Réplication et Dynamique du Génome

  • The chromatin organization and the plasticity of this organization are essential to development programs and the maintenance of differentiation. At each division, chromosomes should be duplicated and also maintain the memory of the specific transcription programs that have been previously established in the embryo. Our objective is to understand how DNA replication can be integrated to transcriptional controls during development. We also characterize the DNA replication initiation complexes and analyze how epigenetic mechanisms control the organization of chromatin domains for replication.

    At each cell division, chromosomes must be duplicated and the memory of the previously established specific transcription programs maintained. DNA replication is a precisely regulated process that starts at dozen of thousands of sites that are dispersed along the genome and are called DNA replication origins (Méchali, 2010, Fragkos et al, 2015). Errors in this process can cause loss or gain of genetic material that will lead to genome instability, a hallmark of cancer cells.

    Our objectives are to characterize the genetic and epigenetic nature of DNA replication origins, to understand how they are integrated in chromatin domains and transcriptional programs, and to identify new factors involved in initiation of DNA replication.


    Membres

    Philippe Coulombe
    Coulombe Philippe
    James Hutchins
    Hutchins James
    Marcel Mechali
    Mechali Marcel
    Isabelle Peiffer
    Peiffer Isabelle
    Hanna Emlein
    Emlein Hanna
    Antoine Aze
    Aze Antoine
    Romain Charton
    Charton Romain
    Faezeh Forouzanfar
    Forouzanfar Faezeh
    Olivier Ganier
    Ganier Olivier
    Paulina Prorok
    Prorok Paulina
    Ahsan Rizvi
    Rizvi Ahsan
    Joëlle Nassar
    Nassar Joëlle

    Publications

    Author Correction: RNAs coordinate nuclear envelope assembly and DNA replication through ELYS recruitment to chromatin.

    Aze A, Fragkos M, Bocquet S, Cau J, Méchali M

    2018 - Nat Commun, 9(1):581

    Demander l'article complet29402910

    The gastrula transition reorganizes replication origin selection in Caenorhabditis elegans

    Rodríguez-Martínez, M., Pinzón, N., Ghommidh, C., Beyne, E., Seitz, H., Cayrou, C., Méchali, M.

    2017 - Nature Structural & Molecular Biology, 24(3):290-299

    Demander l'article complet28112731

    Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication

    Brustel J, Kirstein N, Izard F, Grimaud C, Prorok P, Cayrou C, Schotta G, Abdelsamie AF, Déjardin J, Méchali M, Baldacci G, Sardet C, Cadoret JC, Schepers A, Julien E.

    2017 - EMBO J., 36(18):2726-2741

    Demander l'article complet28778956

    Developmental determinants in non-communicable chronic diseases and ageing

    Bousquet J, Anto JM, Berkouk K, Gergen P, Pinto Antunes J, Augé P, Camuzat T, Bringer J, Mercier J, Best N, Bourret R, Akdis M, Arshad SH, Bedbrook A, Berr C, Bush A, Cavalli G, Charles MA, Clavel-Chapelon F, Gillman M, Gold DR, Goldberg M, Holloway JW, Iozzo P, Jacquemin S, Jeandel C, Kauffmann F, Keil T, Koppelman GH, Krauss-Etschmann S, Kuh D, Lehmann S, Lodrup Carlsen KC, Maier D, Méchali M, Melén E, Moatti JP, Momas I, Nérin P, Postma DS, Ritchie K, Robine JM, Samolinski B, Siroux V, Slagboom PE, Smit HA, Sunyer J, Valenta R, Van de Perre P, Verdier JM, Vrijheid M, Wickman M, Yiallouros P, Zins M.

    2015 - Thorax, 70(6):595-7

    Demander l'article complet25616486
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    Publications de l'équipe

  • Genetic and epigenetic nature of DNA replication origins

    Until recently, only few DNA replication origins had been identified in metazoan cells. In contrast to Saccharomyces cerevisiae, origins in multicellular organisms do not share a common genetic sequence. The availability of new high-throughput methods allowed us to carry out genome-wide analysis of metazoan DNA replication origins. We have now mapped DNA replication origins in mouse embryonic stem cells (ES) cells (Cayrou, Coulombe et al, 2011; Cayrou et al, 2012 ; Cayrou et al, 2015), and also in Drosophila cells (Cayrou, Coulombe et al, 2011). We also mapped replication origins in vivo in C. elegans embryos (Rodriguez et al, 2017).
    We found that DNA replication origins are located at precise sites along the genome, but do not share a strict consensus sequence, in contrast to bacterial or S. cerevisiae DNA replication origins. However, we did identify at least one common, repeated consensus element in Drosophila, mouse and human cells that we called OGRE, for Origin G-rich Repeated Element (Cayrou et al, 2012, Cayrou et al, 2015 and Figure 1). This motif is unexpectedly G-rich, in contrast to the AT richness of bacterial and yeast DNA replication origins. We also showed that OGREs can form G quadruplexes (G4s), and that DNA synthesis initiates at a short distance downstream of these elements (Cayrou, Coulombe et al, 2011; Cayrou et al, 2012; Cayrou et al, 2015).

    figure 1
    Figure 1: OGRE/G4 elements and initiation of DNA replication. OGRE/G4 elements are upstream of the initiation site of DNA synthesis (From Cayrou et al, 2011, 2012, 2015).

    We also identified three distinct classes of DNA replication origins, characterized by specific epigenetic marks (Cayrou et al, 2015). These results highlight the plasticity of DNA replication origins according to their chromosomal contexts. Our DNA combing experiments also showed that replication origins are flexible, because only a minor fraction of all potential DNA replication origins is activated at each cell cycle in a given cell, in an apparent stochastic manner. The excess of potential DNA replication origins appears to be an important genome safeguard mechanism to ensure that all sequences are duplicated during the cell cycle. It might also permit to choose the DNA replication origins to be activated according to the pattern of gene expression in a given cell.

    DNA replication origins and cell identity

    The arrangement of DNA replication origins could be associated with the organization of chromosomal domains in function of the cell fate and identity, a process linked to development. We provided a first piece of evidence for this model several years ago by demonstrating that DNA replication origins are developmentally regulated in X. laevis (Hyrien and Méchali, 1993 ; Hyrien et al, 1995). We then demonstrated the correlation between the activation of transcription and the specific location of some DNA replication origins (Danis et al, 2004). Moreover, we spotted the coupling between DNA replication and gene expression during the differentiation of pluripotent teratocarcinoma cells into neural cells (Fisher et al, 2003; Gregoire et al, 2006). A similar link between DNA replication origin activity was observed when we mapped DNA replication origins in a living animal, C. Elegans (Rodriguez et al, 2017)
    We also demonstrated that the position of DNA replication origins can change in function of the cell identity. Indeed, an extensive reprogramming of DNA replication origin organization occurs when a nucleus from a differentiated cell is exposed to an embryonic context (Lemaitre et al, 2005 ; Ganier et al, 2011). Specifically, when nuclei from differentiated X. laevis cells are incubated with X. laevis egg extracts, their chromosome structure and replication origin organization are remodeled, a process that we demonstrated to be topoisomerase II-dependent (Cuvier et al, 2008). Moreover, this process parallels the transcriptional reprogramming of differentiated nuclei (Ganier et al, 2011).

    DNA replication origin complexes

    The third objective of our laboratory is to characterize in greater depth the replication initiation complex, and to understand how DNA replication origins are organized and activated. DNA replication origin recognition and activation occur through the multi-step assembly of several replication factors. The first complex is the pre-replication complex (pre-RC) that permits the assembly of the DNA helicase MCM2-7. ORC is the first known protein to assemble on DNA replication origins, and then serves as a landing pad to assemble CDC6 and CDT1 that are used to recruit the DNA helicase MCM2-7 (Figure 3).

    Figure 2
    Figure 2: Multi-step assembly of the replication initiation complex (From Fragkos et al, 2015)

    We isolated CDT1 using a specific screening method in X. laevis, and demonstrated that it binds to DNA replication origins in an ORC-dependent manner (Maiorano et al, 2000). Further work showed that geminin binds to and inhibits CDT1 (Tada et al, 2001; Maiorano et al, 2004). We then confirmed that the CDT1-geminin complex acts as an ON/OFF switch at DNA replication origins (Lutzmann et al, 2006). Once the helicase is assembled at DNA replication origins, CDT1 is displaced and is no longer required for the further stages of DNA synthesis (Maiorano et al, 2004). If the ratio of CDT1 to geminin is altered, for example by increasing CDT1 level, the cell undergoes another round of replication (Maiorano et al, 2005). To avoid this abnormal re-initiation of replication, CDT1 is degraded during the S-phase of the cell cycle. However, it is synthesized again during the G2 and M phases to prepare initiation in the next cell cycle. We identified a new CDT1 regulatory domain that could be involved in the mechanism to prevent premature DNA replication origin licensing before the next cell cycle (Coulombe et al, 2013). Mutations in this domain increase re-replication and CDT1 oncogenic properties.
    Our laboratory is currently using different proteomic approaches to identify and characterize new proteins involved in the regulation of initiation of DNA replication.

    Dissociation of replication complexes

    The dissociation of replication complexes is an essential phenomenon that occurs at mitosis entry to allow the clearing of replication complexes from chromosomes. We found that replication protein A (RPA) is not phosphorylated during the whole S phase, but its 34kDa subunit is hyperphosphorylated in mitosis (Francon et al, 2004). RPA34 hyperphosphorylation correlates with its disassembly from chromatin and is critically required for proper chromosome assembly and segregation at mitosis (Cuvier et al, 2006). The ORC complex is also necessary to recruit the kinase CDK1 that phosphorylates RPA34 and allows the disassembly of replication foci before mitosis can occur (Cuvier et al, 2006). We also found that topoisomerase II couples termination of DNA replication with the clearing of the replication complexes at the end of S phase (Cuvier et al, 2008).
    The MCM2-7 DNA helicase is another protein complex that must be removed from chromatin after replication. MCM2-7 is a hexamer that forms a ring around DNA, a process regulated in a stepwise manner in X. laevis egg extracts (Maiorano et al, 2000). At the end of replication, this ring should be opened to allow its removal from chromatin. We have shown that MCM-BP, a protein that binds to MCM2-7, allows the disassembly of the MCM2-7 complex in late S phase. MCM-BP inhibition delays mitosis and causes mitotic defects (Nishiyama et al, 2011). We proposed that MCM-BP plays a key role in the mechanism by which pre-RC is cleared from replicated DNA in vertebrate cells.

    Interconnections between DNA replication and DNA repair

    We have characterized MCM8 and MCM9, two new MCM family members that are involved in DNA replication and are only present in multicellular organisms. MCM8 is an ATP-dependent DNA helicase that binds to chromatin after the pre-RC assembly and acts at the replication fork (Maiorano et al, 2006). In X. laevis, MCM9 helps CDT1 in the loading of the MCM2-7 helicase at DNA replication origins (Lutzmann et Mechali, 2008). MCM9 might prevent CDT1 inhibition by geminin, which is present in excess in X. laevis egg extracts, during the licensing reaction.
    We also generated Mcm8 and Mcm9 knockout mice (Lutzmann et al, 2012), and found that they are viable but sterile. Moreover, Mcm8-/- and Mcm9-/- cells are highly sensitive to replication stress and DNA damage because of defective homologous recombination. Both meiotic and somatic recombination are affected. Consistent with this, we also found that MCM8- and MCM9-defective mice develop ovarian cancer. We also demonstrated that MCM8 and MCM9 form a stable complex in the DNA mismatch repair (MMR) reaction. This complex has a DNA helicase activity that could be involved in the resection of the damaged piece of DNA (Traver et al, 2015).

    Current research

    • We are characterizing DNA  domains are organized for DNA replication.
    • We wish to understand the epigenetic mechanisms leading to the formation of DNA replication initiation complexes.
    • We are also interested in uncovering new factors involved in the regulation of the initiation complex using in vitro systems derived f rom X. laevis eggs and cultured mammalian cells.

    We welcome enthusiastic post-doctoral scientists willing to share our interest in these areas.