Identification of TSG101 Functional Domains and p21 Loci Required for TSG101-Mediated p21 Gene Regulation

TSG101 (tumor susceptibility gene 101) is a multi-domain protein known to act in the cell nucleus, cytoplasm, and periplasmic membrane. Remarkably, TSG101, whose location within cells varies with the stage of the cell cycle, affects biological events as diverse as cell growth and proliferation, gene expression, cytokinesis, and endosomal trafficking. The functions of TSG101 additionally are recruited for viral and microvesicle budding and for intracellular survival of invading bacteria. Here we report that the TSG101 protein also interacts with and down-regulates the promoter of the p21CIP1/WAF1tumor suppressor gene, and identify a p21 locus and TSG101 domains that mediate this interaction. TSG101 deficiency in Saos-2 human osteosarcoma cells was accompanied by an increased abundance of p21 mRNA and protein and the retardation of cell proliferation. A cis-acting element in the p21 promoter that interacts with TSG101 and is required for promoter repression was located using chromatin immunoprecipitation (ChIP) analysis and p21-driven luciferase reporter gene expression, respectively. Additional analysis of TSG101 deletion mutants lacking specific domains established the role of the central TSG101 domains in binding to the p21 promoter and demonstrated the additional essentiality of the TSG101 C-terminal steadiness box (SB) in the repression of p21 promoter activity. Neither binding of TSG101 to the p21 promoter nor repression of this promoter required the TSG101 N-terminal UEV domain, which mediates the ubiquitin-recognition functions of TSG101 and its actions as a member of ESCRT endocytic trafficking complexes, indicating that regulation of the p21 promoter by TSG101 is independent of its role in such trafficking.TSG101（肿瘤易感基因101，tumor susceptibility gene 101）是一种多结构域蛋白，已知可在细胞核、细胞质及周质膜中发挥功能。值得关注的是，TSG101在细胞内的定位随细胞周期阶段动态变化，其参与调控的生物学过程涵盖细胞生长与增殖、基因表达、胞质分裂以及内体运输等多个维度。此外，TSG101的功能还可被病毒与微泡出芽过程招募，并参与入侵细菌的胞内存活。本研究首次报道，TSG101蛋白还可与p21CIP1/WAF1肿瘤抑制基因的启动子结合并下调其活性，同时鉴定出介导该相互作用的p21基因座区域与TSG101结构域。在Saos-2人骨肉瘤细胞中缺失TSG101后，细胞内p21 mRNA与蛋白的丰度显著升高，细胞增殖亦受到阻滞。本研究分别通过染色质免疫沉淀（ChIP，chromatin immunoprecipitation）分析与p21驱动的荧光素酶报告基因表达实验，定位到了p21启动子中与TSG101结合且对启动子抑制作用必需的顺式作用元件。进一步针对携带特定结构域缺失的TSG101突变体开展分析，证实了TSG101中央结构域在结合p21启动子过程中的核心作用，并证明TSG101 C端稳定盒（SB，steadiness box）对于抑制p21启动子活性同样不可或缺。值得注意的是，TSG101与p21启动子的结合以及对该启动子的转录抑制均无需其N端UEV结构域——该结构域介导TSG101的泛素识别功能，以及其作为ESCRT内吞运输复合物成员的相关作用——这表明TSG101对p21启动子的调控不依赖于其在内吞运输过程中的功能。