Extended Data Figure 10: Nanog induces PGC-like fate - a model.
a, ChIP-seq data tracks9,30,39 at the Prdm1 and Prdm14 loci for NANOG in ESCs, D1 and D2 EpiLCs (EpiLCs were collected after 3h with 200 ng/ml Dox for Nanog expression). Boxed are putative enhancer elements. The Prdm1 enhancer is enriched for H3K4me1 in D2 EpiLCs and gains H3K27ac in PGCLCs. The Prdm14 enhancer shows enrichment for H3K4me1 in ESCs and EpiLCs and becomes enriched for H3K27ac in ESCs and PGCLCs but not in EpiLCs. Note that these enhancer marks follow the expression pattern of Prdm1 or Prdm14, respectively. RPM = Reads per Million. Related to Fig. 4d.
b, ChIP-qPCR validation of NANOG ChIP-seq data with GOF-GFP D2 EpiLCs before and 3h after PGCLC induction by Nanog expression (+Dox). NANOG is enriched at putative enhancer regions, which are close to Prdm1 and Prdm14. Error bars indicate s.d. (n=2 technical replicates each from 2 biological replicates).
c, ESC lines with luciferase reporter plasmids with a genomic region, which does not show any enhancer marks and NANOG binding, and indicated Dox-inducible transgenes served as a negative control. Luciferase activity, measured in ESCs, D2 EpiLCs and 24h after PGCLC induction (EpiLC aggregations), was normalised to protein quantity (luc/pro). Mean values +/− s.d. (n=3 technical replicates each from 2 biological replicates).
d, Biological replicate experiment for the luciferase assay with the Prdm1 enhancer as shown and described in Fig. 4e. Luciferase activity, measured in ESCs, D2 EpiLCs and 24h after PGCLC induction, was normalised to protein quantity (luc/pro). Mean values +/− s.d. (n=3 technical replicates); colour code is shown in (c); reference for p-values (two-sided/unpaired t-test): EpiLC aggregations −Dox; **p<0.01; *p<0.05.
e, Biological replicate experiment for the luciferase assay with the Prdm14 enhancer as shown and described in Fig. 4g. Luciferase activity, measured in ESCs, D2 EpiLCs and 12/24h after PGCLC induction, was normalised to protein quantity (luc/pro). Mean values +/− s.d. (n=3 technical replicates); colour code is shown in (c); reference for p-values (two-sided/unpaired t-test): EpiLC aggregations −Dox 24h; **p<0.01; *p<0.05.
f, Model showing the role of NANOG during PGCLC induction in vitro. D1 EpiLCs are not competent to become PGCLCs, but retain the capability to revert to an ES-like state via 2i LIF and/or Nanog overexpression. D2 EpiLCs differentiate into PGCLCs upon Nanog expression. NANOG binds to putative enhancer elements of Prdm1 and Prdm14 to activate their transcription, which is sufficient to induce the PGCLC fate. This effect can be antagonized by SOX2, which co-binds the Prdm1 enhancer.