A hormone response element upstream from the human alcohol dehydrogenase gene ADH2 consists of three tandem glucocorticoid receptor binding sites.
The 5'-flanking region of the human gene encoding beta-alcohol dehydrogenase (ADH2) was shown by DNase I footprinting to contain three tandem binding sites for purified glucocorticoid receptor. The three binding sites lie very close together between nucleotide (nt) positions -245 and -171 with respect to the transcription start point. DNase I footprinting using a rat liver nuclear extract indicated a lack of protection of the glucocorticoid receptor binding sites, but protection of a sequence between nt -209 and -191 which partially overlaps the glucocorticoid receptor binding sites I and II. This site has homology with the known binding site for hepatocyte nuclear factor 1 (HNF1). ADH2 promoter DNA fragments containing various lengths of 5'-flanking sequences were fused upstream from the gene encoding chloramphenicol acetyltransferase (cat) and transfected into the HepG2 human hepatoma cell line. The resulting cat expression was subject to induction by dexamethasone in constructions containing ADH2 DNA between nt -272 and -171. This indicates that the glucocorticoid receptor binding sites identified by footprint analysis function as a glucocorticoid response element (GRE) in a liver cell line. Heterologous ADH-cat fusions, in which the ADH2-GRE was fused to the adenovirus major late promoter, exhibited glucocorticoid induction of cat expression in CV-1B cells when cotransfected with a glucocorticoid receptor expression vector. Glucocorticoid regulation in CV-1B was observed when either all three glucocorticoid receptor binding sites (sites 0, I, II) or the two distal sites (sites 0, I) were present. Overall, these results indicate that the ADH2 gene possesses a functional GRE which can potentially regulate expression transcriptionally.(ABSTRACT TRUNCATED AT 250 WORDS)
Winter
L A
LA
Department of Biochemistry, Colorado State University, Fort Collins 80523.
Stewart
M J
MJ
Shean
M L
ML
Dong
Y
Y
Poellinger
L
L
Okret
S
S
Gustafsson
J A
JA
Duester
G
G
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003907:Dexamethasone / Q000494:pharmacology; D004742:Enhancer Elements, Genetic; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D000426:Alcohol Dehydrogenase