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-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.
- SourceAvailable from: Maike Stam
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- "Multiple 853-bp repeats, ;100 kb upstream, are required for high b1 expression (Stam et al., 2002a). The presence of repeated sequences in enhancers and other regulatory elements appears to influence transcription in other systems as well (Chandler et al., 2002; Greene et al., 2007; Shadley et al., 2007; Romney et al., 2008; Espley et al., 2009). Intriguingly, two of the newly identified b1 regions that seem to have regulatory activity contain repeated sequences as well (Figure 4), in a direct (;43 kb upstream) and an inverted orientation (;15 kb upstream). "
ABSTRACT: This work examines the involvement of chromatin looping in the transcriptional regulation of two epialleles of the maize (Zea mays) b1 gene, B-I and B'. These two epialleles are tissue-specifically regulated and are involved in paramutation. B-I and B' are expressed at high and low levels, respectively. A hepta-repeat approximately 100 kb upstream of the transcription start site (TSS) is required for both paramutation and high b1 expression. Using chromosome conformation capture, we show that the hepta-repeat physically interacts with the TSS region in a tissue- and expression level-specific manner. Multiple repeats are required to stabilize this interaction. High b1 expression is mediated by a multiloop structure; besides the hepta-repeat, other sequence regions physically interact with the TSS as well, and these interactions are epiallele- and expression level-specific. Formaldehyde-assisted isolation of regulatory elements uncovered multiple interacting regions as potentially regulatory.The Plant Cell 04/2009; 21(3):832-42. DOI:10.1105/tpc.108.064329 · 9.58 Impact Factor
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ABSTRACT: Flavin-containing monooxygenase 3 (FMO3) is important for oxidative xenobiotic metabolism, but regulation of the FMO3 gene remains poorly understood. FMO3 is not expressed in HepG2 cells, a commonly employed model for hepatic gene regulation studies. Transcription factor transient expression and treatment with histone deacetylase or DNA methylase inhibitors identified decreased hepatic nuclear factor (HNF) 4alpha levels and DNA hypermethylation as mechanisms suppressing HepG2 FMO3 expression. The absence of major deficiencies in transcriptional machinery suggested that within limits, the HepG2 model is suitable for the study of FMO3 regulation. DNA-protein binding studies with HepG2 cell and hepatic tissue nuclear protein extracts and reporter construct transient expression experiments were performed to characterize FMO3 sequences from position -494 to -439 (domain I), previously demonstrated to significantly impact promoter function. Although both HNF3beta and CCAAT enhancer-binding protein (C/EBP) were observed to specifically interact with this element using HepG2 cell nuclear proteins, only C/EBP DNA-protein interactions were observed using adult liver nuclear proteins. No specific DNA/protein interactions were observed using fetal liver nuclear proteins. Mutation of a putative HNF3beta element had no effect on FMO3 promoter activity, while mutagenesis of a distinct, but overlapping C/EBP element resulted in a 55% reduction in activity. Furthermore, promoter activity was regulated as a function of defined C/EBPbeta liver activating protein:liver inhibitory protein ratios through this same element. Chromatin immunoprecipitation demonstrated C/EBPbeta binding to the FMO3 domain I element in intact cells and adult liver tissue. These results are consistent with C/EBPbeta being important for regulating hepatic FMO3 expression.Biochemical pharmacology 08/2008; 76(2):268-78. DOI:10.1016/j.bcp.2008.05.002 · 4.65 Impact Factor