A minimal DNA sequence within the ARF promoter represses p16INK4A expression via long-range chromatin interaction [Capture-C]

Loss of function of CDKN2A/B, also known as INK4/ARF [encoding for p16INK4A, p15INK4B, and p14ARF (mouse p19Arf)]), confers susceptibility to cancers, whereas its up-regulation during organismal aging provokes cellular senescence and tissue degenerative disorders. To better understand molecular regulation of the locus, we knocked P2A-mCherry into the C-terminus of p16INK4A, and this construct faithfully recapitulated the endogenous transcription. Using this reporter cell line, we performed a noncoding tiling pooled screening targeting open chromatin regions spanning the approximately 500-kb region in the topological associated domain that includes p16INK4A. In both Cas9- and dCas9-KRAB–mediated screenings, we identified a 42-bp DNA sequence within exon 1ß of the ARF gene, as the repressive element controlling p16INK4A expression. Chromatin conformation capture indicated that inactivating this element abolished the physical interaction between p15INK4B and ARF, thereby abolishing p16INK4A repression. This study has revealed a new mechanism of transcriptional regulation of p16INK4A by long-range chromatin interaction. Overall design: To understand how certain DNA segments in the ARF promoter could regulate the p16INK4A promoter (which is located approximately 13 kb away in the genome) at the long-range chromatin interaction level, we deployed a recently developed multiplex assay of high-resolution chromatin conformation called Capture-C. When we used oligonucleotides to hybridize with the p16INK4A promoter in a 3C library, we detected strong enrichment of sequences at the bait site, confirming the efficiency of the capture. We then identified the ARF promoter as a strong cis-interaction region associated with the p16INK4A promoter by using two different oligonucleotides. Similarly, when we used oligonucleotides to hybridize with the p15INK4B promoter, we also detected strong interaction with the ARF promoter.