Description du sujet de thèse

Genomes are organised in an orderly manner according to several scales of DNA folding in all organisms. The organisation of chromatin in the nucleus is functionally important for controlling, in particular, the gene expression programme, DNA replication, repair and segregation. Defects in this organisation are implicated in diseases and cancers. SMC complexes, particularly cohesin, actively participate in this phenomenon by forming intra-chromatid DNA loops, thereby ensuring dynamic regulation of gene expression. Recent studies suggest that transcription may influence the dynamics of these loops, particularly by modulating the positioning of cohesin. In this context, transcription by RNA polymerase I (Pol I), responsible for the production of ribosomal RNAs within the nucleolus, may be an ideal model for exploring this coupling.

We propose to study how Pol I transcriptional activity can modulate chromatin architecture via cohesin. To do this, we will use a mutant identified in the laboratory, which we call SuperPol because it increases Pol I transcriptional activity, as well as the small anti-cancer molecule BMH-21, which specifically inhibits Pol I activity and induces premature transcription terminations (PTTs). We hypothesise that modulating Pol I activity using SuperPol and BMH-21 alters the anchoring or blocking points of cohesin. By comparing the effects of high (SuperPol) and inhibited (BMH-21) transcriptional states, we will seek to determine how the dynamics of Pol I elongation regulate the formation and stability of DNA loops. This project will elucidate a fundamental mechanism linking ribosomal transcription and genome organisation.

The project requires in-depth expertise in molecular biology and genetics of the model organism Saccharomyces cerevisiae in order to explore the links between ribosomal transcription and three-dimensional genome organisation. The candidate must be proficient in classical experimental approaches, such as DNA, RNA and protein manipulation (cloning, site-directed mutagenesis, PCR, RT-qPCR, nucleic acid extraction and purification, Northern blot and protein analysis), as well as the construction and characterisation of mutant strains, in particular the SuperPol mutant. Solid experience in chromatin biology is essential. The project also requires advanced imaging and fluorescence microscopy skills. On the analytical side, bioinformatics skills are essential to integrate multi-omic and imaging analysis methods. Knowledge of ImageJ software is particularly required. At the conceptual level, the project requires a thorough understanding of ribosomal transcription regulation, cohesin mechanisms of action, and genome folding principles. It also requires the ability to integrate molecular and cellular observations in order to propose mechanistic models explaining how Pol I activity influences DNA organisation. Finally, cross-disciplinary skills in managing experimental protocols, critically interpreting results, writing and scientific communication are essential. A strong aptitude for teamwork and promoting collective well-being in the workplace will be appreciated in order to maintain and develop good working conditions.

Contexte de travail

Understanding how living organisms function by deciphering their mechanisms, dynamics and behaviour is the ambition of the Centre for Integrative Biology (CBI) in Toulouse. The CBI was created in 2016 as a Research Federation under the supervision of the French National Centre for Scientific Research (CNRS) and the University of Toulouse (UT). Today, the CBI brings together more than 400 people, spread across its support services, platforms and around 40 teams in three research units: Microbiology (LMGM), Molecular, Cellular and Developmental Biology (MCD) and Animal Cognition (CRCA). Its teams, platforms and services are composed of researchers, teacher-researchers, doctoral and post-doctoral students, administrative and technical staff from the CNRS, as well as from the University and INSERM, making it one of the most important scientific research centres in France. The CBI benefits from a highly collaborative and innovative scientific environment thanks to its location on the main campus of the University of Toulouse. The CBI is a member of the Biology and Biotechnology for Health network (sFR B2S), dedicated to the coordinated dissemination of knowledge and information between different research centres in the field of biology and health research.
Within the CBI, the PhD student will join the Dynamic Organisation of the Nucleus team led by Frédéric Beckouët and Olivier Gadal. The team seeks to answer the central question: How do RNA polymerase and SMC complexes influence the three-dimensional configuration of the genome, using Saccharomyces cerevisiae as a model. This is achieved by combining approaches:
– Genetic, to generate mutants and analyse their impacts
– Biochemical and structural, to explore the mechanisms of SMCs and RNA polymerases
– Molecular, using techniques such as Hi-C and ChIP to study transcription and chromatin folding
– Cellular, using advanced microscopy (video, super-resolution, electron) and innovative methods such as correlative imaging (CLEM)