair (MMR); Very short patch (VSP) repair; Transcription; Epigenetics; Bacteria; Cell cycle control

Lead
Méthylation et maintenance du génome bactérien.
Lay summary
Le génome de nombreux organismes peut être méthylé. De telles modifications épigénétiques ont des conséquences multiples, y compris sur: (i) l’expression de gènes codant pour des protéines nécessaires à la multiplication cellulaire, (ii) le maintien de l’intégrité du génome. Le premier objectif de ce projet de recherche fondamentale est d’identifier de nouveaux mécanismes de régulation épigénétique de l’expression des gènes conservés dans la famille des Alphaprotéobactéries, qui inclue des bactéries environementales, mais aussi des bactéries pathogènes pour l’humain, les animaux ou les plantes. Pour ce faire, des méthodes de génétique classique, de génomique et d’épigénomique seront utilisées. Le second objectif de ce projet est de comprendre comment les bactéries empêchent la formation de mutations au niveau de bases méthylées dans leur génome, en utilisant Caulobacter crescentus comme système modèle. Nous caractériserons les systèmes de réparation des mésappariements de l’ADN chez cette bactérie en utilisant notamment des approches cellulaires par l’utilisation de microscopes de nouvelle génération à très haute résolution. Globalement, les résultats de ce projet devraient permettre de mieux comprendre le rôle et les conséquences de la méthylation de l’ADN chez les Alphaprotéobactéries, mais aussi chez les organismes plus complexes où l’épigénétique est associée au développement de nombreuses maladies.

Direct link to Lay Summary

Last update: 21.04.2017

Active participation

Self-organised

Title	Year

Number	Title	Start	Funding scheme

Methylation is a DNA modification frequently found in all kingdoms of life. Epigenetics has been mostly studied in eukaryotes such as fungi, plants and humans, where it has now become evident that it plays important roles during embryogenesis, cellular differentiation, genomic imprinting and cancer development. Methylated cytosines (m5C and m4C) and adenines (m6A) are also frequently found in bacterial genomes due to the activity of restriction-modification systems acting as immigration controllers preventing horizontal gene transfers, but also of orphan DNA methyl-transferases (MTases) involved in DNA mismatch repair and/or in regulating gene expression to control a variety of processes including pathogenesis and virulence. Our previous work on the cell cycle-regulated DNA MTase CcrM that is found in most Alphaproteobacteria, has uncovered a tight connection between adenine methylation and cell cycle control in the model C. crescentus bacterium. Still, too little is known on which genes are directly regulated by DNA methylation in Alphaproteobacteria and on epigenetic regulators that can sense DNA methylation marks to modulate gene expression. The first aim of this proposal is to address this issue directly. We will use (epi)genomics and classical molecular biology methods to identify genes regulated by DNA methylation in the environmental bacterium Caulobacter crescentus and in the distantly-related Agrobacterium tumefaciens plant pathogen. We will then select the most interesting genes, preferentially regulated in a CcrM-dependent manner in all three bacteria, to identify novel and conserved epigenetic regulators in C. crescentus using forward genetics or biochemical screens. Considering that the expression of the majority of the genes suspected to be regulated by DNA methylation is cell cycle-regulated and/or encoding proteins involved in DNA metabolism in C. crescentus, we hope to uncover novel cell cycle regulators that we will characterize in more details to reveal their mechanism(s) of action and their function(s) in Alphaproteobacteria.Although DNA methylation plays important roles in so many organisms, it comes with a serious drawback: m5C can be accidentally deaminated into thymines (T). Resulting TG DNA mismatches must be corrected before they get replicated or they can turn into permanent mutations. Bacteria use Vsr endonucleases during the Very Small Patch (VSP) repair process to eliminate these mismatches but the regulation and the specificity of this process remain unclear, especially in bacteria that have multiple cytosine MTases and multiple Vsr-like proteins like C. crescentus. The second aim of this proposal is to characterize and visualize the VSP process in live C. crescentus cells using molecular genetics and state-of-the-art microscopy experiments. Considering that VSP repair also requires MutS and MutL, two proteins also involved in the repair of DNA mismatches escaping from the proofreading activity of the replicative DNA polymerase (MMR), we will study the potential competition or cooperation between MMR and VSP repair in C. crescentus cells. Defects in DNA mismatch repair are a concern for human health as they can promote the incidence of antibiotic-resistant bacterial strains or of several cancers and neurodegenerative diseases.