Blanc SVSE 6 - Sciences de la vie, de la santé et des écosystèmes : Génomique, génomique fonctionnelle, bioinformatique, biologie systémique

Spatiotemporal program of replication of the human genome – REFOPOL

How human cells copy their genome

Chromosomes are faithfully duplicated each time a cell divides into two daughter cells. This fundamental process to all living beings has a considerable medical and societal impact. In human it starts at tens of thousands «replication origins« whose location and activation time we try to identify exactly.

Understanding the logic of the chromosome duplication program

Our goal is to identify the location and activation time of each of these sites by determining the local direction of copying on the whole set of chromosomes. The results allow us to understand in each cell type the link between chromosomal organisation in the nucleus, gene expression, temporal order of copying different parts of the chromosomes and points of fragility in this process.

Our goal is to identify the location and activation time of each of these replication start points by determining the local copying direction on the whole set of human chromosomes. To do this we isolate DNA fragments while they are being copied and establish their nucleotide sequence so as to reconstruct the process. This very difficult task has become possible thanks to new sequencing techniques that allow large scale, genome-wide studies, to progresses in bionformatics that allow to manipulate huge amounts of data, and to mathematical modelling efforts that allow to interpret these data and understand the dynamics of the process.

We have shown that the genome can be subdivided into one to two thousands domains that are copied in a characteristic manner. The borders of these domains are the first to be copied. Copying then progresses towards their centre, through ordered activation of (about thirty) replication origins scattered along the domain. Each of these domains corresponds to a chromatin globule within which the chromatin filament is coiled upon itself. Thus the different parts of a domain frequently interact with themselves but seldom interact with other, even adjacent domains. Inside these insulated domains genes position near the borders and are oriented so that they are transcribed in the same direction as they are replicated. Finally we observed that the copy is more faithful at the borders than at the center and that during evolution the last copied parts change faster than the first copied parts. Our results therefore unveil a physico-biological structuration of chromosomes conserved through mammals and birds into functional units of coordinated replication, gene expression and chromosomal filament organisation.

Work in progress will allow to provide the scientific community a set of data allowing any researcher to know in which direction any part of the genome is copied in a variety of cell types (normal or cancerous, embryonic of differentiated, young or aging, etc...). It will be possible to confront these data with other data available now or in the future on innumerable aspects of chromosomal functioning: rearranged regions in cancers, position and expression of genetic-disease associated genes, regions conserved or not between human and other animal species. In the long term we will better understand how the DNA copying process interacts with all other aspects of DNA function and how it shapes the genome during evolution.

1. Chen CL, Duquenne L, Audit B, Guilbaud G, Rappailles A, Baker A, Huvet M, d'Aubenton-Carafa Y, Hyrien O, Arneodo A, Thermes C (2011) Replication-associated mutational asymmetry in the human genome. Mol Biol Evol 28: 2327-2337

2. Guilbaud G, Rappailles A, Baker A, Chen CL, Arneodo A, Goldar A, d'Aubenton-Carafa Y, Thermes C, Audit B, Hyrien O (2011) Evidence for Sequential and Increasing Activation of Replication Origins along Replication Timing Gradients in the Human Genome. Plos Comput Biol 7: e1002322.

3. Ma E, Hyrien O, Goldar A (2012) Do replication forks control late origin firing in Saccharomyces cerevisiae? Nucleic Acids Res 40: 2010-2019.

4. Baker A, Audit B, Chen CL, Moindrot B, Leleu A, Guilbaud G, Rappailles A, Vaillant C, Goldar A, Mongelard F, d'Aubenton-Carafa Y, Hyrien O, Thermes C, & Arneodo A (2012) Replication fork polarity gradients revealed by megabase-sized U-shaped replication timing domains in human cell lines. PLoS Comput. Biol. 8(4): e1002443.

5. Audit B, Baker A, Chen CL, Rappailles A, Guilbaud G, Julienne H, Goldar A, d'Aubenton-Carafa Y, Hyrien O, Thermes C, Arneodo A (2012) Multi-scale analysis of genome wide replication timing profiles using wavelets. Nature Protocols, en révision.

6. A. Baker, B. Audit, S. C.-H. Yang, J. Bechhoefer, A. Arneodo, Inferring where and when replication initiates from genome-wide replication timing data, Phys. Rev. Lett. 108 (2012) 268101.

7. B. Audit, L. Zaghloul, A. Baker, A. Arneodo, C.-L. Chen, Y. d’Aubenton-Carafa & C. Thermes. Megabase replication domains along the human genome : Relation to chromatin structure and genome organisation in Epigenetics: Development and Disease, édité par T. K. Kundu (Springer, New York, 2012).

8. B. Moindrot, B. Audit, P. Klous, A. Baker, C. Thermes, W. de Laat, P. Bouvet, F. Mongelard, A. Arneodo, 3D chromatin conformation correlates with replication timing and is conserved in resting cells, Nucleic Acids Res. (2012) in press.

Human DNA replication initiates from a large number of sites called replication origins according to a spatiotemporal program modulated by the type of tissue and/or by the development stage. Understanding how this program is controlled is a major goal in the field but to date, very few origins have been characterized. Origin specification appears to rely on ill-defined features of chromatin rather than just on recognition of simple DNA sequence motifs.
During the HUGOREP ANR project (2006-2008), we determined the replication timing profile of the entire human genome by massive sequencing of replication intermediates. This, in association with bioinformatic analyses showed that a third of the human genome is organized in "N-domains", i.e. ~1Mb segments whose borders contain particularly efficient replication origins and which show a characteristic N-shaped nucleotide compositional skew profile. This skew is thought to arise from an asymmetry of mutation/repair processes between the leading and lagging strands of the replication fork and to reflect the mean polarity of the forks. Detailed analysis of the temporal gradient of replication that spreads from the borders to the center of N-domains and DNA combing experiments showed that replication first initiates at master origins located at N-domain borders and specified by a DNA-sequence encoded, open chromatin structure, followed by a cascade of less efficient, secondary initiations within N-domains.
These results constitute an important breakthrough in understanding the human replication program, but they call for several experimental validations concerning the existence of master and secondary origins, their efficiency, and the remarkable organisation of fork polarity gradients between master origins. Here, we propose to experimentally determine the average polarity of replication forks throughout the human genome by sequencing highly purified Okazaki fragments by a technique that keeps strandedness information. This will not only map origins with high resolution but also measure their efficiency and allow further testing of the initiation cascade model. Abrupt changes in replication fork polarity will identify efficient origins and smooth changes will reveal zones of inefficient initiations that otherwise would be difficult to discern but are key to understanding the spatiotemporal pattern of origin activation genome wide. To our knowledge, sequencing Okazaki fragments has never been considered or reported elsewhere. This method is more direct, more informative and more original than other previously used large scale approaches. We will use both physical modeling of nucleosome dynamics and experiments aimed at revealing the three-dimensional conformation of chromatin domains to establish a link between chromatin structure and fundamental mechanisms of DNA replication within N-domains and in other regions of the genome. These studies will shed new light on the role of chromatin in regulating chromosome replication.
The work will be performed by a consortium of molecular biologists (Partner 1), physicists (Partner 2), bioinformaticians (Partner 3), and biophysicists (Partner 4) who already worked together on previous projects. Our consortium is truly multidisciplinary, highly competitive and motivated and brings together all the competences required for a detailed picture of human genome replication. We are confident that the quality of the consortium that we have built over the years and the solid rooting of our novel approaches in previous experimental and theoretical works will allow us to decipher the enigma of replication origin specification and to understand how thousands of origins are coordinated at the genome-wide scale to ensure the faithful duplication of the human genome.

Project coordinator


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 676,782 euros
Beginning and duration of the scientific project: - 36 Months

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