Multiple elements within the glucocorticoid regulatory unit of the rat alpha 1-acid glycoprotein gene are recognition sites for C/EBP

Abstract

Sequences between -106 and -42, located immediately downstream of the glucocorticoid response element, are essential for efficient glucocorticoid-stimulated expression of the alpha 1-acid glycoprotein (AGP) gene. We have used mobility shift assays with oligonucleotides bearing wild type and mutated sequences from segmented portions of this region to characterize the specific interaction of similar binding factors from rat liver and HTC rat hepatoma cell nuclear extracts. One of these factors, AGP nuclear factor 2 (ANF-2), appears capable of dual interaction with the homologous recognition sites, HA (-133 to -104) and HB (-81 to -72), which overlap and are located downstream of the glucocorticoid response element, respectively. Using an affinity matrix containing the HB sequence we have isolated ANF-2 from rat liver nuclear extracts. On the basis of immunological evidence rat liver ANF-2 was confirmed to be highly related and probably identical to CCAAT/enhancer-binding protein (C/EBP). Methylation protection analyses with partially purified, rat liver ANF-2 confirmed HA and HB as recognition sites for C/EBP-related factors and are consistent with the location of a third interaction site for these transactivating proteins at HX (-102 to -93). We propose that the sequences HA, HX, and HB, spanning residues -113 to -72 of the AGP promoter, might serve as recognition sites for a family of C/EBP-like nuclear factors that coordinate the glucocorticoid-mediated induction of the AGP gene.