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

Ruiqi Han, Li Li, Alejandro Piñeiro Ugalde, Arieh Tal, Zohar Manber, Eric Pinto Barbera, Veronica Della Chiara, Ran Elkon, Reuven Agami

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. 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 EnhAP1-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 EnhAP1-OIS1 effect on the senescence phenotype. 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.