Fig. 4: In vitro germline competence is correlated with the partial decommissioning of PGCLC enhancers.

a E14 WT and Otx2^−/− ESC were differentiated into d2 EpiLC, d4 EpiSC or d8 EpiSC, which were then differentiated into PGCLC. PGCLC were quantified as the percentage of CD15⁺CD61⁺ cells within d4 EB. Bar plots: mean percentage of PGCLC ± SD from at least two biological replicates. b Average levels of chromatin marks measured in WT and Otx2^−/− d2 EpiLC within 1 kb of the indicated enhancers. c H3K4me1 and H3K4me2 levels for Group I and II PGCLC enhancers in d2 EpiLC, d4 EpiSC, and d8 EpiSC differentiated from E14 WT or Otx2^−/− ESC. P-values were calculated using paired two-sided Wilcoxon tests. Scales: RPGC. d CpG methylation for Group I and II PGCLC enhancers in d2 EpiLC differentiated from WT or Otx2^−/− ESC. P-values were calculated using paired two-sided Wilcoxon tests. Scale: percentage of methylated CpGs. e WT ESC and ESC lines enabling overexpression of NANOG-HA (blue) or PRMD14-HA (red) were differentiated into d2 EpiLC and EpiSC. D2 EpiLC and EpiSC were differentiated into PGCLC with (+Dox) or without (ctrl) Doxycycline and in the absence of growth factors. As a positive control, WT d2 EpiLC and EpiSC were differentiated with growth factors (WT + GF). Bar plots: mean percentage of PGCLC (CD15⁺CD61⁺ cells) in d4 EB ± SD from two biological replicates. f PRDM14-HA and NANOG-HA ChIP-seq signals in EpiLC and EpiSC in which PRDM14-HA or NANOG-HA were overexpressed. P-values were calculated using paired two-sided Wilcoxon tests. Scales: RPGC. g ESC enabling inducible overexpression of NANOG-HA were differentiated into d2 EpiLC and EpiSC. Cells were left untreated or treated with Doxycycline and H3K27ac ChIP-seq experiments were performed. P-values were calculated using paired two-sided Wilcoxon tests. Scales: RPGC. h NANOG-HA (two replicates in EpiSC), H3K27ac, and H3K4me2 signals in untreated (-Dox) and Dox-treated (i.e. NANOG-HA overexpression) EpiLC and EpiSC are shown for the Klf5 and Esrrb loci. Deleted PGCLC enhancers are highlighted in blue and other PGCLC enhancers in gray. In (b–c), the H3K4me1 data for WT d2 EpiLC and d8 EpiSC corresponds to the second replicates from Fig. 3c.