Abstract
The liver alpha-1-acid glycoprotein (AGP) gene promoter contains several positive cis-acting sequences that are involved in the hormone regulation of its expression. We have characterized a new functionally important sequence located at −155 to −143 upstream from the glucocorticoid regulatory element (GRE, −120 to −105). At least three nuclear proteins bind to this sequence (CTGTGGGAACAG), called the upstream regulatory element (URE). One of these proteins, AGP nuclear factor 4 (ANF-4), is the major component of the DNA-protein complex we detected in footprint and electrophoresis mobility shift assay (EMSA) experiments using rat liver, HTC(JZ-l) rat hepatoma cell extracts and affinity-purified proteins. Another is C/EBPβ, which also binds to three elements downsteam from the GRE. The third protein is shown to have a molecular weight of 102 kD. Deletions and site-directed mutagenesis demonstrated that this complex of proteins is involved in the positive hormonal regulation of AGP gene transcription. Binding experiments revealed that ANF-4 and C/EBPβ binding sites are partially overlapping and require the palindromic structure of the URE for high-affinity binding. Southwestern (DNA-protein blot analysis) and cross-linking experiments with nuclear exptracts from rat liver and HTC(JZ-l) rat hepatoma cells, revealed two identical constitutive binding activities with molecular masses of 66 and 102 kD.
We concluded that this transcription complex is composed of three distinct proteins, ANF-4, C/EBPβ, and a 102-kD protein, and that they play an important role for the hormone regulation of AGP.
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