TY - JOUR
T1 - 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
AU - Potter, James J.
AU - Mezey, Esteban
AU - Cornelius, Peter
AU - Crabb, David W.
AU - Yang, Vincent W.
N1 - Funding Information:
We thank Drs. Steven McKnight, Robert Christy, and Mario Chojkier for their generous supply of plasmids and/or proteins. We also thank Ms. Ann Sheppe for her excellent assistance in preparing the manuscript. This work was supported by U.S. Public Health Service Grants AA00626 (to E.M.) and AA06434 (to D.W.C.) from the National Institute on Alcohol Abuse and Alcoholism and the Alcoholic Beverage Medical Research Foundation (to V.W.Y. and J.J.P.). P.C. was the recipient of a National Research Service Award from the National Institutes of Health. V.W.Y. is an American Gastroenterological Association/Sandoz Research Scholar.
PY - 1992/6
Y1 - 1992/6
N2 - 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).
AB - 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).
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U2 - 10.1016/0003-9861(92)90529-6
DO - 10.1016/0003-9861(92)90529-6
M3 - Article
C2 - 1586166
AN - SCOPUS:0026650358
SN - 0003-9861
VL - 295
SP - 360
EP - 368
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -