UBR7 is a novel E3 ubiquitin ligase for H2BK120 and acts as a tumor-suppressor in breast cancer [ChIP-Seq]

Plant Homeo Domain (PHD) is a versatile chromatin reader/effector module which recognizes methylated, acetylated or unmodified histone substrates and regulates cellular gene expression programs. Although PHD domains shows selective epigenetic recognition of methylated, acetylated and unmodified histone substrates, there has been no previous report on its catalytic function regulating malignant transformation of cells. Here we report that PHD finger of UBR7 (Ubiquitin Protein Ligase E3 Component N-Recognin 7 (Putative)), in isolation or in context of full length protein, harbors E3 ubiquitin ligase activity towards monoubiquitination of histone H2B at lysine 120 . Knockdown of UBR7 in MCF10a and breast cancer cells decreased H2BK120ub both at the global levels and on specific genes. Conversely, overexpression of wild type, but not catalytic mutant, rescued H2BK120ub levels. Low UBR7 expression was associated with basal-like and triple negative breast cancers as well as showed poor expression in metastatic tumors. Consistently, UBR7 loss resulted in invasion properties, induced epithelial-to-mesenchymal transition and promoted metastasis. Conversely, ectopic expression of UBR7 reduced cell growth, invasion and tumor growth in mouse fat pad. Mechanistically, UBR7 reduced H2BK120ub gene body of cell-adhesion related genes as well as gene expression including on CDH4 gene. Importantly, rebuilding CDH4 levels rescued invasion phenotypes seen in UBR7-low cells. Collectively, our results establish that UBR7 PHD has novel H2B ubiquitin ligase activity and it suppresses tumor growth in basal-like breast cancers. Overall design: Examination of 7 different histone modifications in Wild Type and UBR7-shRNA MCF10A Cell Line

植物同源结构域（Plant Homeo Domain，PHD）是一类多功能的染色质阅读/效应模块，可识别甲基化、乙酰化或未修饰的组蛋白底物，并调控细胞基因表达程序。尽管PHD结构域能够选择性地识别甲基化、乙酰化及未修饰的组蛋白底物，但此前尚无关于其催化功能调控细胞恶性转化的相关报道。本研究报道，UBR7（泛素蛋白连接酶E3组分N识别蛋白7（推定））的PHD指结构域，无论是单独存在还是以全长蛋白的形式存在，均具有E3泛素连接酶活性，可介导组蛋白H2B在赖氨酸120位点的单泛素化修饰。在MCF10A细胞与乳腺癌细胞中敲低UBR7，会使全局及特定基因位点的H2BK120ub修饰水平下降。反之，过表达野生型UBR7（而非催化突变体）可恢复H2BK120ub的修饰水平。UBR7低表达与基底样型及三阴性乳腺癌相关，且在转移性肿瘤中表达量显著降低。一致的是，UBR7缺失会增强细胞侵袭能力，诱导上皮间质转化（epithelial-to-mesenchymal transition），并促进肿瘤转移。反之，外源过表达UBR7则可抑制细胞增殖、侵袭以及小鼠脂肪垫内的肿瘤生长。从机制上来看，UBR7会降低细胞黏附相关基因的基因体区域H2BK120ub修饰水平，并下调包括CDH4基因在内的相关基因的表达。重要的是，重建CDH4的表达水平可挽救UBR7低表达细胞中出现的侵袭表型。综上，本研究结果证实UBR7的PHD结构域具有全新的H2B泛素连接酶活性，并可抑制基底样型乳腺癌的肿瘤生长。整体实验设计：检测野生型与UBR7短发夹RNA（shRNA）转染的MCF10A细胞系中的7种不同组蛋白修饰。