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Fig. 2 | Genome Biology

Fig. 2

From: Functional CRISPR screen identifies AP1-associated enhancer regulating FOXF1 to modulate oncogene-induced senescence

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 EnhAP1-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 EnhAP1-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 EnhAP1-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 EnhAP1-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-indRASG12V 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 EnhAP1-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-indRASG12V cells were transfected with the indicated enhancer constructs. Endogenous motif represents the original sequence of EnhAP1-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

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