DNA REPLICATION IS REQUIRED FOR THE TRANSCRIPTIONAL SWITCH DURING MOUSE EMBRYONIC STEM CELL DIFFERENTIATION

WAISMAN, ARIEL; COSENTINO, SOLEDAD; BARAÑAO, LINO; GUBERMAN, ALEJANDRA; SOLARI, CLAUDIA MARÍA; FRANCIA, MARCOS; VÁZQUEZ ECHEGARAY, CAMILA; BRIVANLOU, ALI; PETRONE, MARÍA VICTORIA; MIRIUKA, SANTIAGO

Congreso; LXI REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA; 2016

A central question in developmental biology is how cells adopt different fates during differentiation. Mouse embryonic stem cells (mESCs) provide a good in vitro model to study this, since their differentiation recapitulates early embryonic development. Here, we aimed to gain insight on how transcriptional programs are switched during differentiation, with the hypothesis that the epigenetic transformation underlying gene expression changes is coupled to processes that normally reorganize the struc- ture of chromatin, such as DNA replication. We have previously shown that inhibition of DNA replication when synchronized cultures of mESCs are set to differentiate to epiblast-like cells (EpiLCs) severely abrogates the transcriptional switch (TS) associated with this cell transition. However, inhibition of DNA synthesis is known to activate the DNA damage response (DDR), raising the possibility that failure to differentiate was connected to this process and not to replication itself. In this work, we evaluated the role of DDR in the TS repression upon DNA replica-tion inhibition. We show that inhibition of DNA synthesis with mechanistically unrelated drugs activates DDR, as judged by Chk1 phosphorylation, p53 stabilization and upregulation of the p53 transcriptional target Mdm2. To comprehensively dissect the role of DDR, we used the CRISPR/Cas9 system to generate a mESC knockout line for p53 (p53 KO). After validation of several clonal lines by DNA sequencing and Western blotting, we studied the effect of replication inhibition in synchronized cultures of p53 KO cells differentiating to EpiLCs. Although we observed a partial rescue in the TS to EpiLCs, KO cells never reached the wild type control levels. We further inhibited DDR upstream of p53, targeting ATR and Chk1 proteins, and observed that TS was still inhibited even in the absence of an active DDR. Our results indicate that DNA replication is a critical process in the TS that takes place during cell differentiation.