Team
ODN
Team manager: Gadal Olivier & Beckouet Fredéric
Presentation
Genomes are dynamically organized, with structures ranging from nucleosomes to chromosome territories, which change throughout the cell cycle. This 3D chromosome folding influences key DNA processes like transcription, repair, and chromosome segregation during mitosis. Loss of this 3D organization is linked to diseases, including cancer. The organization of the genome depends on translocases such as RNA polymerases, DNA polymerases, topoisomerases, and SMC complexes, but the interactions and relative roles of these complexes remain unclear.
Our research focuses on understanding how RNA polymerase and SMC complexes shape the 3D genome. Using Saccharomyces cerevisiae as a model organism, we employ cutting-edge technologies, including:
– Genetic approaches to create and analyze mutant strains
– Biochemical and structural studies to explore the functions of SMC complexes and RNA polymerases, with tools like DNA curtains for chromatin loop analysis
– Molecular biology techniques (3C, Hi-C, ChIP, RNAseq and CRAC) to examine RNA synthesis, maturation, and chromatin folding
– Cell biology methods (super-resolution, fast video microscopy, electron microscopy, CLEM) to study nuclear compartmentalization, gene transcription, and chromatin structure
This research aims to clarify how 3D genome organization impacts cellular processes and to understand the molecular mechanisms behind chromatin folding and gene regulation.
Project 1
The three nuclear RNA polymerases are molecular motors that influence genome structure. RNA polymerase II transcribes most of the genome, as well as all mRNAs and many non-coding RNAs. RNA polymerase III transcribes small RNAs (tRNA, 5S rRNA), while RNA polymerase I transcribes a single gene, the precursor of large ribosomal RNAs (35S rRNA).
To understand how RNA polymerase contributes to genome organization, we are focusing our research on the mechanisms regulating ribosomal RNA synthesis by RNA polymerase I and analyzing their impact on 3D nucleolar organization and genome stability:
We have identified several levels of transcriptional control:
A novel role for the initiation factor Rrn3 in genomic stability and the accumulation of an immature form of pre-rRNA (Normand et al., 2024). Our results suggest that this inhibitory function of rRNA maturation has an impact on genomic stability. We are continuing to characterize this new inhibitory function of Rrn3 on rRNA processing.
A mechanism for attenuating transcription. By characterizing a mutant form of Pol I that increases rRNA production, we were able to show that premature termination of transcription frequently occurs during rRNA synthesis (Azouzi et al., 2023). We propose that this termination regulates rRNA synthesis through attenuation (Azouzi et al., 2021). We will characterize the rearrangements of RNA polymerase subunits and the factors in trans that regulate this process.
A new role for the KKE domain (Dominique et al., 2024). The nucleolus is enriched with a KKE-rich protein domain, known as the intrinsically disordered region (IDR), capable of forming phases in vitro. In collaboration with the Eukarybio team (Henry/Henras), we have recently demonstrated the role of this particular KKE domain in the organization and function of the nucleolus (Dominique et al., 2024). These domains are also present on RNA polymerase I subunits and on certain transcription factors. Nevertheless, how these IDRs regulate transcriptional activity and the 3D organization of the nucleolus remains poorly understood. Our aim is to better understand the role of these IDRs in nucleolar activity and organization.
The tools used for these studies are :
Techniques for quantifying transcript synthesis (CRAC, Run-On, metabolic labeling), analysis of accumulated RNA (Northern) and purification of protein complexes.
Cell biology tools for the study of nuclear compartmentalization in relation to gene transcription include rapid videomicroscopy, super-resolution microscopy on live or fixed cells, and electron microscopy.
We are developing CLEM (Correlative light and electron microscopy) imaging methods to improve the resolution of fluorescence imaging.
Bibliography
Bibliography
– Azouzi, C., Jaafar, M., Dez, C., Abou Merhi, R., Lesne, A., Henras, A.K., and Gadal, O. (2021). Coupling between production of ribosomal RNA and maturation: just at the beginning. Front. Mol. Biosci. 8, 778778.
– Azouzi, C., Schwank, K., Queille, S., Kwapisz, M., Aguirrebengoa, M., Henras, A., Lebaron, S., Tschochner, H., Lesne, A., Beckouët, F., et al. (2023). Ribosomal RNA synthesis by RNA polymerase I is regulated by premature termination of transcription. BioRxiv.
– Dominique, C., Maiga, N.K., Méndez-Godoy, A., Pillet, B., Hamze, H., Léger-Silvestre, I., Henry, Y., Marchand, V., Neto, V.G., Dez, C., et al. (2024). The dual life of disordered lysine-rich domains of snoRNPs in rRNA modification and nucleolar compaction . Nat. Commun.
– Normand, C., Dez, C., Dauban, L., Queille, S., Danché, S., Abderrahmane, S., Beckouet, F., and Gadal, O. (2024). RNA polymerase I mutant affects ribosomal RNA processing and ribosomal DNA stability. RNA Biol. 21, 1–16.
Project 2
DNA loops. These loops are important for the three-dimensional organization of the genome and the regulation of various biological processes. A series of in vitro studies has recently demonstrated that SMC complexes form DNA loops by capturing small loops and expanding them by moving along the DNA using a molecular motor (Ref). Although these studies represent a considerable advance, the mechanisms governing SMC-dependent loop formation remain mysterious. Pioneering studies have shown that the cohesin complex is capable of forming DNA loops in mammals during interphase. These loops are important for organizing the genome into topological domains (TADs), regulating gene transcription or controlling VDJ recombination. Recently, we demonstrated that cohesin-dependent DNA loops also organize the mitotic chromosome of S. cerevisiae. We are therefore using the yeast S. cerevisiae, taking advantage of the panel of technologies (Hi-C, Chip, microscopy and genetics) to highlight the molecular mechanisms controlling cohesin-dependent loop formation.
QUESTIONS :
What mechanisms inhibit loop expansion?
L’anneau de cohésine n’agit pas seul. Son activité ATPase et son association avec l’ADN sont régulées par un ensemble de protéines auxiliaires : Scc2, Pds5, Wpl1 Scc1 et l’acétyl transférase Eco1. L’utilisation de Hi-C chez les mammifères a démontré que la dissociation des cohésines par la protéine Wpl1 atténue l’expansion des boucles d’ADN. En utilisant Hi-C, nous avons démontré que ce mécanisme est conservé chez S. cerevisiae. Nos études récentes ont également montré que l’expansion des boucles d’ADN est inhibée par d’autres mécanismes indépendants de Wpl1 impliquant Pds5 (Bastié et al., 2022), Eco1 (Dauban et al., 2020) ou la cohésion des chromatides sœurs (Bastié et al., 2024). Un de nos objectifs est de comprendre comment ces protéines auxiliaires exercent leur activité inhibitrice sur l’expansion des boucles d’ADN.
What are the mechanisms governing DNA loop expansion?
The Scc2 factor was originally identified as being necessary for the association of cohesins on DNA. Recently, it has been shown that Scc2 is also required for the expansion of DNA loops along chromosomes. Scc2 is thought to promote loop expansion by stimulating the ATPase activity required for cohesin translocation on DNA. We are currently investigating the mechanisms regulating Scc2 function during the cell cycle.
Interaction between cohesin and RNA polymerase
Cohesion mainly localizes between genes in convergent orientation. Based on this observation, it has been proposed that the expansion of DNA loops is caused by RNA polymerase pushing cohesin along the DNA. Another model proposes that RNA polymerase acts as a barrier to loop expansion. Finally, another model proposes that cohesin loads mainly between converging genes. Our laboratory studies the role of transcription in regulating genome organization (Chapard et al., 2023).
What is the nature of the interaction between cohesin and DNA loops?
The cohesin complex was originally identified for its role in sister chromatid cohesion (SCC) during mitosis. Data from Kim Nasmyth’s laboratory demonstrated that cohesin ensures SCC by encompassing sister DNAs within its ring (topological interaction). The nature of the DNA/cohesin interaction involved in loop formation and maintenance remains a mystery to this day. Our team aims to identify the nature of the interaction between cohesin and DNA loops, and to determine how auxiliary factors regulate it.
Bibliography
– Bastié, N., Chapard, C., Dauban, L., Gadal, O., Beckouët, F., and Koszul, R. (2022). Smc3 acetylation, Pds5 and Scc2 control the translocase activity that establishes cohesin-dependent chromatin loops. Nat. Struct. Mol. Biol. 29, 575–585.
– Bastié, N., Chapard, C., Cournac, A., Nejmi, S., Mboumba, H., Gadal, O., Thierry, A., Beckouët, F., and Koszul, R. (2024). Sister chromatid cohesion halts DNA loop expansion. Mol. Cell 84, 1139–1148.e5.
– Chapard, C., Bastie, N., Cournac, A., Gadal, O., Koszul, R., and Beckouet, F. (2023). Transcription promotes discrete long-range chromatin loops besides organizing cohesin-mediated DNA folding. BioRxiv.
– Dauban, L., Montagne, R., Thierry, A., Lazar-Stefanita, L., Bastié, N., Gadal, O., Cournac, A., Koszul, R., and Beckouët, F. (2020). Regulation of Cohesin-Mediated Chromosome Folding by Eco1 and Other Partners. Mol. Cell 77, 1279–1293.e4.
– The dual life of disordered lysine-rich domains of snoRNPs in rRNA modification and nucleolar compaction. Dominique C, Maiga NK, Méndez-Godoy A, Pillet B, Hamze H, Léger-Silvestre I, Henry Y, Marchand V, Gomes Neto V, Dez C, Motorin Y, Kressler D, Gadal O, Henras AK, Albert B. Nat Commun. 2024 Oct 31;15(1):9415. doi: 10.1038/s41467-024-53805-1. PMID: 39482307
– RNA polymerase I mutant affects ribosomal RNA processing and ribosomal DNA stability. Normand C, Dez C, Dauban L, Queille S, Danché S, Abderrahmane S, Beckouet F, Gadal O. RNA Biol. 2024 Jan;21(1):1-16. doi: 10.1080/15476286.2024.2381910. PMID: 39049162
– Sister chromatid cohesion halts DNA loop expansion. Bastié N, Chapard C, Cournac A, Nejmi S, Mboumba H, Gadal O, Thierry A, Beckouët F, Koszul R. Mol Cell. 2024 Mar 21;84(6):1139-1148.e5. doi: 10.1016/j.molcel.2024.02.004. PMID: 38452765
– Smc3 acetylation, Pds5 and Scc2 control the translocase activity that establishes cohesin-dependent chromatin loops. Bastié N, Chapard C, Dauban L, Gadal O, Beckouët F, Koszul R. Nat Struct Mol Biol. 2022 Jun;29(6):575-585. doi: 10.1038/s41594-022-00780-0. PMID: 35710835
– Regulation of Cohesin-Mediated Chromosome Folding by Eco1 and Other Partners. Dauban L, Montagne R, Thierry A, Lazar-Stefanita L, Bastié N, Gadal O, Cournac A, Koszul R, Beckouët F. Mol Cell. 2020 Mar 19;77(6):1279-1293.e4. doi: 10.1016/j.molcel.2020.01.019. PMID: 32032532
– Quantification of the dynamic behaviour of ribosomal DNA genes and nucleolus during yeast Saccharomyces cerevisiae cell cycle. Dauban L, Kamgoué A, Wang R, Léger-Silvestre I, Beckouët F, Cantaloube S, Gadal O. J Struct Biol. 2019 Nov 1;208(2):152-164. doi: 10.1016/j.jsb.2019.08.010. PMID: 31449968
Funding
Affiliation
