Purification and identification of a tissue-specific repressor involved in serum amyloid A1 gene expression.
We have previously demonstrated that the 5'-flanking regions from the rat serum amyloid A1 (SAA1) promoter are necessary and sufficient to confer specific cytokine-induced expression in cultured hepatoma cells. Deletion analysis identified a tissue-specific repressor (TSR) regulatory element, located 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 mobility shift assays, an intense DNA-protein complex was detected in nuclear extracts from HeLa and several other nonliver tissues. This TSR binding activity, however, was undetectable in extracts from liver or liver-derived cells. The distribution of TSR binding activity is therefore consistent with its regulatory role in repressing SAA1 expression in a tissue-specific manner. In this study, we purified TSR protein from HeLa nuclear extracts and showed that it has a molecular mass of approximately 50 kDa. Surprisingly, protein sequencing and antibody supershift experiments identified TSR as transcription factor AP-2. Subsequent functional analysis showed that forced expression of AP-2 in HepG2 cells could indeed inhibit conditioned medium-induced 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 derepression of the SAA1 promoter, presumably by neutralizing the inhibitory effects of the endogenous wild-type AP-2. Our results therefore demonstrate a novel function for AP-2 in the transcriptional repression of SAA1 promoter. Together with its tissue distribution, AP-2 may contribute to SAA1's highly liver-specific expression pattern by restricting its expression in nonliver cells.
Ren
Y
Y
Department of Biochemistry, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
Reddy
S A
SA
Liao
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WS
D000595:Amino Acid Sequence; D000818:Animals; D016207:Cytokines / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000502:physiology; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D051381:Rats; D012097:Repressor Proteins / Q000302:isolation & purification*; D000685:Serum Amyloid A Protein / Q000235:genetics*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000302:isolation & purification* / Q000502:physiology
D016207:Cytokines; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D000685:Serum Amyloid A Protein; D050656:Transcription Factor AP-2; D014157:Transcription Factors