Landscape of enhancer disruption and functional screen in melanoma [HiChIP]

The high mutation rate across the whole melanoma genome provides a major challenge in stratifying true driver events from the background mutations. Many non-coding recurrent events, such as those occurred in enhancer, can shape tumor evolution, emphasizing the importance in systematically deciphering enhancer disruptions in melanoma. Here, we leveraged 297 melanoma whole-genome sequencing (WGS) samples to prioritize highly recurrent regions (HRRs). By performing a genome-scale CRISPR interference (CRISPRi) screen on HRR-associated enhancers in melanoma cells, we identified 66 significant hits which could have tumor-suppressive roles. These functional enhancers show unique mutational patterns independent of classical significantly mutated genes in melanoma. Target gene analysis for the essential enhancers revealed many known and hidden mechanisms underlying melanoma development. We demonstrated that a super enhancer element could modulate melanoma cell proliferation by targeting MEF2A and another distal enhancer was able to sustain PTEN tumor-suppressive potential via long-range interaction. Our study established a catalogue of crucial enhancers and their target genes in melanoma development and progression, which illuminates the identification of novel mechanism of dysregulation for melanoma driver genes and new therapeutic targeting strategy. To delineate the potential target genes for functional enhancers, we performed in situ Hi-C followed by chromatin immunoprecipitation (HiChIP) of enhancer mark H3K27ac on A375 cells.

全黑色素瘤基因组的高突变率，使得从背景突变中甄别真正的驱动突变事件成为一大核心挑战。诸多复发性非编码变异（例如发生于增强子（enhancer）区域的变异）可调控肿瘤演化进程，凸显了系统性解析黑色素瘤中增强子功能异常的重要研究价值。本研究依托297例黑色素瘤全基因组测序（whole-genome sequencing, WGS）样本，对高复发性区域（highly recurrent regions, HRRs）进行优先级筛选。通过在黑色素瘤细胞中针对HRR相关增强子开展全基因组规模的CRISPR干扰（CRISPR interference, CRISPRi）筛选，本研究鉴定出66个具备潜在抑癌功能的显著命中位点。这些功能性增强子展现出独特的突变特征，且其突变模式独立于黑色素瘤中的经典显著突变基因。针对核心增强子开展靶基因分析后，本研究揭示了诸多参与黑色素瘤发生发展的已知及潜在全新机制。本研究证实，一处超级增强子（super enhancer）元件可通过靶向MEF2A调控黑色素瘤细胞增殖，而另一处远端增强子则可通过远程染色质相互作用维持PTEN的抑癌功能活性。本研究构建了黑色素瘤发生发展进程中关键增强子及其靶基因的研究目录，为解析黑色素瘤驱动基因的失调新机制以及开发新型治疗靶向策略提供了重要理论支撑。为明确功能性增强子的潜在靶基因，本研究在A375细胞中先开展了原位Hi-C实验，随后针对增强子标记H3K27ac完成了HiChIP（高通量染色质免疫沉淀偶联Hi-C技术）实验。