The role of CCAAT/enhancer-binding protein in the differential transcriptional regulation of a family of human liver alcohol dehydrogenase genes.
The transcription factor CCAAT/enhancer-binding protein (C/EBP) was found to selectively trans-activate one member of the human class I alcohol dehydrogenase (ADH) gene family. A comparison of the promoters for the three human class I ADH genes ADH1, ADH2, and ADH3 indicated a very similar pattern of binding sites (sites A-F) for rat liver nuclear proteins located between -10 and -210 base pairs (bp). In all three promoters site A consisted of two binding sites for the transcription factor C/EBP closely flanking both sides of the TATA box, but C/EBP bound with much greater affinity to site A of ADH2. C/EBP also bound at two locations which coincide with site D (-120 bp) and site E (-160 bp) of all three promoters. Cotransfection studies of human hepatoma cells using ADH-cat fusions and a C/EBP expression plasmid indicated that the human ADH2 promoter responded well to C/EBP trans-activation whereas the human ADH1 and ADH3 promoters, which bind C/EBP weakly, responded poorly. Individual mutations in several ADH2 nuclear factor-binding sites allowed the identification of four functional C/EBP-binding sites, i.e. two in site A as well as one each in sites D and E. Also, the ADH2 TATA box was found to be dispensable for C/EBP induction. Compared to ADH2 and ADH3, site A in ADH1 contains four extra base pairs between the two C/EBP motifs, and deletion of these nucleotides increased the C/EBP responsiveness of ADH1 presumably by changing the spacing of the two C/EBP motifs. Thus, sequence divergence of human class I ADH gene family members has led to forms which vary in their responsiveness to C/EBP. We suggest that C/EBP contributes to liver-specific expression of the human class I ADH gene family by selectively inducing the ADH2 gene via a TATA-independent mechanism during liver development.
Stewart
M J
MJ
Department of Biochemistry, Colorado State University, Fort Collins 80523.
Shean
M L
ML
Paeper
B W
BW
Duester
G
G
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000502:physiology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D000426:Alcohol Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase