The upstream stimulatory factor binds to and activates the promoter of the rat class I alcohol dehydrogenase gene.
The gene encoding rat class I alcohol dehydrogenase (ADH) is expressed primarily in the liver. Recent studies in our laboratories indicate that multiple cellular factors present in the rat liver interact with various regions of the promoter of this gene. One of the regions contains the sequence 5'-CACATG-3' that has an "E box" homology to which a number of transcription factors containing the basic helix-loop-helix motif bind. We now demonstrate that the human transcription factor, upstream stimulatory factor (USF), a basic helix-loop-helix-containing protein, binds to and activates the promoter of the rat class I ADH gene. Electrophoretic mobility shift assays of labeled oligonucleotide containing the 5'-CACATG-3' sequence within the ADH promoter revealed the formation of multiple DNA-protein complexes when nuclear extracts obtained from adult rat liver were used. The binding of proteins to the DNA could be competed away with an oligonucleotide specifying a sequence within the adenovirus major late promoter (MLP) that had previously been shown to bind USF. Similar complexes were observed when electrophoretic mobility shift assays of labeled MLP oligonucleotide were performed with rat liver nuclear extracts. Conversely, nuclear extracts isolated from HeLa cells, cells known to have abundant USF, contain factors that interact with the sequence present in the ADH promoter. This interaction could be competed efficiently by the MLP oligonucleotide. USF synthesized in an in vitro transcription and translation system also binds to the ADH promoter as well as to the MLP. In addition, antiserum directed against USF recognizes factors present in the rat liver nuclear extracts that interact with the ADH promoter. Furthermore, transcription directed from both the ADH and the adenovirus major late promoters was inhibited by an oligonucleotide representing the USF-binding site within the ADH promoter in a cell-free in vitro transcription system. Lastly, an ADH promoter-reporter gene construct was transactivated by an eukaryotic expression vector containing USF in HepG2 cells co-transfected with the two constructs. These experiments demonstrate that USF is present in the rat liver and that it binds to and activates the promoter of the rat class I ADH gene in a sequence-specific manner.
Potter
J J
JJ
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Cheneval
D
D
Dang
C V
CV
Resar
L M
LM
Mezey
E
E
Yang
V W
VW
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA; D004268:DNA-Binding Proteins; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; D004247:DNA; D000426:Alcohol Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase