Fig. 2

Functional CRISPR screen discovers a novel enhancer required for OIS. a Schematic representation of the set-up of our functional screen. b Results of the CRISPR screen. sgRNAs are sorted by the enrichment score based on the ratio between their prevalence in the BJ + 4-OHT and BJ + DMSO control populations (measured 4 weeks after 4-OHT treatment). Y-axis shows Z scores of the mean sgRNA enrichment scores (calculated over the four replicates of the screen). Colored in red are two sgRNAs, sgRNA-AP1⁶⁹ and sgRNA-AP1⁷¹, that target the same enhancer, called here Enh^AP1-OIS1 c Individual transductions of sgRNA- AP1⁶⁹ and sgRNA-AP1⁷¹ validated that they cause OIS bypass. sgRNA targeting p53 was used as a positive control and a non-targeting (NT) sgRNA was used as a negative control. d Targeting Enh^AP1-OIS1 by either sgRNA-AP1⁶⁹ or sgRNA-AP1⁷¹ caused OIS bypass as measured by β-gal staining, a canonical mark for senescence (p53ko used as a positive control). Data shown represent mean (SD), n = 4. *p < 0.05. e Targeting Enh^AP1-OIS1 by either sgRNA-AP1⁶⁹ or sgRNA-AP1⁷¹ resulted in enhanced proliferation as measured by BrdU staining (p53ko used as a positive control). Data shown represent mean (SD), n = 4. *p < 0.05. f Measurement of eRNA production at Enh^AP1-OIS1 in cells with the indicated sgRNAs. eRNA levels are significantly decreased upon mutagenesis of the AP1 binding site caused by either sgRNA-AP1⁶⁹ or sgRNA-AP1⁷¹. Data shown represent mean (SD), n = 3. *p < 0.05. g BJ-indRAS^G12V cells were transfected with the indicated plasmids and treated with DMSO or 4-OHT for 72 h. pGL3 constructs contain firefly luciferase reporter gene with the corresponding enhancer (none for pGL3-promoter, two different orientations for Enh^AP1-OIS1). Relative luciferase activity is calculated by dividing the firefly luciferase activity to that of Renilla luciferase. Normalized luciferase activity is calculated by dividing the relative luciferase activity to that of pGL3-promoter for each condition. Data shown represent mean (SD), n = 6. *p < 0.05. h BJ-indRAS^G12V cells were transfected with the indicated enhancer constructs. Endogenous motif represents the original sequence of Enh^AP1-OIS1, in vitro mutation construct represents mutagenesis of the AP1 consensus motif, and MU44835851 represents mutant construct bearing a C > A mutation as indicated. The cells were treated with 4-OHT for 48 h before transfection. Data shown represent mean (SD), n = 3. *p < 0.05