Differential regulation of human hepatic flavin containing monooxygenase 3 (FMO3) by CCAAT/enhancer-binding protein beta (C/EBPbeta) liver inhibitory and liver activating proteins.

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.