Characterization of a novel transcription complex required for glucocorticoid regulation of the rat alpha-1-acid glycoprotein gene.
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-1) rat hepatoma cell extracts and affinity-purified proteins. Another is C/EBP beta, which also binds to three elements downstream 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/EBP beta 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 extracts from rat liver and HTC(JZ-1) 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/EBP beta, and a 102-kD protein, and that they play an important role for the hormone regulation of AGP.
Institute of Chemistry, School of Medicine, University of Brescia, Italy.
R
R
Ingrassia
G F
GF
Savoldi
A
A
Caraffini
M
M
Tironi
C
C
Poiesi
P
P
Williams
A
A
Albertini
D
D
Di Lorenzo
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism; D004586:Electrophoresis; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000502:physiology*; D006023:Glycoproteins / Q000235:genetics*; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D014157:Transcription Factors; D014158:Transcription, Genetic
D022762:CCAAT-Enhancer-Binding Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D006023:Glycoproteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D004247:DNA