Purification and characterization of the serum amyloid A3 enhancer factor

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secret ed mainly by the liver. In response to acute inflammation, its expression m ay be induced up to 1000-fold, primarily as a result of a 200-fold increase in the rate of SAA gene transcription. We showed previously that cytokine- induced transcription of the SAA3 gene promoter requires a transcriptional enhancer that contains three functional elements: two CCAAT/enhancer-bindin g protein (C/EBP)-binding sites and a third site that interacts with a cons titutively expressed transcription factor, SAA3 enhancer factor (SEF), Each of these binding sites as well as cooperation among their binding factors is necessary for maximum transcription activation by inflammatory cytokines . Deletion or site-specific mutations in the SEF-binding site drastically r educed SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa nuclear extracts to near homogene ity by using conventional liquid chromatography and DNA affinity chromatogr aphy, Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 6 5 kDa, Protein sequencing and antibody supershift experiments identified SE F as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression vector with SAA3-luciferase reporter resulted in approximately a 5-fold inc rease in luciferase activity. Interestingly, interleukin-1 treatment of SEF -transfected cells caused dramatic synergistic activation (31-fold) of the SAA3 promoter. In addition to its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of the alpha(2)-macroglobulin and A alpha-fibrinogen genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes.