DS0402 - Décryptage des fonctions biologiques élémentaires et de leur intégration

New molecular insights into the regulation of the spatiotemporal program of DNA replication – OriMolMech

Submission summary

The faithful duplication of vertebrate genomes at each S phase is under the control of a spatiotemporal program that regulates both the positioning and the timing of firing of about 100 000 replication starting points also called replication origins. Recent genome wide mappings of this spatiotemporal program have brought important information on potential mechanisms involved in this regulation. We now propose to go deeper into the understanding of molecular processes involved by focusing on two major axes.

The strongest overlap between replication origins and a specific feature found genome wide was the association of origins with G-quadruplex (G4) motifs. By using a genetic approach on two model origins, we recently showed that G-quadruplexes (G4s) are important regulators of replication origins in vertebrates. However, we also demonstrated on one model origin that the G4 has to cooperate with a flanking cis-element suggesting a regulation involving at least two modules. The first axis aims at understanding when and how G4 are involved in origin function and to elucidate the nature and function of cooperative cis-elements. Identification of the cooperative element will be done on two chicken model origins and then will be extended to human model origins. Statistical methods will be developed to identify and/or validated enriched motifs next to G4s. New statistical methods to compare profiles of enrichment in origin signal in different genetic contexts will be developed in order to pinpoint potential important trans-factors involved in replication origin dynamics.

The second axis will address the question of how early replicating domains can be formed and then will explore the impact of early DNA replication timing on chromatin formation, nuclear organization and gene expression. The DNA replication timing program is emerging as a system that integrates genome regulation, genome 3-D organization with genome stability. Early replication is organized and associated with active gene expression, while late replication is less organized and associated with increased mutation rates. Altogether, these observations have led to a model (the Replication Domain Model) in which each replication timing domain is a unit of regulation influencing when the replicons within its boundaries could fire. An alternative model is that early timing regions result fortuitously from the more or less synchronous firing of a cluster of replicons each having its own individual timing elements nearby. We have already identified a combination of cis-elements that can drive the formation of an independent replicon and advance locally the replication timing of late replicating regions. We will test the later hypothesis (the Replicon based model) by inserting our model autonomous replicon at two sites spaced by 30 Kb in a region naturally replicated in late S-phase. Firstly, we will test how the dynamic of replication is perturbed in the modified region. DNA replication has been considered as a window of opportunity to change the chromatin landscape since chromatin undergoes destabilization and re-assembly behind the replication forks on the two daughter strands. One hypothesis is that early replication would favor the assembly of more open chromatin structure. An attractive feature of this model is its self-reinforcing nature but also it provides a mechanism by which replication timing and chromatin context could spread. The chromatin structure of the 30 Kb modified region will be tested and compared with the wild type unmodified allele. These genetic studies will be complemented by the development of new statistical methodologies to integrate our genome-wide maps of origins with recent chromosome conformation capture data. The challenge here will be to provide epigenetic signatures for replication origins that integrate the 3D structure of the nucleus.

Project coordinator

Madame Marie-Noelle PRIOLEAU (Institut Jacques Monod)

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.

Partner

IJM Institut Jacques Monod
LBBE - UCBL Laboratoire de Biométrie et Biologie Evolutive

Help of the ANR 499,928 euros
Beginning and duration of the scientific project: September 2015 - 48 Months

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