β-naphthoflavone-induced upregulation of CYP1B1 expression is mediated by the preferential binding of aryl hydrocarbon receptor to unmethylated xenobiotic responsive elements

Abstract

Human cytochrome P450 1 (CYP1) enzymes are transcriptionally induced by specific xenobiotics through a mechanism that involves the binding of aryl hydrocarbon receptors (AhR) to target xenobiotic responsive element (XRE) sequences. To examine the effect of DNA methylation on the AhR-mediated pathway, reverse transcription-quantitative PCR analysis was performed. β-naphthoflavone (βNF)-induced CYP1B1 expression was found to be potentiated by pre-treatment of human HepG2 liver cancer cells with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, but not HuH7 cells. It was hypothesized that this increase is mediated by the demethylation of CpG sites within XRE2/XRE3 sequences, suggesting that methylation of these sequences inhibits gene expression by interfering with the binding of AhR to the target sequences. To test this hypothesis, a novel method combining the modified chromatin immunoprecipitation of AhR-XRE complexes with subsequent DNA methylation analysis of the XRE regions targeted by activated AhR was applied to both liver cancer cell lines treated with βNF. XRE2/XRE3 methylation was found to be exclusively observed in the input DNA from HepG2 cells but not in the precipitated AhR-bound DNA. Furthermore, sub-cloning and sequencing analysis revealed that the two XRE sites were unmethylated in the samples from the AhR-bound DNA even though the neighboring CpG sites were frequently methylated. To the best of our knowledge, the present study provides the first direct evidence that ligand-activated AhR preferentially binds to unmethylated XRE sequences in the context of natural chromatin. In addition, this approach can also be applied to assess the effects of DNA methylation on target sequence binding by transcription factors other than AhR.

Keywords: DNA methylation; aryl hydrocarbon receptor; cytochrome P450 family 1 subfamily B member 1; xenobiotic responsive element; β-naphthoflavone.

Figure 1

Basal and induced mRNA expression levels of CYP1B1, AhR and Arnt as determined using quantitative reverse transcription-quantitative PCR. (A-F) HepG2 and HuH7 cells were treated with either PBS, DMSO, βNF, DAC alone or D+β. CYPB1 expression in (A) HepG2 and (B) HuH7 cells. AhR expresion in (C) HepG2 and (D) HuH7 cells. Arnt expression in (E) HepG2 and (F) HuH7 cells. The vertical axes indicate relative transcription levels normalized to those of ACTB (plotted as a ratio to levels in PBS-treated cells). Transcript levels are expressed as means ± SD. Statistically significant differences between the two groups are depicted as ^*P<0.05. ACTB, β-actin; XRE, xenobiotic response element; AHR, aryl hydrocarbon receptor; Arnt, AhR nuclear translocator; CYP1B1, cytochrome P450 family 1 subfamily B member 1; βNF, β-naphthoflavone; DAC, 5-aza-2'-deoxycytidine; D + β, DAC + βNF.

Figure 2

DNA methylation status of the eight XRE sites detected in the two liver cancer cell lines. XRE sites and their proposed nucleotide positions in the CYP1B1 gene as depicted in the map. DNA methylation status of each XRE site is presented as one of the following four categories: Fully methylated; highly methylated; lowly methylated; and rarely methylated (mostly unmethylated). XRE, xenobiotic response element; CYP1B1, cytochrome P450 family 1 subfamily B member 1.

Figure 3

DNA methylation status of the XRE2/XRE3 sequences as examined using the COBRA method in the (A) HepG2 and (B) HuH7 liver cancer cell lines. Methylated XRE2/XRE3 fragments were digested using the restriction endonuclease HpyCH4IV, whereas unmethylated XRE2/XRE3 fragments were not. IP(-) indicates the result of input DNA obtained from HepG2 or HuH7 cells after treatment with βNF alone and/or DAC. IP(+) indicates the results of precipitated AhR-bound XRE2/XRE3 sequences obtained from HepG2 or HuH7 cells after the aforementioned treatments. XRE, xenobiotic response element; COBRA, combined bisulfite restriction analysis; IP, immunoprecipitation; U, unmethylated; M, methylated; CYP1B1, cytochrome P450 family 1 subfamily B member 1; βNF, β-naphthoflavone; DAC, 5-aza-2'-deoxycytidine.

Figure 4

DNA methylation status around the XRE2/XRE3 sequences as determined by sequencing the individual copies of the gene in the two liver cancer cell lines. (A) Nucleotide sequence of XRE2/XRE3 in the CYP1B1 enhancer region (DDBJ/EMBL/GenBank, NG_0088386). The numbers on the left of each line represent the distance from the putative transcription start site (bp). The amplified region and individual CpG sites are indicated by underlined text and bold text, respectively. (B) Sequencing electropherograms from representative clones comprising either methylated or unmethylated XRE2/XRE3 sequences. Arrows indicate CpG dinucleotides. (C) Methylation profiles around the XRE2/XRE3 sequences in HepG2 and HuH7 cells after treatment with βNF. Each line represents a DNA sequence of an individual clone. IP(-) indicates the methylation status of individual clones derived from input DNA. IP(+) shows the methylation status of clones from precipitated AhR-bound DNA. CpG dinucleotides in the XRE2/XRE3 region are depicted as circles horizontally aligned in the 5'-3' direction. White and black circles represent unmethylated and methylated CpG sites, respectively. Shaded circles represent the CpG sites where the methylation status could not be determined in this study. Two CpG dinucleotides within the XRE2 and XRE3 sequences are enclosed by square boxes. XRE, xenobiotic response element; CYP1B1, cytochrome P450 family 1 subfamily B member 1; IP, immunoprecipitate; βNF, β-naphthoflavone.