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

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

Congreso; LXI Congreso de la Sociedad Argentina de Investigaciones Clínicas; 2016

A central question in developmental biology is howcells adopt different fates during differentiation. Mouseembryonic stem cells (mESCs) provide a good in vitromodel to study this, since their differentiation recapitulatesearly embryonic development. Here, we aimed to gaininsight on how transcriptional programs are switched dur-ing differentiation, with the hypothesis that the epigenetictransformation underlying gene expression changes iscoupled to processes that normally reorganize the struc-ture of chromatin, such as DNA replication. We havepreviously shown that inhibition of DNA replication whensynchronized cultures of mESCs are set to differentiateto epiblast-like cells (EpiLCs) severely abrogates the tran-scriptional switch (TS) associated with this cell transition.However, inhibition of DNA synthesis is known to activatethe DNA damage response (DDR), raising the possibilitythat failure to differentiate was connected to this processand not to replication itself. In this work, we evaluatedthe role of DDR in the TS repression upon DNA replica-tion inhibition. We show that inhibition of DNA synthesiswith mechanistically unrelated drugs activates DDR, asjudged by Chk1 phosphorylation, p53 stabilization andupregulation of the p53 transcriptional target Mdm2. Tocomprehensively dissect the role of DDR, we used theCRISPR/Cas9 system to generate a mESC knockout linefor p53 (p53 KO). After validation of several clonal linesby DNA sequencing and Western blotting, we studiedthe effect of replication inhibition in synchronized culturesof p53 KO cells differentiating to EpiLCs. Although weobserved a partial rescue in the TS to EpiLCs, KO cellsnever reached the wild type control levels. We furtherinhibited DDR upstream of p53, targeting ATR and Chk1proteins, and observed that TS was still inhibited even inthe absence of an active DDR. Our results indicate thatDNA replication is a critical process in the TS that takesplace during cell differentiation.