Transcriptional regulation of the human CYP1B1 gene - Evidence for involvement of an aryl hydrocarbon receptor response element in constitutive expression

The cytochrome P450 1B1 gene (CYP1B1) is expressed constitutively and is in ducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the human breast a denocarcinoma cell line MCF-7 but not in the human hepatoma cell line HepG2 , Genomic DNA isolated from both cell lines was digested with the methylati on-sensitive restriction enzyme isoschizomers MspI and HpaII, and subjected to Southern analysis with a probe for the CYP1B1 promoter/enhancer region. Although differences were observed in methylation patterns for the CYP1B1 gene from MCF-7 and HepG2 cells, treatment with the demethylating agent 5-a zacytidine (10 mu M for 6 days) did not activate CYP1B1 mRNA expression in HepG2 cells. Furthermore, treatment with the histone deacetylase inhibitor trichostatin A (100 nM for 24 h) did not activate CYP1B1 mRNA expression in HepG2 cells. Comparative analysis of the constitutive expression of lucife rase/1B1 reporter constructs containing a series of deletions in the 5' enh ancer region indicated that in MCF-7 cells the region from -987 to -732 (re lative to the transcription start site) was necessary for maximal levels of activity. Mutation of the aryl hydrocarbon receptor response elements (dio xin response elements) in this region showed that the dioxin response eleme nts located at -833 is essential for constitutive gene expression in MCF-7 cells. In HepG2 cells, reporter gene activity was at least equal or greater than the activity observed in MCF-7 cells, which is in marked contrast to the expression of the native CYYP1B1 gene. Taken together these findings in dicate that the observed cell-specific differences in CYP1B1 constitutive e xpression are not mediated by DNA promoter/enhancer methylation, but are li kely due to either 1) inaccessibility of the 5'-enhancer region in HepG2 ce lls to transcriptional activators due to a higher order chromatin structure that does not involve histone acetylation, or 2) the action of a repressor protein at cis-elements located outside of the -2296 to +25 region examine d with the CYP1B1 reporter constructs. Furthermore, at least one of the dio xin response elements in the enhancer region is required for constitutive e xpression of CYP1B1.