Fig. 2: ORFtag interrogates protein function with high specificity.
From: Proteome-scale tagging and functional screening in mammalian cells by ORFtag
a, Overlap between activator (green), repressor (blue) and PTGR (yellow) hits. b, Enrichment of screen hits for human homologous genes with annotated DNA binding, activation, repressive domains and RNA-binding proteins. Additionally, activator screen hits are enriched for the ORFeome activator hits, although with only a limited overlap (n = 15; Extended Data Fig. 3d). Enrichment was assessed using a one-tailed Fisher’s exact test (alternative = ‘greater’), with intronic protein-coding genes as the background. TF, transcription factor. c, Top enriched protein domains, biological process and cellular component GO terms for activator, repressor and PTGR hits. Enrichment was assessed using a one-tailed Fisher’s exact test (alternative = ‘greater’), with intronic protein-coding genes as the background. P values were adjusted for multiple testing using the FDR method. miRNA, microRNA; reg., regulation. d, Independent validation of select screen hits. GFP intensity measured by flow cytometry in reporter cell lines stably expressing the indicated full-length proteins fused to TetR (activator, repressor) or λN (PTGR); one-sided Wilcoxon test (alternative = ‘greater’ for activator, and ‘less’ for repressor/PTGR hits), ***P ≤ 2.2 × 10−308. The sample size was 25,000 cells for each validation, except for N4bp1 (n = 5,766) and Trim8 (n = 3,775). Refer to Fig. 1d for the position of the hits in the volcano plot. Box plots show the median (line), upper and lower quartiles (box) ± 1.5 × interquartile range (whiskers); outliers are not shown. e, Schematic view of Zfp574 rapid depletion using AID. Western blot analysis demonstrates the rapid depletion of Zfp574 following treatment with IAA. This result was consistently observed in two independent experiments. f, Cell viability timecourse in the presence (−IAA, in gray) or absence of Zfp574 (+IAA, in red). Shown are two biological replicates. g, MA plot showing PRO-seq fold changes (log2) after 6 h depletion of Zfp574. Significantly up- (0) or downregulated (39) genes are highlighted in red. h, PRO-seq fold changes (log2) of not-bound (n = 12,381) versus Zfp574 promoter-bound genes (n = 105) after Zfp574 depletion; two-sided Wilcoxon test. Box plots show the median (line), upper and lower quartiles (box) ± 1.5 × interquartile range (whiskers); outliers are not shown. i, Zfp574 Cut&Run and PRO-seq screenshots at the Rpl10 locus. j, Enrichment of screen hits for genes that were identified as part of oncogenic fusions.
