We identified a role of the Greatwall(Gwl)/ENSA/PP2A-B55 pathway in S phase control via the regulation of the stability of Treslin, a master replication factor controlling origin firing. Our preliminary data also suggest an impact of this pathway in the stability of additional proteins controlling replication fork progression. Moreover, because its capacity to modulate Treslin stability, this pathway could participate to late origin firing inhibition upon intra S phase checkpoint activation.
The main objectives of this project are:<br />1- To determine whether, besides Treslin, the Gwl/ENSA/PP2A-B55 pathway is additionally regulating additional proteins controlling replication fork progression<br />2- To characterize the molecular pathway by which the Gwl/ENSA/PP2A-B55 promotes Treslin ubiquitination and degradation <br />3-Check whether the intra S-phase checkpoint could prevent late replication origin firing via the inactivation of Gwl/ENSA and the subsequent PP2A-B55-dependent dephosphorylation and degradation of Treslin.
In the first 18 months of this project, time-lapse microscopy, ENSA knockdown experiments, Cullin-dominant-negative transfection experiments and biochemical identification of the cullin E3 complex involved in Treslin degradation have been performed. The activation of the intra-S phase checkpoint via different genotoxic agents such us UV , Thymidine, Bleomycin, Hydroxyurea treatments has been induced. Finally, the effect of this checkpoint activation in the loading of Treslin on the chromatin has been investigated.
Data obtained during this period allowed the identification of the ubiquitin-ligases involved in Treslin ubiquitination and degradation. The effect of the phosphorylation of this protein in two key sites by CDK has been also determined. Finally, the temporal pattern of phosphorylation and ubiquitination and degradation of this master replication factor during the cell cycle has been established and the impact of its miss-regulation investigated.
This project identified Gwl/ENSA/PP2A-B55 as a new unsuspected cascade controlling DNA replication whose temporal regulation during G1/S is essential for a normal cell cycle progression.
Bianco JN, Bergoglio V, Lin Y-L, Pillaire M-J, Schmitz A-L, Gilhodes J, Lusque A, Mazières J, Lacroix-Triki M, Roumeliotis TI, Choudhary J, Moreaux J, Hoffmann JS, Tourrière H* and Pasero P* (2019) Overexpression of Claspin and Timeless protects cancer cells from replication stress in a checkpoint-independent manner. Nat Commun, 10, 910.
Cell division is a fundamental biological process essential for the development of multicellular organisms and for the maintenance of tissue homeostasis. During the cell cycle, the genome must be entirely replicated once and only once during S-phase. In the subsequent M phase, replicated genome must be precisely partitioned between the daughter cells to maintain their genetic content. From a molecular point of view, cell cycle transitions are controlled by sequential reversible protein phosphorylation and irreversible degradation processes induced by the cyclin/cdk kinases and the ubiquitin-proteolytic system, respectively, which ensures that cell cycle phases occur in a specific sequential order.
S-phase cyclin/cdks trigger DNA replication initiation and define the timing of the DNA replication program, whereas the M-phase cyclinB/cdk1 orchestrates mitotic progression by phosphorylating a myriad of substrates. Recent work from Partner's 1 laboratory identified a new pathway preventing Cdk1 substrate dephosphorylation in mitosis. Notably, they demonstrated that, at mitotic entry, the Greatwall (Gwl) kinase phosphorylates two proteins, Arpp19 and ENSA, turning them into potent inhibitors of the phosphatase PP2A, ensuring in this way the stable phosphorylation of cyclin B/cdk1 substrates and successful mitotic entry. In addition, this partner has recently shown that Gwl controls G1 progression by modulating Akt activity. Notably, Gwl regulates G1 progression by promoting the CRL1ßtrcp-dependent degradation of the PHLPP, the phosphatase responsible of Akt dephosphorylation. Importantly, this laboratory recently discovered that Gwl plays an additional crucial role in the control of S phase by regulating the levels of two proteins involved in replication origin firing and fork protection.
The main objective of this project is to investigate how the Gwl/ENSA pathway regulates fork activation and progression under normal and pathological conditions and thereby ensures a correct S phase.
Since Gwl/ENSA pathway controls G1, S and M phases, the results of this project will constitute a breakthrough not only in the understanding of the mechanisms controlling DNA replication under normal and DNA damage conditions but also in the understanding of cell cycle progression.
This new concept will not only impact scientific community but would also yield important social benefits since recent compelling data identifies Gwl as new target for cancer therapy.
Madame Anna CASTRO (Centre de Recherche en Biologie cellulaire de Montpellier)
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.
CRBM Centre de Recherche en Biologie cellulaire de Montpellier
IJM Instut Jacques Monod
IGH Institut de Génétique Humaine
Help of the ANR 628,131 euros
Beginning and duration of the scientific project: January 2019 - 48 Months