The first 22 base pairs of the proximal promoter of the rat class I alcohol dehydrogenase gene is bipartite and interacts with multiple DNA-binding proteins

Abstract

The rat class I alcohol dehydrogenase (ADH) gene is primarily expressed in the liver. We previously showed that the liver-enriched transcription factor, the CCAAT/ enhancer binding protein (C/EBP), binds to the proximal promoter of the rat class I ADH gene between positions −11 and −22 relative to the start site of transcription. We now demonstrate that another transcription factor, the liver activator protein (LAP), also interacts with the same region of the promoter based on the following observations: (1) LAP synthesized by in vitro transcription and translation of cloned cDNA sequence forms complexes with an oligonucleotide containing the C/EBP-binding sequence within the ADH promoter as determined by the electrophoretic mobility shift assay (EMSA), (2) purified LAP interacts with the proximal ADH promoter when analyzed by the DNase I protection assay, and (3) an ADH promoter-reporter gene construct containing the C/EBP-binding site is transactivated by an eukaryotic expression vector containing the LAP sequence. EMSA of an oligonucleotide containing the first 22 base pairs (between positions −1 and −22) of the ADH promoter with rat liver nuclear extracts (RLNE) resulted in the formation of two major complexes. Complex 1 was competed away by a heterologous oligonucleotide containing a C/EBP-binding site within the promoter of the adipocyte 422 (aP2) gene, while complex 2 was not. Additional competition experiments with the ADH or 422 (aP2) oligonucleotide using either RLNE or extracts from 3T3-L1 adipocytes demonstrated that complex 1 contains either C/EBP or LAP, while complex 2 contains a DNA-binding protein that binds to a novel sequence 5′-TGGCCCAGTT-3′ between positions −1 and −10 of the ADH promoter. Ultraviolet cross-linking between RLNE and a labeled oligonucleotide containing the above sequence indicates that this protein, designated EDBP (for enhancer-site downstream binding protein), has an estimated molecular weight of 47 kDa, which is larger than that reported for either C/EBP (42 kDa) or LAP (36 kDa).