Received for publication October 23, 2006.
Revised March 8, 2007.
Accepted for publication March 9, 2007.

Identification and functional analysis of a novel human CYP2E1 far upstream enhancer

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 kbp portion of the CYP2E1 promoter was associated with 5- to 10-fold greater luciferase activity. This upstream region contained five direct repeats of 59 bp that increased thymidine kinase-driven luciferase reporter activity in HepG2 cells over 5-fold regardless of orientation. Electrophoretic mobility shift assays (EMSAs) identified sequence-specific nuclear protein binding to the 59 bp repeats that was dependent upon 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 (TRPS1) factor, which also binds a GATA sequence, was eliminated. Rather, competitive EMSA using known binding sequences for the orphan nuclear receptors, steroidogenic factor-1 (SF1 or NR5A1) and fetoprotein transcription factor (FTF or NR5A2) implicated an NR5A member in binding a sequence overlapping the canonical GATA. Chromatin immunoprecipitation (ChIP) assay demonstrated in vivo binding of NR5A2 to the enhancer sequence in human hepatocytes. The enhancer sequence is conserved within the human population, but appears species specific. The identification of this novel enhancer and its putative mechanism adds to the complexities of human CYP2E1 regulation.

Key words: DNA binding sites, Promoter analysis, Cytochrome P450