Identification of a short form of ubiquitin-specific protease 3 that is a repressor of rat glutathione S-transferase gene expression

The transcription rate and protein expression from both GSTA2 (glutathione S-transferase A2) and albumin genes decrease in rat liver after IL-6 (interleukin 6) plus DEX (dexamethasone) treatment of primary hepatocytes or after LPS (lipopolysaccharide)-induced acute-phase response in animals. The down-regulation is associated with the induced expression of a nuclear protein (termed IL6DEX-NP for IL-6/DEX-induced nuclear protein) that binds to a specific site on the promoter of GSTA2, leading to a decrease in transcriptional activity. IL6DEX-NP is not similar to other transcription factors, and, for identification, we functionally cloned it from a rat liver library using a yeast one-hybrid screen based on DNA-binding activity. The cloned sequence was a truncated form of USP3 (ubiquitin-specific protease 3) and the truncated USP3 protein in a yeast extract bound to DNA containing the IL6DEX-NP recognition sequence. Using 5′- and 3′-RACE (rapid amplification of cDNA ends), the complete sequence of USP3 was found in liver from LPS-treated rats. However, using Western blot analysis, only truncated forms of USP3 could be identified in nuclear extracts from LPS-treated rat livers. A GSTA2 promoter–reporter gene plasmid and USP3-expressing plasmids were transfected into rat hepatoma cells. Expression of the short form of USP3, but not the full-length protein, abolished expression from the reporter gene. Chromatin immunoprecipitation localized USP3 to the GSTA2 promoter in rat hepatocytes in vivo. We believe that the short form of USP3 is IL6DEX-NP and that it may play an important role in the negative regulation of proteins during the acute-phase response.