Chromatin Conformation Dynamics are essential to Control the Transcription of INK4/ARF locus

The INK4/ARF (CDKN2A/B) locus has been identified as a hotspot for susceptibility to genetic changes associated with a vast variety of cancers and aging related diseases including coronary artery disease and type II diabetes. Using chromatin Capture-C technologies, we discovered a distal enhancer upstream INK4/ARF looped to CDKN2A, ARF and CDKN2B in human fibroblast cells IMR90 and cancer cell lines HCT116 and SEM, but absent in human fibroblast cell-derived induced pluripotent stem cells, which maintained an epigenetically silenced INK4/ARF locus. Bi-allelic deletion of the ~1.4 Kb core enhancer sequence by CRISPR/Cas9 technology in human HCT116 cells abrogated the long-range chromatin interaction, thereafter disrupting gene expression of these tumor suppressor genes. Moreover, cross-species conservation of the distal enhancer elements between human and mouse was observed. Using combinational strategies including histone modifier chromatin immunoprecipitation, chromatin conformation capture, and CRISPR/Cas9 genome editing tools, we identified a distal genomic segment containing cis-acting elements required for transcriptional regulation of INK4/ARF. This series includes the Capture-C samples.