Fig. 3: ZFP266 impedes activation of pluripotency genes via its KRAB domain.

A Diagram of Zfp266 Wt and mutants. A red bar indicates a silent mutation that confers sgRNA resistance. KRAB domain deletion mutants with (ΔKRAB + L) and without a linker (ΔKRAB) do not have the sgRNA target sequence. DV-AA, DEW-AAA, MLE-AAA mutants have alanine substitutions in the indicated critical amino acids in the KRAB domain. Triple mutant contains all the alanine substitutions. B Nanog-GFP MEF reprogramming with MKOS piggyBac transposons and BFP or Wt Zfp266 cDNA overexpression, imaged at day 15. Red; mOrange, Green; Nanog-GFP. Data represent an average of 3 independent experiments. Error bars indicate SEM, significance calculated using an  unpaired two-tailed t-test. C Cas9 Nanog-GFP MEF reprogramming with MKOS piggyBac transposons, Zfp266 sgRNA expression as well as cDNA overexpression of BFP, sgRNA resistant Zfp266, or sgRNA resistant Zfp266 with mutations in KRAB domain, imaged at day 15. Red; mOrange, Green; Nanog-GFP. D Mean Nanog-GFP⁺ colony numbers of C. Data represent an average of 3 independent experiments. Error bars indicate SEM, p-values calculated using  a one-way ANOVA test. E RNA-Seq volcano plot of Zfp266 KO vs Wt MEF, day 3, 5, and 7 of reprogramming. Up-regulated and down-regulated genes in KO cells are shown to the right and left of the plot, respectively (cut-off FDR < 0.05, log2FC > |1 | ). ESC- and MEF-associated genes (FDR < 0.05, log2FC > |1| in ESCs vs MEFs) are highlighted in green and red. Graphs below volcano plots show the number of ESC-associated, MEF-associated and other genes within D3, D5, D7 reprogramming differentially expressed genes (DEGs). F GO enrichment analysis of upregulated and downregulated genes in Zfp266 KO reprogramming. G Principal component analysis of Zfp266 Wt and KO RNA-Seq samples. Blue dots indicate Wt samples, red dots indicate KO samples, three samples per timepoint. Source data are provided as a Source Data file.