Replication program and genome instability


Chunlong Chen Team Leader Tel:

DNA replication must adapt to changes in chromatin organization associated with cell differentiation and development, whose deregulation can challenge genome stability and leads to mutations, cancer and many other genetic diseases. However, despite intensive studies, the mechanisms that coordinate where and when replication initiates in the human genome remain poorly known. Our team focuses on using cutting-edge high-throughput genomic approaches and genome-wide data analyses to study the spatio-temporal replication program of the human genome and its impact on genome stability, in particular to address the following questions:

  • What determines the replication program, i.e. the position, the time of firing and the efficiency of replication origins in the human genome?
  • How this program is regulated and associated with gene transcription and chromatin organization?
  • How deregulation of these programs challenges genome stability and leads to human diseases?
Figure 1: Le programme Spatio-temporel de réplication du génome humain révélé par Ok-Seq (la directionalité des fourches de réplication, RFD) et Repli-Seq (le « Timing » de réplication). Les segments ascendants et descendants sur les profils RFD indiquent les emplacements des zones d’initiation et terminaison de réplication, respectivement.
Figure 1: Spatio-temporal replication program of the human genome revealed by Ok-Seq (Replication Fork Directionality, RFD) and Repli-Seq (Replication Timing). The ascending and descending segments on the RFD profiles indicate the locations of replication initiation and termination zones, respectively.

In collaboration with experimental biologists, we have developed a method (Repli-Seq) and generated one of the first high-resolution replication timing profiles of the human genome (Fig. 1). Studies of these profiles from different human cell types have allowed us to reveal that the genome is organized in megabase replication domains associated with higher order chromatin structural units. By evolutionary analyses, we have also established that replication is a major process driving genome mutational landscape in normal and cancer cells. We are now applying Repli-Seq technique to analyze the replication dynamics from cells upon replication stress to study how deregulation of the replication program challenges genome stability, in particular, common fragile site activity (Fig. 2).

Screenshot 2019-09-19 at 14.53.31
Figure 2: OK-Seq, Repli-Seq (for Non-Treated and Aph treated cells), and GRO-Seq profiles are shown along regions around the indicated genes hosting Common Fragile Sites (CFSs) fine-mapped by FISH (black bars) in lymphocytes. The initiation and termination zones are highlighted by orange and purple boxes. Replication initiation poor regions nested in large gene body leads to under-replication upon fork slowing and causes CFS instability (Brison 2019).

More recently, we have developed a new method to study the replication program based on the deep sequencing of Okazaki fragments (OK-Seq). This allows us to determine the fractions of rightward- (R) and leftward- (L) moving forks at each locus, and to construct the complete profiles of replication fork directionality (RFD = R-L) along the genome. A transition from rightward- to leftward-moving forks occurs when crossing a replication origin position, leading to an upward transition in the RFD profile (Fig. 1). The quality and novelty of the data, leads to new insights into the replication landscape of the human genome and to further unravel the links between replication, gene expression, epigenetic modification and 3D genome organization in normal and cancer cells. We further used these unique data to study the replication-transcription conflict and its link to genome instability.

Screenshot 2019-09-20 at 15.55.10
Figure 3: Study human replication program at single molecule/cell resolution. (a) Map early initiation events in human cells by a high-throughput single-molecule approach, called Optical Replication Mapping (ORM) that combines the Bionano Genomics approach to mapping long individual DNA molecules (green labels) with in vivo fluorescent nucleotide pulse-labeling (red) (Wang 2020). (b) Heatmap shows the single cell replication timing (scRT) profiles based on single cell copy number variation (scCNV) detection. The pseudo bulk RT computed from the scRT and the population RT have a very high correlation (Pearson correlation R=0.9), and the cell-to-cell heterogeneity of replication within a cell population can be further investigated.

Moreover, we have developed new genome-wide approaches to study replication program at single molecule/cell resolution (Fig. 3), in order to further study the intrinsic (between alleles) and extrinsic (cell to cell) variation in replication and to further investigate the relation between cell-to-cell heterogeneity of replication and the cell-to-cell heterogeneity in gene transcription and chromatin organization.

Our study on DNA replication and genome instability will provide the important bases for further understanding its role during development and aging, and how its deregulation contributes to tumorigenesis and to human diseases.


GitHub page of the team:

Key publications

Year of publication 2020

Aurore Sanchez, Céline Adam, Felix Rauh, Yann Duroc, Lepakshi Ranjha, Bérangère Lombard, Xiaojing Mu, Mélody Wintrebert, Damarys Loew, Alba Guarné, Stefano Gnan, Chun-Long Chen, Scott Keeney, Petr Cejka, Raphaël Guérois, Franz Klein, Jean-Baptiste Charbonnier, Valérie Borde (2020 Nov 17)

Exo1 recruits Cdc5 polo kinase to MutLγ to ensure efficient meiotic crossover formation.

Proceedings of the National Academy of Sciences of the United States of America : DOI : 202013012
Gnan Stefano, Liu Yaqun, Spagnuolo Manuela, Chen Chun-Long (2020 Aug 31)

The impact of transcription-mediated replication stress on genome instability and human disease

Genome Instability & Disease : 1 : 207-234 : DOI : 10.1007/s42764-020-00021-y
Alexy Promonet, Ismaël Padioleau, Yaqun Liu, Lionel Sanz, Anna Biernacka, Anne-Lyne Schmitz, Magdalena Skrzypczak, Amélie Sarrazin, Clément Mettling, Maga Rowicka, Krzysztof Ginalski, Frédéric Chedin, Chun-Long Chen, Yea-Lih Lin, Philippe Pasero (2020 Aug 10)

Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites.

Nature communications : 3940 : DOI : 10.1038/s41467-020-17858-2
Wang W., Klein K., Proesmans K., Yang H., Marchal C., Zhu X., Borrman T., Hastie A., Weng Z., Bechhoefer J., Chen C.L., Gilbert D.M., Rhind N. (2020 Aug 1)

Genome-Wide Mapping of Human DNA Replication by Optical Replication Mapping Supports a Stochastic Model of Eukaryotic Replication Timing

bioRxiv : 2020.08.24.263459 : DOI : 10.1101/2020.08.24.263459

Year of publication 2019

Olivier Brison, Sami El-Hilali, Dana Azar, Stéphane Koundrioukoff, Mélanie Schmidt, Viola Nähse, Yan Jaszczyszyn, Anne-Marie Lachages, Bernard Dutrillaux, Claude Thermes, Michelle Debatisse, Chun-Long Chen (2019 Dec 15)

Transcription-mediated organization of the replication initiation program across large genes sets common fragile sites genome-wide.

Nature communications : 5693 : DOI : 10.1038/s41467-019-13674-5

Year of publication 2017

Klein K., Wang W., Borrman T., Chan S., Zhang D., Weng Z., Hastie A., Chen C., Gilbert D.M., Rhind N. (2017 Jan 1)

Genome-Wide Identification of Early-Firing Human Replication Origins by Optical Replication Mapping

bioRxiv : 214841 : DOI : 10.1101/214841
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