Within the team, we are developing and coordinating 3 areas of research and one area of collaboration:

Project 1: Replisome dynamics: Characterization of DNA polymerase pause sites during replication in the human genome, role of Pif1 helicase and impact on genome stability.

Our work aims to identify the molecular mechanisms of replication fork breaks, using human cells. This work should enable us to better understand how these replication defects, known as fork stoppers, can contribute to the development of cancers associated with genomic instability. The question now is whether replication pauses are due to: i) either stochastic events, i.e. they occur at location X at time Y and can vary from one cell cycle to another, or ii) they take place at a specific location which, if coordinated with other molecular processes, would enable the genome to be organized and thus reduce the risk of genetic instability.

To carry out this mapping of pause sites, we postulate that loci that impede the progression of replication forks show a longer association with the replisome compared with sites where there are no pauses. These break sites must therefore present a strong association with replicative DNA polymerases. We can therefore observe the distribution of DNA polymerase delta across the genome in replicating asynchronous cells using NGS approaches (ChIP-seq approach with Pol Delta antibody).

Profile obtained from sequencing with anti-pol epsilon and delta antibodies and from the Input sample

We are also using NGS and biochemical approaches to determine the role of the Pif1 helicase during replication and the impact of deregulation of this enzyme on genome stability. During the S-phase, replication forks may encounter several types of structures on chromatin that can impede fork progression. These obstacles can have several origins, for example: a) the formation of a DNA secondary structure, b) the formation of a DNA-Protein(s) complex c) a chromatin modification and/or finally d) transcription itself. Helicases of the Pif1 family are present in virtually all eukaryotes. Pif1 is required for mitochondrial DNA stability. It also appears to be involved in the resolution of specific structures such as G-quadruplexes (in vitro experiment). Finally, Rrm3 (homologous to Pif1) is known in S. cerevisae to facilitate fork progression across obstacles. Conflicts with transcription can have two origins: i) the association of transcription proteins with chromatin, or ii) the formation of transcription-derived RNA-DNA hybrids known as R-loops. The aim of this research project is therefore to characterize the role of Pif1 in the replication process and its impact on genome stability. In other words, we’d like to know whether the absence or overexpression of Pif1 induces replicative stress. In the human K562 line, we have obtained Pif1 KO clones using CRISP/Cas9 approaches, as well as a Pif1 overexpression line using PiggyBAC approaches, in which an HA tag has been added to the protein. On these two lines, we have initiated an initial experiment to compare the timing of replication.

Examples of replication timing profiles obtained for the Pif1 KO line and the Pif1 Surexpression line. In blue, the WT line, in red the genetically modified line. Green lines indicate an advance in timing for the mutant in the regions concerned, pink lines indicate delays.

We can already observe that the absence or overexpression of Pif1 deregulates replication timing. These effects are exacerbated by creating additional replicative stress in the presence of Aphidicolin. Finally, we observed a slowdown in cell growth for these different lines.

Project 2: Impact of CDK4-CDK6 inhibitors on replication and consequent genome stability

Palbociclib is a promising therapeutic molecule in the fight against cancer, as it belongs to the family of “CDK4 and 6 inhibitors”. However, resistance to these molecules can occur in cancer patients. So-called “pRb-negative” cancers are considered resistant to these molecules: cells continue to proliferate despite treatment. However, we have recently shown that these cells continue to proliferate, but have greater difficulty in carrying out DNA replication. This molecule therefore induces “replicative stress”. We are now trying to understand at what level this molecule interferes with the replication process. Using molecular combing approaches, we have observed a reduction in the number of active replication origins, but also an acceleration in the rate of replisome synthesis. This treatment has an effect on the molecular composition of the Initiation Precomplex of replication origins.

Project 3: Impact of Parylation on replication control

PARylation, via the PARP1 enzyme and NAD+, is the molecular process by which a multi-branched chain of ADP-Ribose (PAR) is synthesized at the protein (post-translational modification) or DNA level. This poly-ADP-ribose chain enables the recruitment of other proteins possessing PAR-binding peptide motifs. PARylation may thus be involved in several molecular processes such as chromatin remodeling, transcription, DNA repair and the cell cycle. PARP1’s involvement in DNA repair is the best described today. Briefly, PARP1 acts as a break detector, as it can interact with DNA thanks to the two zinc finger motifs this protein possesses in one of its domains. With this interaction, PARP1 then synthesizes a PAR chain, enabling the subsequent recruitment of proteins involved in DNA repair to the break site.

The involvement of PARP1 in DNA replication is very partially established, but the associated molecular mechanisms are not yet clear. It has been observed, in earlier work using fluorescence microscopy approaches (Coll et al., 1997), that PARP1 appears to be co-localized at sites of DNA synthesis but also at the initiation of this synthesis during the S phase of the cell cycle. Other work seems to show the presence of PARP1 on arrested replisomes (Ronson et al., 2018). We performed experiments in which cells were treated with PARPi or PARGi, inhibitors of the PARP and PARG enzymes respectively. We observed that cells have a strongly disrupted replication timing. EdU incorporation kinetics were performed to determine the effect of this type of treatment on DNA synthesis and replicative stress proteins. A decrease in EdU incorporation was observed at the second hour of treatment. With regard to replicative stress proteins, we once again observed, from the second hour, the appearance of phosphorylation of ChK1, a major protein in the response to replicative stress. Taken together, these preliminary data clearly indicate the direct role of PARylation in the replication process.

PARP inhibition by Olaparib and PARG inhibition by a specific inhibitor have an impact on the replication timing program in K562 cells. (left) Example of a replication timing profile visualized via START-R. The grey dots correspond to the probes, each covering a region of human chromosome 16 (hg18). The y-axis indicates early replication (positive values) and late replication (negative values). (right)Analysis of differences in replication timing between the conditions indicated. The number of regions with delayed or advanced replication is also indicated.

Project 4: Analysis of the temporal program of replication and collaborations.

We have created an App-Shiny to study the temporal program of replication throughout the genome (doi: 10.5281/zenodo.3243339 and https://thomasdenecker.github.io/START-R/index.html ).

copie d’écran du logiciel

To contact a member of the team by e-mail: name.surname@ijm.fr

• Barbara Ben Yamin, Sana Ahmed-Seghir, Junya Tomida, Emmanuelle Despras, Caroline Pouvelle, Andrey Iurchenko, Jordane Goulas, Raphael Corre, Quentin Delacour, Nathalie Droin, Philippe Dessen, Didier Goidin, Sabine S. Lange, Maria Teresa Mitjavila-Garcia, Giuseppe Baldacci, Sergey Nikolaev, Jean-Charles Cadoret*, Richard D Wood* and Patricia L Kannouche*. DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability. EMBO J. 2021 Nov 2;40(21):e104543. *Co-last authors
• Courtot L, Bournique E, Maric C, Guitton-Sert L, Madrid-Mencía M, Pancaldi V, Jean-Charles Cadoret*, Hoffmann JS*, Bergoglio V*. Low Replicative Stress Triggers Cell-Type Specific Inheritable Advanced Replication timing. Int J Mol Sci. 2021 May 7;22(9):4959. doi: 10.3390/ijms22094959. PMID: 34066960; PMCID: PMC8125030. * co-senior and co-corresponding authors
• Djihad Hadjadj, Thomas Denecker, Eva Guérin, Su-Jung Kim, Fabien Fauchereau, Giuseppe Baldacci, Chrystelle Maric, Jean-Charles Cadoret. Efficient, quick and easy-to-use DNA replication timing analysis with START-R suite. NAR genomics & bioinformatics, 2020 journal 2, vol2.
• Djihad Hadjadj, Su-jung Kim, Thomas Denecker, Laura Ben Driss, Jean-Charles Cadoret, Chrystelle Maric, Giuseppe Baldacci, Fabien Fauchereau. A hypothesis-driven approach identifies CDK4 and CDK6 inhibitors as candidate drugs for treatments of adrenocortical carcinomas. Aging, Albany NY). 2017 Dec 26;9(12):2695-2716
• Julien Brustel, Nina Kirstein, Fanny Izard, Stanimir Dulev, Charlotte Grimaud, Chrystelle Cayrou, Gunnar Schotta, Marcel Mechali, Giuseppe Baldacci, Claude Sardet, Nizar N. Batada, Jean-Charles Cadoret*, Aloys Schepers* & Eric Julien*. Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication. EMBO J. 2017 Sep 15;36(18):2726-2741, * co-senior and co-corresponding authors & Commentaire dans F1000
• Fernandez-Vidal, Anne *; Guitton-Sert*, Laure; Jean-Charles Cadoret*;  et al. A role for DNA polymerase theta in the timing of DNA replication. Nature Communications Volume: 5 Article Number:4285 JUL 2014 * first co-authors, Selected for a research highlight in Nature Reviews Molecular Cell Biology 15, 499 5)
• Briu LM, Maric C, Jean-Charles Cadoret. Replication Stress, Genomic Instability, and Replication timing: A Complex Relationship. Int J Mol Sci. 2021 Apr 30;22(9):4764. doi: 10.3390/ijms22094764. PMID: 33946274; PMCID: PMC8125245.

software : logiciel START-R : https://github.com/thomasdenecker/START-R

Publications

Reviews

Book chapter

Manuel de Sciences fondamentales pour l’ostéopathe, chapitre sur la biologie moléculaire, Edition DE BOECK, parution août 2018, Auteur de deux chapitres : Biologie moléculaire et Biologie cellulaire

Software

Logiciel START-R : https://github.com/thomasdenecker/START-R[/vc_column_text][/vc_tta_section][vc_tta_section title=”Thèses” tab_id=”1645024967580-08355b73-0edc”][vc_column_text]• Lina-Marie Briu (depuis 2020)
• Su-Jung Kim ( 2017-2021) → Post-doctorat à l’Institut Gustave Roussy
• Djihad Hadjad (2014-2018) → Poste de Maître de conférence à l’Université de Paris

•  Lina-Marie Briu (since 2020)
• Su-Jung Kim ( 2017-2021) → Post-doc at Institut Gustave Roussy
• Djihad Hadjad (2014-2018) → Lecturer at Université de Paris

• Erika Brunet, Institut Imagine, Paris.
• Robert Feil, IGMM, Montpellier.
• Françoise Dantzer et Daan Noordermeer, BSC, Strasbourg et I2BC Gif/yvette.
• Cyril Ribeire, IGH, Montpellier.
• Corrado Santocanale, Centre for Chromosome Biology, Galway University
•  Valérie Bergoglio, Cancer Research Center of Toulouse : stress réplicatif et réplication
• Patricia Kannouche, Institut Gustave Roussy, Villejuif
• Eric Julien, IRCM, Montpelliers
• Philippe Pasero Institut de génétique humaine, Montpellier
• Benoit Miotto, Institut Cochin, Paris USPX9 et réplication
• Laure Crabbé , I2BC, Gif/ Yvette
• Maria Gomez, Madrid, Espagne
• Frédéric Chedin, UC Davis, USA : Programme de professeur invité

• 2022-2025: INCa project: Alteration of the temporal program of replication in T lymphocytes with ALK translocation
• 2022-2024: Emergence project (IDEX Université de Paris) with Karine Dubrana’s team, CEA Fontenay aux Roses
• 2020-2022 : IDEX “Dynamique Recherche” AAP winner
• 2018 : Winner of a Visiting Professorship: Fred Chedin UC-Davis USA
• 2018 : GEFLUC research grant: Impact of replicative DNA polymerase pauses on genome stability.
• 2016-2019 : INCa project: Role of translesional DNA polymerases in the replication process.
• 2015-2016 : Ligue contre le cancer Île de France: Alterations of DNA replication in Cancers
• 2015-2018 : ANR blanche: SPUR; Specialized Polymerase involved in Unchallenged Replication
• 2016 : Donation Mme Larzat