Extended Data Fig. 6: Rescue of Zfp462 KO ESCs with ZFP462 full-length and ZFP462 C-terminal truncation.
a) Heatmap shows Spearman correlation between three independent ATAC-seq experiments in WT and Zfp462 KO ESCs. b) Scatter plot shows pairwise correlation of gene expression changes between Zfp462 KO vs WT and G9a/Glp dKO vs WT. coefficient of determination (R²): black - all genes, red – differentially regulated genes in Zfp462 KO vs WT (padj. ≤ 0.05, LFC ≥ 1). c) Cartoon depicts CRISPR strategy to knock-in coding sequences for ZFP462 full-length and ZFP462 NT+Mid proteins at the Zfp462 gene locus. Insertions were targeted in-frame with exon 3. d) Bar plot shows RT-qPCR analysis of Zfp462 RNA transcript levels in WT, KO and rescue ESCs. n = 2 independent biological replicates. e) Western blot analysis shows ZFP462 protein levels in WT, Zfp462 KO and rescue ESCs. LMNB1 is used as loading control. f) Alkaline phosphatase staining of WT and Zfp462 KO and rescue ESCs (scale bar = 100μm). g) Heatmaps of ATAC-seq signal ratios at ZFP462 and REST peaks of Zfp462 KO vs WT (KO/WT) and Zfp462 rescue ESCs (ZFP462 FL/WT) or (ZFP462 NT+Mid/WT). Each row represents a 10 kb window centred on peak midpoints, sorted by KO/WT enrichment ratio (n = average distribution of two ATAC-seq replicates). h) Line plots show RT-qPCR analysis of lineage marker expression during neural differentiation (n = two replicates) in WT, Zfp462 KO and rescue ESCs. Expression levels are shown relative to ESCs (2i/S/L).
