Promoters for the human alcohol dehydrogenase genes ADH1, ADH2, and ADH3: interaction of CCAAT/enhancer-binding protein with elements flanking the ADH2 TATA box.
The human ADH1, ADH2, and ADH3 genes are closely related members of a gene family which are differentially expressed during liver development. To begin examining the mechanism of this tissue-specific and stage-specific expression, the 5'-flanking nucleotide (nt) sequences of the three genes were determined and the transcription start point (tsp) were identified. Sequences of all three genes indicated a high degree of homology (greater than 80% nt sequence identity) from the AUG translation start codon to about nt -780 relative to the tsp. Transient transfection assays of a set of plasmids containing various lengths of ADH 5'-flanking DNA fused to cat were performed in the HepG2 and Hep3B human hepatoma cell lines. The results indicated that the ADH2 promoter-proximal region was transcriptionally active in the absence of upstream sequences. To identify potential cis-acting elements in the ADH2 promoter-proximal region, a DNase I footprinting assay using a rat liver nuclear extract was used. Protection occurred in several locations including one, between nt -51 and -10, which shares homology with known binding sites for a previously identified rat-liver transcription factor called CCAAT/enhancer binding protein (C/EBP). Purified C/EBP was shown by footprint analysis to bind at two distinct sites in the ADH2 promoter located at nt -51 to -31 and -21 to -10. The TATA-box promoter element at nt -30 to -22 was not protected by C/EBP, but was partially protected by a factor in the rat liver nuclear extract. Thus, it is possible that the flanking C/EBP molecules may create a novel binding pocket for TFIID, the TATA-binding general transcription factor for RNA polymerase II. Alternatively, the C/EBP molecules may block access to the TATA box, and stimulate transcription of ADH2 by interacting with some component(s) other than TFIID.
Stewart
M J
MJ
Department of Biochemistry, Colorado State University, Fort Collins 80523.
McBride
M S
MS
Winter
L A
LA
Duester
G
G
D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D003850:Deoxyribonuclease I; D006801:Humans; D008113:Liver Neoplasms; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D000426:Alcohol Dehydrogenase; D003850:Deoxyribonuclease I