Chaque division cellulaire requiert une duplication précise du génome. Des dizaines de milliers de sites d’initiation de la réplication d’ADN (origines de réplication) sont impliqués dans la réplication complète du génome humain. L’activation des origines de réplication est régulée précisément et des études génomiques extensives ont démontré la présence de caractéristiques génomiques associées à l’activation des origines de réplication. Le complexe de pré-réplication (pre-RC) est la base de l’initiation de la réplication et consiste en deux sous-complexes majeurs : l’ « origin recognition complex » (ORC) qui interagit directement avec l'ADN et est nécessaire pour recruter le second sous-complexe, les hélicases Mcm2 7, qui sont responsables de l'initiation de la réplication. La régulation de l’assemblage du pre-RC est bien étudiée, mais les caractéristiques de la chromatine qui déterminent le positionnement du pre-RC sur le génome restent peu connues. Les études génomiques par immuno-précipitation de la chromatine et séquençage à haut débit (ChIP-seq) des pre-RCs sont rares et jusqu’à aujourd’hui seulement disponibles pour ORC. Du fait que Mcm2-7 migre de son site de chargement initial, il est crucial d'obtenir des informations sur le positionnement des Mcm2-7 pour la compréhension complète de la régulation de la réplication. Ce travail présente la première analyse génomique par méthode ChIP-seq des deux sous-unités majeures du pre-RC, ORC et Mcm2-7, dans la lignée cellulaire de lymphome de Burkitt Raji infectée par le virus d’Epstein-Barr (EBV). La présence du génome d’EBV permet d'avoir un contrôle interne de la qualité de nos expériences, en comparant les positions de pre-RC déterminées avec des positions du pre-RC précédemment publiées. Sur le génome humain, les résultats de séquençage du pre-RC corrèlent bien avec des zones de réplication active. De façon intéressante, les zones de terminaison de la réplication étaient spécifiquement bas en pre-RC, spécialement en Mcm2-7. La localisation des sites d'initiation de la réplication identifiés est généralement bien corrélée avec les sites de transcription active. En effet, des sites d’assemblage du pre-RC de haute affinité sont localisés préférentiellement en voisinage de sites de transcription active, ce qui est possiblement dû à l’accessibilité de la chromatine dans ces régions. La fixation de Mcm2-7 fluctue de façon dépendante du cycle cellulaire, ce qui suggère des translocations de Mcm2 7 en G1, probablement dépendantes de la machinerie active de la transcription. Ces résultats indiquent que les positions de ORC et Mcm2-7 sont principalement dépendantes de l’accessibilité de la chromatine avec un accès privilégié dans la chromatine active et Mcm2 7 étant le déterminant majeur de l’initiation de la réplication. Au sein de l'hétérochromatine, ORC est enrichi dans des zones associées avec l'histone modifié H4K20me3. Cependant, cet enrichissement est moins important pour les Mcm2-7. En utilisant un système de réplication basé sur des plasmides, nous avons démontré que l’association d'ORC et H4K20me3 favorise l’assemblage du pre-RC et l’initiation de la réplication. Cette observation suggère que l’interaction ORC-chromatine est le déterminant majeur de la régulation de la réplication d’ADN au sein de l’hétérochromatine. En conclusion, cette étude propose deux mécanismes différents de la régulation de l'assemblage du pre-RC dépendants de l’environnement de la chromatine. / With every cell division, the genome needs to be faithfully duplicated. Tens of thousands of DNA replication initiation sites (origins of replication) are involved in replicating the human genome. Origin activation is precisely regulated and extensive genome-wide studies found association of origin activation to several different genomic features. The pre-replication complex (pre RC) is the basis for replication initiation and consists of two major subcomponents: the origin recognition complex (ORC) binds DNA and is required for loading of the second component, Mcm2-7 helicases, which initiate DNA replication. Regulation of pre-RC assembly is well studied, however, chromatin features driving pre RC positioning on the human genome remain largely unknown. Genome-wide pre-RC chromatin immunoprecipitation experiments followed by sequencing (ChIP-seq) studies are rare and so far only performed for ORC. As Mcm2-7 can translocate from their initial loading site, information about Mcm2-7 positioning are required for full understanding of DNA replication regulation.This work presents the first genome-wide ChIP-seq analysis of the two major pre-RC subcomponents ORC and Mcm2-7 in the Epstein-Barr virus (EBV) infected Burkitt’s lymphoma cell line Raji. Successful ChIPs were validated on the EBV genome by comparing obtained pre RC positions with already existing pre-RC ChIP-on chip data. On the human genome, pre-RC sequencing results nicely correlated with zones of active replication. Interestingly, zones of replication termination were specifically depleted from pre-RC components, especially from Mcm2 7. Active DNA replication is known to correlate with active transcription. Indeed, strong pre-RC assembly preferentially occurred at sites of active transcriptional regulation, presumably determined by chromatin accessibility. Strong Mcm2-7 binding thereby fluctuated cell cycle-dependently, arguing for Mcm2-7 translocations during G1, possibly depending on the active transcriptional machinery. These results indicate ORC and Mcm2-7 positions being mainly dependent on chromatin accessibility in active chromatin, with Mcm2-7 being the major determinant of replication initiation. In heterochromatin, ORC was enriched at H4K20me3 sites, while Mcm2-7 enrichment was less prominent. Employing a plasmid-based replication system, ORC association to H4K20me3 was proven to promote successful pre-RC assembly and replication initiation, situating direct ORC-chromatin interactions being the major determinant for DNA replication regulation in heterochromatin. Taken together, this study proposes two different modes of pre-RC assembly regulation depending on chromatin environment.

The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G1 phase assembly of pre-replicative complexes. Only the Orc1-5 subunits are required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. In comparison to other eukaryotic Orc6 proteins, budding yeast Orc6 appears to be quite divergent. Two-hybrid analysis revealed that Orc6 only weakly interacts with other ORC subunits. In this assay Orc6 showed a strong ability to self-associate, although the significance of this dimerization or multimerization remains unclear. Imaging of Orc6-eYFP revealed a punctate sub-nuclear localization pattern throughout the cell cycle, representing the first visualization of replication foci in live budding yeast cells. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations from metazoans. An essential role for Orc6 in DNA replication was identified by depleting the protein before and during G1 phase. Surprisingly, Orc6 was required for entry into S phase after pre-replicative complex formation, in contrast to what has been observed for other ORC subunits. When Orc6 was depleted in late G1, Mcm2 and Mcm10 were displaced from chromatin, the efficiency of replication origin firing was severely compromised, and cells failed to progress through S phase. Depletion of Orc6 late in the cell cycle indicated that it was not required for mitosis or cytokinesis. However, Orc6 was shown to be associated with proteins involved in regulating these processes, suggesting that it may act as a signal to mark the completion of DNA replication and allow mitosis to commence.

The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G1 phase assembly of pre-replicative complexes. Only the Orc1-5 subunits are required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. In comparison to other eukaryotic Orc6 proteins, budding yeast Orc6 appears to be quite divergent. Two-hybrid analysis revealed that Orc6 only weakly interacts with other ORC subunits. In this assay Orc6 showed a strong ability to self-associate, although the significance of this dimerization or multimerization remains unclear. Imaging of Orc6-eYFP revealed a punctate sub-nuclear localization pattern throughout the cell cycle, representing the first visualization of replication foci in live budding yeast cells. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations from metazoans. An essential role for Orc6 in DNA replication was identified by depleting the protein before and during G1 phase. Surprisingly, Orc6 was required for entry into S phase after pre-replicative complex formation, in contrast to what has been observed for other ORC subunits. When Orc6 was depleted in late G1, Mcm2 and Mcm10 were displaced from chromatin, the efficiency of replication origin firing was severely compromised, and cells failed to progress through S phase. Depletion of Orc6 late in the cell cycle indicated that it was not required for mitosis or cytokinesis. However, Orc6 was shown to be associated with proteins involved in regulating these processes, suggesting that it may act as a signal to mark the completion of DNA replication and allow mitosis to commence.