Identification de déterminants impliqués dans la différenciation des cellules souches embryonnaires
Stem cells have captured public’s attention in the last years, thanks to their involvement in cancer therapies and also their huge theoretical potential in the regenerative medicine field. In order to translate this new technology to the clinic, a better understanding of their regulatory mechanisms is still needed. It is well established that mouse embryonic stem cell (ESC) fate is highly regulated by core pluripotency factors. However, it is conceivable that novel self-renewal or differentiation regulators are not yet described. To investigate this possibility, we used a viral-based approach to generate a collection of ESC with nested chromosomal deletions called DelES (Deletion in ES cells). This library contains more than a thousand independent ESC clones highly enriched in chromosomal deletions which together cover ~25% of the mouse genome. Using this resource, we conducted an embryoid body (EB) differentiation screen and showed that several clones were having an abnormal EB formation phenotype. Complementation studies later identified Rps14-a ribosomal protein (RP) coding gene- as a novel haploinsufficient gene in EB formation from undifferentiated ESC. Further analyses of our screen results showed a strong bias for a subset of small subunit ribosomal protein genes which are critical for ESC differentiation but not for their self-renewal activity. Interestingly, the most severe differentiation phenotypes were found with ribosomal proteins associated to the ribosome’s mRNA exit site, namely Rps5, Rps14 and Rps28. While RP gene imbalance often leads to a p53 response that can be corrected by p53 suppression, ESC clones with decreased expression of mRNA exit site RP genes were surprisingly insensitive to p53 reduction, but were rescued by BAC or cDNA complementation, thus confirming the causative nature of these genes in the ESC phenotype. Finally, polysomal profiling and RNA-Seq studies showed that Rps5 deleted ESC exhibit an abnormal mesodermal gene signature. Together, our work presents a highly valuable resource for functional genomic studies in ESC and also highlights a novel p53-independent role linked to RP gene imbalance. Our results shed light on the relevance of these subunits for the developmental transition of ESC from a pluripotent to a differentiated state.
Embryonic stem cells Ribosomal proteins