Triple-negative breast cancer (TNBC) is the most therapeutically recalcitrant form of breast cancer, which is due in part to the paucity of targeted therapies. A systematic analysis of regulatory elements that extend beyond protein coding genes could uncover avenues for therapeutic intervention. To this end, we analyzed the regulatory mechanisms of TNBC-specific transcriptional enhancers together with their non-coding enhancer RNA (eRNA) transcripts. The functions of the top 30 eRNA-producing super-enhancers were systematically probed using high-throughput CRISPR-interference assays coupled to RNA-seq that enabled unbiased detection of target genes genome-wide. Generation of high resolution Hi-C chromatin interaction maps enabled annotation of the direct target genes for each super-enhancer, which highlighted their proclivity for genes that portend worse clinical outcomes in TNBC patients. Illustrating the utility of this dataset, deletion of an identified super-enhancer controlling the nearby PODXL gene or specific degradation of its enhancer RNAs led to profound inhibitory effects on target gene expression, cell proliferation, and migration. Furthermore, loss of this super-enhancer suppressed tumor growth and metastasis in TNBC mouse xenograft models. Single-cell RNA-seq and ATAC-seq analyses demonstrated the enhanced activity of this super-enhancer within the malignant cells of TNBC tumor specimens compared to non-malignant cell types. Collectively, this work examines several fundamental questions about how regulatory information encoded into eRNA-producing super-enhancers drives gene expression networks that underlie the biology of triple-negative breast cancer.

中文翻译：

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转录的超级增强子的 CRISPR 筛选确定了三阴性乳腺癌进展的驱动因素


三阴性乳腺癌 （TNBC） 是治疗上最难治疗的乳腺癌形式，部分原因是缺乏靶向治疗。对超越蛋白质编码基因的调节元件进行系统分析可以揭示治疗干预的途径。为此，我们分析了 TNBC 特异性转录增强子及其非编码增强子 RNA （eRNA） 转录本的调控机制。使用与 RNA-seq 偶联的高通量 CRISPR 干扰测定系统地探测前 30 种产生 eRNA 的超级增强子的功能，从而能够在全基因组范围内对靶基因进行无偏检测。高分辨率 Hi-C 染色质相互作用图的生成能够注释每个超级增强子的直接靶基因，这突出了它们对预示着 TNBC 患者临床结果较差的基因的倾向。说明该数据集的实用性，删除控制附近 PODXL 基因的已鉴定的超级增强子或其增强子 RNA 的特异性降解导致对靶基因表达、细胞增殖和迁移的严重抑制作用。此外，这种超级增强子的缺失抑制了 TNBC 小鼠异种移植模型中的肿瘤生长和转移。单细胞 RNA-seq 和 ATAC-seq 分析表明，与非恶性细胞类型相比，这种超级增强子在 TNBC 肿瘤标本恶性细胞内的活性增强。总的来说，这项工作研究了几个基本问题，即编码到产生 eRNA 的超级增强子中的调节信息如何驱动基因表达网络，这些网络是三阴性乳腺癌生物学的基础。