Replication stress primes a trophectoderm fate in embryonic stem cells

Abstract Early embryogenesis features rapid cell cycles that impose substantial replication stress (RS). How RS shapes lineage potential remains unclear. Using mouse embryonic stem cells (ESC) as an inner cell mass model, we show that RS rapidly and durably induces trophoectoderm (TE) regulators, including Cdx2, Gata3, Eomes, Elf5, Hand1 and Tfap2c. This program requires ATR checkpoint signaling as ATR inhibition suppresses RS-induced TE gene expression without restoring pluripotency markers, indicating an active, checkpoint-driven fate response rather than passive identity loss. Additionally, RS activates JNK, increasing c-JUN abundance and phosphorylation. Blocking JNK attenuates TE induction, placing AP-1 as a cooperating pathway. The TE bias persists after stress withdrawal, promoting acquisition of a TE-like phenotype and enhancing ESC contribution to extraembryonic compartments in embryo-like structures. Quantitative chromatin proteomics reveals eviction of nucleosomes and condensin along with enrichment of DNA repair factors and ATR cofactors, consistent with a chromatin-competent and checkpoint-responsive state, permissive for lineage rewiring. These findings define an ATR- and JNK/c-JUN-dependent stress response axis that redirects embryonic cell identity toward TE, linking RS to developmental plasticity. If reactivated in the adult, this pathway could lead to somatic fate shifts that may predispose to malignancy.