Abstract
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.
Highlights
TSG101 (Tumor susceptibility gene 101) was identified initially in mouse cells in a genetic screen for loci that modulate neoplastic transformation of NIH3T3 fibroblasts and tumor formation in nude mice [1]
We report that domains located near the center of the TSG101 protein enable TSG101 to interact with to a 200 bp region of the p21 promoter, that such binding represses p21 promoter activity, and that repression of p21 promoter activity is independent of the ubiquitin E2 variant (UEV) domain that is central to TSG101 function in endocytic trafficking
The results reported here demonstrate that the cell cycle regulator and tumor suppressor gene p21 is negatively regulated by interaction of the TSG101 protein with the p21 promoter, leading to consequent suppression of p21-mediated cell growth retardation by adventitiously expressed TSG101
Summary
TSG101 (Tumor susceptibility gene 101) was identified initially in mouse cells in a genetic screen for loci that modulate neoplastic transformation of NIH3T3 fibroblasts and tumor formation in nude mice [1]. More than 15 years of study has revealed that TSG101 affects multiple cellular functions beyond growth and proliferation; these include gene expression, cytokinesis, and endosomal trafficking [5,6,7,8,9]. As a component of the ESCRT-1 complex, TSG101 regulates endosomal trafficking of ubiquitinated proteins by interacting with PTAP or PSAP motifs of these proteins through its UEV domain [8,13]. Increased abundance of TSG101 protein has been observed in cancer cells [6,21], and the additional ability of antisense RNA or siRNA directed against TSG101 to alter cellular functions [8] implies that autoregulation of TSG101 protein stability by the SB does not prevent the effects of perturbations that affect negatively TSG101 expression at the pre-translational level. We report that domains located near the center of the TSG101 protein enable TSG101 to interact with to a 200 bp region of the p21 promoter, that such binding represses p21 promoter activity, and that repression of p21 promoter activity is independent of the UEV domain that is central to TSG101 function in endocytic trafficking
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