Ecole doctorale "LOGIQUE DU VIVANT

Ecole Doctorale COMPLEXITE DU VIVANT – Fiche Projet CONCOURS
Nom et prénom du directeur de thèse (et si besoin du co-directeur) : Olivier Hyrien (directeur) et Auguste
Genovesio (co-directeur)
Le directeur de thèse et le co-directeur doivent impérativement être habilités à diriger les recherches (HDR)
Coordonnées
Tel : 0144323920 (O.H.) 0144322354 (A.G.)
e-mail : [email protected], [email protected]
Nom et prénom du co-encadrant (non HdR ) (s’il y a lieu) :
Coordonnées Tel :
e-mail :
Nom et prénom du responsable de l’équipe :Olivier Hyrien
Nombre de chercheurs et enseignants-chercheurs statutaires de l’équipe titulaires d’une HDR :
Equipe OH : 1 Equipe AG : 1
Nom et prénom du responsable du laboratoire : Antoine Triller
Intitulé du laboratoire et N° d’unité : Institut de Biologie de l'Ecole Normale Supérieure (IBENS), UMR CNRS 8197, U
Inserm 1024
Spécialité : Bioinformatique, Génomique
Titre du projet de thèse : Imagerie à haut débit de molécules d'ADN réplicatives / High-throughput
imaging of replicating DNA molecules
Résumé du projet de thèse (1 page maximum, en anglais)
Context and aims. The aim of the project is to develop an automated analysis pipeline of single molecules of replicating
DNA visualized by fluorescence microscopy in order to analyze the replication of the genome of several model organisms at
the single-molecule level. The experimental data will be produced by the experimental biology team of O. Hyrien at the
IBENS and their automated analysis will be developed by the team of A. Genovesio at the IBENS. Modelisation of wholegenome replication will involve our long-term collaborators B. Audit (ENS Lyon) and A. Goldar (CEA-Saclay), two
physicists specialized in signal analysis and modelling of DNA replication. The student, who must possess basic knowledge
in applied mathematics and statistics, image analysis and programming (Python) will therefore be integrated into a
multidisciplinary consortium with appropriate competences for the project.
DNA replication is a fundamental process of all living organisms important for medicine and biotechnology. Perturbations
of this process can cause mutations, cancers and other diseases. Many antiviral, antibacterial and anticancer drugs target the
DNA replication process. For these reasons replication "stress", defined by an increased incidence or slowed or stalled DNA
synthesis, has recently attracted much attention.
Stochasticity in DNA replication and the interest of single-molecule studies. Eukaryotic DNA replication starts at
multiple sites called replication origins which are activated at different times during the S phase of the cell cycle. Each
origin emits a pair of replication forks which progress at a relatively constant speed until they merge with converging forks
emitted by adjacent origins. The mechanism that specify the position and activation time of origins in mammalian cells are
incompletely understood. In a cell population, each origin is activated in only a fraction of the chromosomal copies and
within a broad temporal window rather than at a fixed time. Due to this stochasticity, any locus can replicate with some
probability by forks coming from any origin and moving in any direction. Most genomic techniques for studying replication
only provide an average picture of replication in a cell population. Only single-molecule techniques can visualise the
activation of individual origins, the progression of individual forks, and evaluate cell-to-cell heterogeneity and reveal rare
events (e.g. fork stalling) which are masked in the population average. Single-molecule techniques are also required to
reveal local correlations between neighboring origins and forks, which is important to correctly understand and model the
process and its pathologies (e.g. in tumour cells).
Current limitations in single-molecule study of DNA replication. Current methods are slow and laborious for two main
reasons. 1) Visualizing replication fork progression requires metabolic labeling with nucleoside or nucleotide analogs, DNA
purification and stretching on microscope coverslips, and detection of the tracts of incorporated analogues with specific
fluorescent antibodies. 2) Identification and orientation of fibres corresponding to a locus of interest requires fluorescent in
situ hybridization with a complex collection of DNA probes, which can only identify a tiny fraction (<0.05%) of the
molecules. 3) Automation of the process is difficult due to imperfect stretching of the molecules and background signals.
High-throughput visualisation of single replicating DNA molecules. We have recently developed technical innovations
that considerably increase the quality and throughput of visualization of single replicating DNA molecules. 1) Incorporation
of fluorescent dNTPs during metabolic labeling allows to detect replication tracts directly, without any antibody detection.
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Ecole Doctorale COMPLEXITE DU VIVANT – Fiche Projet CONCOURS
2) When DNA is stained with YOYO-1, a fluorescent DNA intercalator, prior to stretching, replicated segments show a
distinctly stronger fluorescence than unreplicated segments of the same DNA molecule. 3) DNA barcoding by incorporation
of fluorescent dNTPs at specific nicking endonuclease (NE) sites allows to visualize a restriction map of each molecule,
opening the opportunity for rapid mapping of the DNA replication intermediates to their locus of origin. 4) The combination
of these innovations allows to simulateneously visualize the replication tracts, backbone and barcode of stretched DNA
molecules in three distinct colours by standard epifluorescence microscopy, with no need for antibody staining and/or FISH
detection. 5) DNA can be stretched in multiple parallel nanochannels rather than on glass surfaces, which increases the
throughput by two orders of magnitude (typically 50,000 molecule images instead of a few hundreds per experiment). These
technical breakthroughs allow us to envision the automated analysis of these three-colour molecular images to create a
genome-wide, single-molecule profile of DNA replication.
Automated analysis of single replicating DNA molecules. The exploitation of the huge datasets that we are generating in
several model organisms requires to automate their analysis. The steps to develop are 1) automatic detection of the ends of
the YOYO-1 stained DNA molecules; 2) tracing a fluorescent intensity profile of each molecule in three colours (YOYO-1,
replicative label and barcode; note that YOYO-1 will detect all replicated DNA whereas the replicative label will detect
DNA replicated during the labeling pulse, allowing to measure individual replication fork speeds; 3) segment these profiles
in replicated/unreplicated and labeled/unlabeled segments; analyze the coincidence of these labels and extract replication
fork speeds; 4) align the barcode of each molecule to the restriction map of the genome of reference so as to determine the
locus of origin of each molecule. Steps 1 and 2 are relatively easy and already advanced. Steps 3 and 4 are more complex
but can take advantage of existing algorithms for analysis of copy number variation, "optical mapping" of single molecules
and sequence alignment.
Modelisation of genome replication. The generation of novel datasets and their automated analysis will allow us to address
several important biological problems in DNA replication. In all eukaryotes, more potential replication origins are licensed
in G1 phase than are actually used in the next S phase. This redundancy of potential origins contributes to their apparently
stochastic usage, and provides a failsafe mechanism to rescue replication downstream of slow forks and to boost S phase
completion. The project proposed here will allow to quantify site-specific and dispersive initiation and replication fork
progression genome-wide in yeast, Xenopus and human cells. This will allow to explore how these parameters relate to each
other and to transcription and genomic instability in the three organisms, which may lead to a more unified view of
eukaryotic DNA replication.
Thèses actuellement en cours dans l’équipe
Nom et Prénom du doctorant
Nom du directeur de thèse
Année de 1ere
inscription et
Ecole Doctorale
Financement pendant la thèse
Francesco de Carli
Xia Wu
Olivier Hyrien
Olivier Hyrien/Ruohong
Xia
2013 CdV
2013 CdV
Bourse UPMC
Bourse du Gouvernement Chinois
Felipe Delestro
Auguste Genovesio
2014 CdV
Bourse PSL
Trois publications récentes du directeur de thèse (du co-directeur ou du co-encadrant s’il y a lieu).Mettre en gras le nom
du directeur de thèse.
Directeur (O. Hyrien) :
1. Goldar A, Arneodo A, Audit B, Argoul F, Rappailles A, Guilbaud G, Petryk N, Kahli M, Hyrien O (2016) Deciphering
DNA replication dynamics in eukaryote cell populations in relation to their averaged chromatin conformations. Sci. Rep., 6,
22469.
2. Petryk N, Kahli M, d'Aubenton-Carafa Y, Jaszczyszyn Y, Shen Y, Sylvain M, Thermes C, Chen CL, Hyrien O (2016)
Replication landscape of the human genome. Nature Comm., 7, 10208.
3. Hyrien O (2015) Peaks cloaked in the mist: The landscape of mammalian replication origins. J. Cell Biol, 208, 147-160.
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Ecole Doctorale COMPLEXITE DU VIVANT – Fiche Projet CONCOURS
Co-directeur (A. Genovesio) :
1.
Shantanu Singh, Anne E Carpenter, and Auguste Genovesio. Increasing the content of high-content screening: An
overview. J Biomol Screen, 19(5):640–650, Apr 2014.
2.
Auguste Genovesio*, Yong-Jun Kwon*, Marc P Windisch*, Nam Youl Kim, Seo Yeon Choi, Hi Chul Kim,
Sungyong Jung, Fabrizio Mammano, Virginie Perrin, Annette S Boese, Nicoletta Casartelli, Olivier Schwartz, Ulf
Nehrbass, and Neil Emans. Automated genome-wide visual profiling of cellular proteins involved in hiv infection.
J Biomol Screen, 16(9):945–958, Oct 2011.
3.
Auguste Genovesio*, Miriam A Giardini*, Yong-Jun Kwon*, Fernando de Macedo Dossin, Seo Yeon Choi, Nam
Youl Kim, Hi Chul Kim, Sung Yong Jung, Sergio Schenkman, Igor C Almeida, Neil Emans, and Lucio H Freitas
Junior. Visual genome-wide rnai screening to identify human host factors required for trypanosoma cruzi
infection. PLoS One, 6(5):e19733, 2011.
Docteurs encadrés par le directeur de thèse ayant soutenu après septembre 2010 et publications relatives à leur sujet de thèse.
Mettre en gras le nom du directeur de thèse et celui du docteur.
Nom Prénom : le Viet Barbara
Date de soutenance : 06/09/12
Durée de thèse (en mois): 48
Ecole Doctorale : CdV
Publications :
1. Gaggioli V*, Le Viet B*, Germe T, Hyrien O (2013) DNA topoisomerase II controls replication origin cluster
licensing and firing time. Nucleic Acids Res., 41, 7313-7331.
* co-premiers auteurs.
Docteurs encadrés par le co-directeur de thèse
Nom Prénom : Hee Chang Kim
Date de soutenance : 11/2011
Durée de thèse (en mois): 36
Ecole Doctorale : N/A (thèse effectuée en Corée)
Direction: Georges Stamon, Auguste Genovesio
Publications :


H. C. Kim and A. Genovesio. Neuron branch detection and description using random walk. Conf Proc IEEE
Eng Med Biol Soc, 2009:1020-1023, 2009
H. C. Kim, G. Stamon, and A. Genovesio. A method for discontinuous neurite reconstruction based on di
usion tensor, hessian eigenvector, and di used gradient vector fields,. In Proceedings of ICIP, 2011
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