Identification and Functional Analysis of a Novel HumanCYP2E1Far 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-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 sequencespecific 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.CYP2E1 is highly expressed in liver, in which it metabolizes a large number of small molecular weight endogenous or exogenous compounds (Lieber, 1999). CYP2E1 converts some chemicals to a more toxic form; for example, it is the major enzyme responsible for the oxidation of acetaminophen to the reactive quinone, NAPQI. The more than 20-fold interindividual variation in human CYP2E1 activity has been attributed to diverse regulatory mechanisms acting at both transcriptional and post-transcriptional levels (Lieber, 1999). CYP2E1 genetic variants have been found within the structural gene and upstream sequences; however, the majority have not demonstrated an in vivo functional impact (Carrière et al., 1996). An exception is a polymorphism in the CYP2E1 promoter CYP2E1*1D, which has been associated with increased in vivo metabolic activity with ethanol intake and obesity (McCarver et al., 1998). In part because of the emphasis on post-transcriptional regulation, knowledge regarding mechanisms responsible for the control of CYP2E1 transcription has lagged. We report here the use of adenovirus representing the CYP2E1*1D allele and the reference sequence CYP2E1*1C to identify a novel enhancer in the far CYP2E1 upstream region (CYP2E1*1C position Ϫ3690 to Ϫ...

The transcription factor GATA-4 regulates cytochrome P4502C19 gene expression

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Tissue- and Expression Level–Specific Chromatin Looping at Maizeb1Epialleles

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