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

Abstract

Background: Functional characterization of non-coding elements in the human genome is a major genomic challenge and the maturation of genome-editing technologies is revolutionizing our ability to achieve this task. Oncogene-induced senescence, a cellular state of irreversible proliferation arrest that is enforced following excessive oncogenic activity, is a major barrier against cancer transformation; therefore, bypassing oncogene-induced senescence is a critical step in tumorigenesis. Here, we aim at further identification of enhancer elements that are required for the establishment of this state.

Results: We first apply genome-wide profiling of enhancer-RNAs (eRNAs) to systematically identify enhancers that are activated upon oncogenic stress. DNA motif analysis of these enhancers indicates AP-1 as a major regulator of the transcriptional program induced by oncogene-induced senescence. We thus constructed a CRISPR-Cas9 sgRNA library designed to target senescence-induced enhancers that are putatively regulated by AP-1 and used it in a functional screen. We identify a critical enhancer that we name Enh^AP1-OIS1 and validate that mutating the AP-1 binding site within this element results in oncogene-induced senescence bypass. Furthermore, we identify FOXF1 as the gene regulated by this enhancer and demonstrate that FOXF1 mediates Enh^AP1-OIS1 effect on the senescence phenotype.

Conclusions: Our study elucidates a novel cascade mediated by AP-1 and FOXF1 that regulates oncogene-induced senescence and further demonstrates the power of CRISPR-based functional genomic screens in deciphering the function of non-coding regulatory elements in the genome.

Keywords: AP1; CRISPR; Enhancers; FOS; FOXF1; Functional screen; Gene regulation; JUN; Oncogene-induced senescence.

Fig. 1

Design of a CRISPR screen targeting AP1 enhancers which are activated upon oncogenic stress. a An example of an enhancer whose activity is induced in response to oncogenic stress. Enhancer activity is inferred from the typical bi-directional transcription of eRNAs (BJ + DMSO indicates proliferating cells and BJ + 4-OHT indicates senescent cells); genomic regions that show DNase hypersensitivity (DHS), as determined by ENCODE, are shown by the gray track). Overall, our GRO-seq analysis identified 1821 regulatory elements (REs; enhancers or promoters) whose activity was induced in BJ cells in face of RAS activation. b De novo motif analysis detected highly significant enrichment of the FOS:JUN (AP1) DNA motif in the REs that were induced upon oncogenic stress. Top: the enriched motif detected in our dataset; bottom: the AP1 motif from the JASPAR DB [49]. c An example for occurrence of an AP1 motif within an enhancer that was induced upon oncogenic stress, that is located close enough to an NGG PAM motif, resulting in Cas9-mediated DNA cleavage that occur within the motif (Cas9 cleavage occurs ~ 3 nt before the PAM). Overall, we identified 398 induced REs with AP1 motif that met this requirement (Cas9 cleavage within a margin of 5 nt with respect to the motif). d Statistical summary of the CRISPR-AP1-EnhLib used in our functional screen

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

Fig. 3

FOXF1 is the target gene regulated by Enh^AP1-OIS1. a UCSC screenshot of GRO-seq analysis of BJ-indRAS^G12V cells. BJ cells were treated with DMSO or 4-OHT for 14 days. Bi-directional transcription is represented by using positive and negative values for expression in the Crick and Watson strands, respectively. The genomic regions of Enh^AP1-OIS1 and FOXF1 are enlarged. Note the enhancement in GRO-seq signal for both Enh^AP1-OIS1 and FOXF1 in BJ + 4-OHT (brown track) compared to BJ + DMSO (blue track). b mRNA levels of FOXF1 are reduced in sgRNA-AP1⁶⁹ and sgRNA-AP1⁷¹ targeted cells under 4-OHT treatment. Data shown represent mean (SD), n = 3. *p < 0.05. c BJ-indRAS^G12V cells transduced with the specified sgRNAs were treated with DMSO or 4-OHT for 14 days; FOXF1, p21, and HRas protein levels were measured by western blot. HSP90 was used as the loading control. The band of FOXF1 is marked with an arrow. ER-HRas indicates the induced version of HRas. d Targeting the FOXF1 and p53 genes caused OIS bypass as measured by β-gal staining. Note the stronger effect of FOXF1ko compared to the effect elicited by targeting Enh^AP1-OIS1 (Fig. 2d). Data shown represent mean (SD), n = 4. *p < 0.05. e Targeting FOXF1 and p53 gene resulted in enhanced proliferation as measured by BrdU staining. Data shown represent mean (SD), n = 4. *p < 0.05

Fig. 4

Enh^AP1-OIS1 and FOXF1 knockouts display expression profiles of senescence bypass. GSEA analysis of expression profiles measured in 4OHT-treated BJ-indRAS^G12V cells targeted by sgRNA-AP1⁶⁹, sgRNA-AP1⁷¹, or sgRNA-FOXF1 compared to the profile of control 4OH-treated cells transduced with non-targeting sgRNA. A list of shared genes within each group is shown

Fig. 5

Model of Enh^AP1-OIS1 regulation of OIS. a In normal BJ fibroblast cells, hyper-activation of RAS induces MAPK signaling cascade, including AP-1 TFs. Activated AP-1 TFs control different cellular functions, including cell proliferation and apoptosis. AP1 is recruited, among other enhancers, to Enh^AP1-OIS1 and stimulates its activity. This, in turn, promotes the expression of the target gene FOXF1, diverting oncogenic signals into the pre-senescent pathway. b Mutagenesis of the AP-1 binding site in Enh^AP1-OIS1 abrogates its enhancer activity and thus leads to decreased expression of FOXF1. This results in compromised induction of OIS and thus cells continue uncontrolled cell proliferation [52]