INO80 governs super-enhancer-mediated oncogenic transcription and tumor growth in melanoma [ChIP-seq_MiSeq]. Homo sapiens

Super-enhancers (SEs) are large genomic regions with high density of enhancer marks. In cancer, SEs are found near oncogenes and dictate cancer gene expression. However, how oncogenic SEs are regulated remains poorly understood. Here, we show that INO80, a chromatin remodeling complex, is required for SE-mediated oncogenic transcription and tumor growth in melanoma. The expression of Ino80, the SWI/SNF ATPase, is elevated in melanoma cells and patient melanomas compared to normal melanocytes and benign nevi. Furthermore, Ino80 silencing selectively inhibits melanoma cell proliferation, anchorage-independent growth, as well as tumorigenesis and tumor maintenance in mouse xenografts. Mechanistically, Ino80 occupies more than 90% of SEs, and its occupancy is dependent on transcription factors such as MITF and Sox9. Ino80 binding reduces nucleosome occupancy and facilitates Mediator recruitment, thus promoting oncogenic transcription. Consistently, genes co-occupied by Ino80 and Med1 are specifically expressed in melanomas compared to melanocytes. Together, our results reveal an essential role of INO80-dependent chromatin remodeling in SE function, and suggest a novel strategy for disrupting SEs in cancer treatment. Overall design: Human melanoma cell line A375 cells were infected with shNT or shIno80, and total RNA was extracted 2, 3, 4 days after infection. The RNA was submitted to RNA-Seq subsequently. For ChIP-Seq, either wild type A375 and SK-MEL-147, or A375 infected with shNT or shIno80, was used for ChIP-Seq for corresponding factors.

超级增强子（Super-enhancers, SEs）是一类携带高密度增强子标记的大型基因组区域。在癌症中，SEs定位于致癌基因附近，并主导癌症相关基因的表达。然而，目前对于致癌性超级增强子的调控机制仍知之甚少。本研究表明，染色质重塑复合物INO80对于黑色素瘤中SEs介导的致癌转录与肿瘤生长是必需的。作为SWI/SNF家族ATP酶的Ino80，其在黑色素瘤细胞及患者黑色素瘤组织中的表达水平，相较于正常黑素细胞与良性痣组织显著升高。此外，沉默Ino80可选择性抑制黑色素瘤细胞的增殖、非锚定依赖性生长，以及小鼠异种移植模型中的肿瘤发生与肿瘤维持。从机制上看，Ino80可结合超过90%的SEs，且其结合依赖于MITF、Sox9等转录因子。Ino80的结合可降低核小体占据率，并促进中介体复合物（Mediator）的招募，进而推动致癌转录过程。一致的是，相较于正常黑素细胞，同时被Ino80与Med1结合的基因在黑色素瘤中特异性表达。综上，本研究结果揭示了依赖INO80的染色质重塑在SEs功能中的关键作用，并为癌症治疗中靶向干扰SEs提供了全新策略。整体实验设计：将短发夹RNA阴性对照（shNT）或靶向Ino80的短发夹RNA（shIno80）感染人黑色素瘤细胞系A375，分别于感染后2、3、4天提取总RNA，随后进行RNA测序（RNA-Seq）。在染色质免疫沉淀测序（ChIP-Seq）实验中，分别采用野生型A375与SK-MEL-147细胞，或感染shNT、shIno80的A375细胞，针对对应靶因子开展ChIP-Seq实验。