Purification and identification of a tissue-specific repressor involved inserum amyloid A1 gene expression

We have previously demonstrated that the 5'-flanking regions from the rat s erum amyloid A1 (SAA1) promoter are necessary and sufficient to confer spec ific cytokine induced expression in cultured hepatoma cells. Deletion analy sis identified a tissue-specific repressor (TSR) regulatory element, locate d between bp -289 and -256, that functioned as a silencer, contributing to the transcription repression on SAA1 promoter in nonliver cells. When this 34-base pair TSR-binding element was used as a probe in electrophoretic mob ility shift assays, an intense DNA-protein complex was detected in nuclear extracts from HeLa and several other nonliver tissues, This TSR binding act ivity, however, was undetectable in extracts from liver or liver-derived ce lls. The distribution of TSR binding activity is therefore consistent with its regulatory role in repressing SAA1 expression in a tissue-specific mann er. In this study, we purified TSR protein from HeLa nuclear extracts and s howed that it has a molecular mass of approximately 50 kDa. Surprisingly, p rotein sequencing and antibody supershift experiments identified TSR as tra nscription factor AP-2. Subsequent functional analysis showed that forced e xpression of AP-2 in HepG2 cells could indeed inhibit conditioned medium-in duced SAA1 promoter activation. Moreover, expression of a dominant-negative mutant of AP-2 in HeLa cells or mutation of the AP-2-binding site led to d erepression of the SAA1 promoter, presumably by neutralizing the inhibitory effects of the endogenous wild-type AP-2. Our results therefore demonstrat e a novel function for AP-2 in the transcriptional repression of SAA1 promo ter. Together with its tissue distribution, AP-2 may contribute to SAA1's h ighly liver-specific expression pattern by restricting its expression in no nliver cells.