Purification and Characterization of the Serum Amyloid A3 Enhancer Factor

Abstract

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to acute inflammation, its expression may 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-binding protein (C/EBP)-binding sites and a third site that interacts with a constitutively 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 reduced 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 homogeneity by using conventional liquid chromatography and DNA affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression vector with SAA3-luciferase reporter resulted in approximately a 5-fold increase 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 ␣ 2 -macroglobulin and A␣-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.

CRTR-1, a Developmentally Regulated Transcriptional Repressor Related to the CP2 Family of Transcription Factors

“…Consistent with the ability of mouse CP2 to activate transcription, human CP2/LBP-1c has been shown to activate transcription from an SV40 promoter (6) and from cellular promoters such as those directing expression of the serum amyloid A3 gene (11). The ability to activate transcription is conserved among other family members; LBP-1b activates transcription from the Ϫ155/Ϫ131 region of the human P450scc promoter (9), LBP-1a activates transcription from a human immunodeficiency virus, type I promoter (8), and chicken CP2 activates transcription from the ␣A-crystallin gene promoter (10).…”

Altered Hippocampal Transcript Profile Accompanies an Age-Related Spatial Memory Deficit in Mice

“…Annotations for each gene and the Welch t-test P-value (uncorrected for multiple testing) are shown at the right of the figure. ers (Rodda et al 2001). Interestingly, CP2/LPB-1c cooperatively interacts with C/EBP transcription factors (Bing et al 1999). CP2/ LBP-1c has been implicated as a susceptibility gene of Alzheimer's disease (Lambert et al 2000;Lendon and Craddock 2001;Taylor et al 2001;Luedecking-Zimmer et al 2003).…”

Fe65, a Ligand of the Alzheimer's β-Amyloid Precursor Protein, Blocks Cell Cycle Progression by Down-regulating Thymidylate Synthase Expression

“…LSF was originally identified as a transcription factor specifically binding to the SV40 major late promoter (19). Numerous results demonstrated that this protein is a housekeeping factor regulating a variegated array of genes, including the human ␣-globin gene (20), the serum amyloid A3 gene (21), and the thymidylate synthase (TS) gene (22). Two recent papers indicate LSF as a genetic determinant of the risk of AD (23,24).…”