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. 2023 Aug 31;51(19):10292–10308. doi: 10.1093/nar/gkad719

Figure 1.

Figure 1.

Principle of the reactivation screen, generation and validation of the DASH reporter cell line. (A) Mouse ES cells can be grown in 2i medium or in serum. A number of genes acquire DNA methylation (black lollipops) and repressive histone marks (large gray circle) in serum, which inhibits their expression. Performing a CRISPR loss-of-function screen in serum should lead to the identification of repressors. (B) The Dazl gene promoter contains a methylated CpG island, the activating histone mark H3K4me3 and the repressive histone mark H3K27me3: it is bivalent. (C) DNA methylation and the ncPRC1.6 complex are already known to repress Dazl expression in serum. We searched for new repressors. (D) We built the DASH reporter cell line, in which two positively selectable markers (mScarlet and HygroR) are inserted into exon 6 of Dazl. The other allele of Dazl in DASH cells is WT. (E) CRISPR KO of Uhrf1 in DASH cells is confirmed by western blotting. (F) LUMA confirms that Uhrf1 KO in DASH cells leads to a global loss of DNA methylation. (G) Uhrf1 KO in DASH cells results in mScarlet reactivation. (H) Uhrf1 KO in DASH cells results in Hygromycin resistance; surviving cells stained with crystal violet.