Figure 3.

Zrsr1^mu and Zrsr2^mu block activation of the 2-cell transcription program induced by Dux expression and impair conversion of induced pluripotent stem cells (iPSC) to a 2 cell-like cells (2CLC) state. (a) Diagram of Zrsr1 and Zrsr2 mutants with their functional domains: ZF: Zinc Finger, RRM: RNA recognition motif, RS: arginine-serine rich. Diagonal red line indicates the position of the mutation. (b) Diagram of the plasmid-expressing Emerald under Zscan4 promoter [37] and of a doxycycline-inducible lentiviral construct [27]; both plasmids were stably integrated into iPSC. (c,d) Diagram of iPSC metastability (top) and enrichment in 2CLCs relative to conventional iPSCs (bottom) produced after Dux expression in control iPSCs but blocked when Zrsr1 or Zrsr2 are mutated (c), as confirmed by flow cytometry (d) using two cell cultures per condition. (e) Relative mRNA expression in WT Dux-induced iPSCs (2CLCs) of genes related to 2-cell embryo genome activation.