A <i>cis</i>-element within the <i>ARF</i> locus mediates repression of <i>p16</i><sup><i>INK4A</i></sup> expression via long-range chromatin interactions.
basic_science · Level V
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- Record sourced from PubMed, PMID 31818950.
- Also identified by DOI 10.1073/pnas.1909720116 and PMC identifier 6936709.
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Abstract
Loss of function of <i>CDKN2A</i>/<i>B</i>, also known as <i>INK4</i>/<i>ARF</i> [encoding p16<sup>INK4A</sup>, p15<sup>INK4B</sup>, and p14<sup>ARF</sup> (mouse p19<sup>Arf</sup>)], 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 <i>p16</i><sup><i>INK4A</i></sup>, a CRISPR screen targeting open, noncoding chromatin regions adjacent to <i>p16</i><sup><i>INK4A</i></sup> was performed in a human <i>p16</i><sup><i>INK4A-P2A-mCherry</i></sup> reporter cell line. We identified a repressive element located in the 3' region adjacent to the <i>ARF</i> promoter that controls <i>p16</i><sup><i>INK4A</i></sup> expression via long-distance chromatin interactions. Coinfection of lentiviral dCas9-KRAB with selected single-guide RNAs against the repressive element abrogated the <i>ARF</i>/<i>p16</i><sup><i>INK4A</i></sup> chromatin contacts, thus reactivating <i>p16</i><sup><i>INK4A</i></sup> expression. Genetic CRISPR screening identified candidate transcription factors inhibiting <i>p16</i><sup><i>INK4A</i></sup> regulation, including ZNF217, which was confirmed to bind the <i>ARF</i>/<i>p16</i><sup><i>INK4A</i></sup> interaction loop. In summary, direct physical interactions between <i>p16</i><sup><i>INK4A</i></sup> and <i>ARF</i> genes provide mechanistic insights into their cross-regulation.