A cis-element within the ARF locus mediates repression of p16 expression via long-range chromatin interactions

Loss of function of CDKN2A/B, also known as INK4/ARF [encoding p16^INK4A, p15^INK4B, and p14^ARF (mouse p19^Arf)], confers susceptibility to cancers, whereas its up-regulation during organismal aging provokes cellular senescence and tissue degenerative disorders. To better understand the transcriptional regulation of p16, a CRISPR screen targeting open, noncoding chromatin regions adjacent to p16 was performed in a human p16 reporter cell line. We identified a repressive element located in the 3' region adjacent to the ARF promoter that controls p16 expression via long-distance chromatin interactions. Coinfection of lentiviral dCas9-KRAB with selected single-guide RNAs against the repressive element abrogated the ARF/p16 chromatin contacts, thus reactivating p16 expression. Genetic CRISPR screening identified candidate transcription factors inhibiting p16 regulation, including ZNF217, which was confirmed to bind the ARF/p16 interaction loop. In summary, direct physical interactions between p16 and ARF genes provide mechanistic insights into their cross-regulation.