DNA replication is required for transcriptional switch during mouse embryonic stem cell differentiation

VAZQUEZ ECHEGARAY C; PETRONE MV; MIRIUKA S; WAISMAN A; COSENTINO S; BARAÑAO L; GUBERMAN A; SOLARI C; FRANCIA M; BRIVANLOU A

Congreso; saic 2016; 2016

A central question in developmental biology ishow cells adopt different fates during differentiation. Mouse embryonic stemcells (mESCs) provide a good in vitro model to study this, since theirdifferentiation recapitulates early embryonic development. Here, we aimed togain insight on how transcriptional programs are switched duringdifferentiation, with the hypothesis that the epigenetic transformationunderlying gene expression changes is coupled to processes that normallyreorganize the structure of chromatin, such as DNA replication. We havepreviously shown that inhibition of DNA replication when synchronized culturesof mESCs are set to differentiate to epiblast-like cells (EpiLCs) severelyabrogates the transcriptional switch (TS) associated with this cellulartransition. However, inhibition of DNA synthesis is known to activate the DNAdamage response (DDR), raising the possibility that failure to differentiatewas connected to this process and not to replication itself. In this work, weevaluated the role of DDR in the TS repression upon DNA replication inhibition.We show that inhibition of DNA synthesis with different drugs activates DDR, asjudged by phosphorylation of Chk1, stabilization of p53 and upregulation of thep53 transcriptional target Mdm2. To comprehensively dissect the role of DDR, weused the CRISPR/Cas9 system to generate a mESC knockout line for p53 (p53 KO).After validation of several clonal lines with DNA sequencing and westernblotting, we studied the effect of replication inhibition in synchronizedcultures of p53 KO cells differentiating to EpiLCs. Although we observed apartial rescue in the TS to EpiLCs, KO cells never reached the wild typecontrol levels. We further inhibited DDR upstream of p53, targeting ATR andChk1 proteins, and observed that TS was still inhibited even in the absence ofan active DDR. Our results indicate that DNA replication is a critical processin the TS that takes place during cell differentiation