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. 2022 Sep 9;149(17):dev201074. doi: 10.1242/dev.201074

Fig. 6.

Fig. 6.

DNA methylation and ZFP57 binding drive H3K9me3 enrichment at Nanog. (A) Analysis of DNA methylation at the Nanog locus, using available datasets in ESCs cultured in FCS+LIF or in 2i+LIF. The position of two ZFP57 motifs is indicated by yellow arrows. (B) Identical analysis but focusing at the two ZFP57 motifs. (C) Analysis of ZFP57 binding by ChIP-qPCR, presented as in Fig. 1C, in the indicated cell lines: E14Tg2a cultured in FCS+LIF (black) or in 2i+LIF (red), triple-negative DNMT knockout ESCs (TKO, blue) and Zfp57 knockout ESCs (ZKO, purple). (D) Analysis of H3K9me3 at Nanog in E14Tg2a, TKO and ZKO ESCs cultured in FCS+LIF, presented as in C. (E) Analysis of NANOG expression in E14Tg2a, TKO and ZKO ESCs. The differential distribution in TKO and ZKO versus E14Tg2a was assessed with Kolmogorov–Smirnov tests (P<2.2e−16). (F) Analysis of NANOG and GATA6 expression after 7 days of directed differentiation of E14Tg2a, TKO and ZKO into primitive endoderm. Differences in distribution levels were assessed with Kolmogorov–Smirnov tests (P<2.2e−16 for both TKO and ZKO versus E14Tg2a). (G) Representative immunostaining of E14Tg2a, TKO and ZKO subjected to a directed differentiation protocol into primitive endoderm. Insets show individual signals, and main panels merged signals.