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 combinatihttps://www.ncbi.nlm.nih.gov/geo/submission/update/?acc=GSE119947#onal 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.

INK4/ARF（CDKN2A/B）基因座已被鉴定为与多种癌症及衰老相关疾病（涵盖冠状动脉疾病与II型糖尿病）相关遗传改变的易感热点区域。本研究采用染色质捕获-C（chromatin Capture-C）技术，在人成纤维细胞系IMR90以及癌细胞系HCT116和SEM中，发现INK4/ARF基因座上游存在一个远端增强子，该增强子可与CDKN2A、ARF及CDKN2B形成染色质环；而在人成纤维细胞来源的诱导多能干细胞（induced pluripotent stem cells）中则未存在该染色质环相互作用，此类细胞维持着表观遗传沉默状态的INK4/ARF基因座。我们通过CRISPR/Cas9技术对人HCT116细胞中约1.4 Kb的核心增强子序列实施双等位基因敲除，该操作阻断了远距离染色质相互作用，进而扰乱了这些抑癌基因的转录表达。此外，本研究还观察到人与小鼠的远端增强子元件存在跨物种保守性。通过组蛋白修饰染色质免疫沉淀、染色质构象捕获以及CRISPR/Cas9基因组编辑等联合策略，我们鉴定出一段包含调控INK4/ARF转录所需顺式作用元件的远端基因组片段。