Abstract
Both transcriptional and post-transcriptional CYP2E1 regulatory mechanisms are known, resulting in 20-fold or greater variation in CYP2E1 expression. To evaluate functional regulatory elements controlling transcription, CYP2E1 promoter constructs were used to make adenovirus vectors containing CYP2E1 promoter-driven luciferase reporters for analyses in both primary human hepatocytes and HepG2 cells. A 1.2-kilobase pair portion of the CYP2E1 promoter was associated with 5- to 10-fold greater luciferase activity. This upstream region contained five direct repeats of 59 base pairs (bp) that increased thymidine kinase-driven luciferase reporter activity in HepG2 cells more than 5-fold, regardless of orientation. Electrophoretic mobility shift assays (EMSAs) identified sequence-specific nuclear protein binding to the 59-bp repeats that was dependent on a 17-bp sequence containing a canonical GATA binding site (WGATAR). Competitive and supershift EMSA identified the participation of GATA4, another GATA family member or GATA-like factor, and a third factor unrelated to the GATA family. Involvement of the tricho-rhino-phalangeal syndrome-1 factor, which also binds a GATA sequence, was eliminated. Rather, competitive EMSA using known binding sequences for the orphan nuclear receptors, steroidogenic factor-1 (or NR5A1), and fetoprotein transcription factor (or NR5A2) implicated an NR5A member in binding a sequence overlapping the canonical GATA. Chromatin immunoprecipitation assay demonstrated in vivo binding of NR5A2 to the enhancer sequence in human hepatocytes. The enhancer sequence is conserved within the human population but seems species-specific. The identification of this novel enhancer and its putative mechanism adds to the complexities of human CYP2E1 regulation.
Footnotes
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This investigation was supported by Public Health Service grant AA11636 and support from the Children's Research Institute, Children's Hospital of Wisconsin.
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Portions of this work were presented at the Federation of American Societies Experimental Biology annual meeting, Washington D.C. (2004), and at the 27th annual meeting of the Research Society on Alcoholism, Vancouver, BC, Canada (2004).
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.031302.
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ABBREVIATIONS: FBS, fetal bovine serum; ChIP, chromatin immunoprecipitation; CT, threshold cycle; EMSA, electrophoretic mobility shift assay; FTF, fetoprotein transcription factor; HNF, hepatocyte nuclear factor; NR5A, nuclear receptor family 5 group A; PSA, prostate-specific antigen; Ptk, promoter of thymidine kinase gene; RT-PCR, real-time polymerase chain reaction; SF1, steroidogenic factor 1; TRPS1, tricho-rhino-phalangeal type I protein; bp, base pair; PCR, polymerase chain reaction; MOI, multiplicity of infection; PBS, phosphate-buffered saline; IP, immunoprecipitate; LRH, liver receptor homolog; ANOVA, analysis of variance.
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↵1 Current affiliation: Department of Internal Medicine, Wayne State University, Detroit, Michigan.
- Received October 6, 2006.
- Accepted March 9, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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