β‑naphthoflavone‑induced upregulation of <em>CYP1B1</em> expression is mediated by the preferential binding of aryl hydrocarbon receptor to unmethylated xenobiotic responsive elements

Miura,Toshitaka; Onodera,Ryo; Terashima,Jun; Ozawa,Shogo; Habano,Wataru

doi:10.3892/etm.2021.10846

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

Authors:
- Toshitaka Miura
- Ryo Onodera
- Jun Terashima
- Shogo Ozawa
- Wataru Habano
View Affiliations

Affiliations: Division of Pharmacodynamics and Molecular Genetics, Department of Clinical Pharmaceutical Sciences, School of Pharmacy, Iwate Medical University, Shiwa, Iwate 028‑3694, Japan
Published online on: October 6, 2021 https://doi.org/10.3892/etm.2021.10846
Article Number: 1410
Copyright: © Miura et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

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.

View Figures

View References

1	Zanger UM, Turpeinen M, Klein K and Schwab M: Functional pharmacogenetics/genomics of human cytochromes P450 involved in drug biotransformation. Anal Bioanal Chem. 392:1093–1108. 2008.PubMed/NCBI View Article : Google Scholar
2	Pelkonen O, Turpeinen M, Hakkola J, Honkakoski P, Hukkanen J and Raunio H: Inhibition and induction of human cytochrome P450 enzymes: Current status. Arch Toxicol. 82:667–715. 2008.PubMed/NCBI View Article : Google Scholar
3	Wolf CR, Mahmood A, Henderson CJ, McLeod R, Manson MM, Neal GE and Hayes JD: Modulation of the cytochrome P450 system as a mechanism of chemoprotection. IARC Sci Publ. 139:165–173. 1996.PubMed/NCBI
4	Kress S and Greenlee WF: Cell-specific regulation of human CYP1A1 and CYP1B1 genes. Cancer Res. 57:1264–1269. 1997.PubMed/NCBI
5	Krusekopf S, Roots I, Hildebrandt AG and Kleeberg U: Time-dependent transcriptional induction of CYP1A1, CYP1A2 and CYP1B1 mRNAs by H⁺/K⁺ -ATPase inhibitors and other xenobiotics. Xenobiotica. 33:107–118. 2003.PubMed/NCBI View Article : Google Scholar
6	Fujii-Kuriyama Y and Mimura J: Molecular mechanisms of AhR functions in the regulation of cytochrome P450 genes. Biochem Biophys Res Commun. 338:311–317. 2005.PubMed/NCBI View Article : Google Scholar
7	Lund AH and van Lohuizen M: Epigenetics and cancer. Genes Dev. 18:2315–2335. 2004.PubMed/NCBI View Article : Google Scholar
8	University of California: Santa Cruz Genomics Institute. UCSC Genome Browser. Available from: http://genome.ucsc.edu/index.html.
9	Ivanov M, Kacevska M and Ingelman-Sundberg M: Epigenomics and interindividual differences in drug response. Clin Pharmacol Ther. 92:727–736. 2012.PubMed/NCBI View Article : Google Scholar
10	Fisel P, Schaeffeler E and Schwab M: DNA methylation of ADME genes. Clin Pharmacol Ther. 99:512–527. 2016.PubMed/NCBI View Article : Google Scholar
11	Beedanagari SR, Taylor RT, Bui P, Wang F, Nickerson DW and Hankinson O: Role of epigenetic mechanisms in differential regulation of the dioxin-inducible human CYP1A1 and CYP1B1 genes. Mol Pharmacol. 78:608–616. 2010.PubMed/NCBI View Article : Google Scholar
12	Nakajima M, Iwanari M and Yokoi T: Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells. Toxicol Lett. 144:247–256. 2003.PubMed/NCBI View Article : Google Scholar
13	Yao EF and Denison MS: DNA sequence determinants for binding of transformed Ah receptor to a dioxin-responsive enhancer. Biochemistry. 31:5060–5067. 1992.PubMed/NCBI View Article : Google Scholar
14	Swanson HI, Tullis K and Denison MS: Binding of transformed Ah receptor complex to a dioxin responsive transcriptional enhancer: Evidence for two distinct heteromeric DNA-binding forms. Biochemistry. 32:12841–12849. 1993.PubMed/NCBI View Article : Google Scholar
15	Skene PJ and Henikoff S: An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife. 6(e21856)2017.PubMed/NCBI View Article : Google Scholar
16	Nishimura M, Ueda N and Naito S: Effects of dimethyl sulfoxide on the gene induction of cytochrome P450 isoforms, UGT-dependent glucuronosyl transferase isoforms, and ABCB1 in primary culture of human hepatocytes. Biol Pharm Bull. 26:1052–1056. 2003.PubMed/NCBI View Article : Google Scholar
17	Habano W, Gamo T, Sugai T, Otsuka K, Wakabayashi G and Ozawa S: CYP1B1, but not CYP1A1, is downregulated by promoter methylation in colorectal cancers. Int J Oncol. 34:1085–1091. 2009.PubMed/NCBI View Article : Google Scholar
18	Tsuchiya Y, Nakajima M and Yokoi T: Critical enhancer region to which AhR/ARNT and Sp1 bind in the human CYP1B1 gene. J Biochem. 133:583–592. 2003.PubMed/NCBI View Article : Google Scholar
19	Sambrook J and Russell DW: Isolation of high-molecular-weight DNA from mammalian cells using proteinase K and phenol. In: Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor Laboratory Press, New York, NY, pp6.4-6.12, 2001.
20	Xiong Z and Laird PW: COBRA: A sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 25:2532–2534. 1997.PubMed/NCBI View Article : Google Scholar
21	Warnecke PM, Stirzaker C, Song J, Grunau C, Melki JR and Clark SJ: Identification and resolution of artifacts in bisulfite sequencing. Methods. 27:101–107. 2002.PubMed/NCBI View Article : Google Scholar
22	Habano W, Gamo T, Terashima J, Sugai T, Otsuka K, Wakabayashi G and Ozawa S: Involvement of promoter methylation in the regulation of Pregnane X receptor in colon cancer cells. BMC Cancer. 11(81)2011.PubMed/NCBI View Article : Google Scholar
23	Shehin SE, Stephenson RO and Greenlee WF: Transcriptional regulation of the human CYP1B1 gene. Evidence for involvement of an aryl hydrocarbon receptor response element in constitutive expression. J Biol Chem. 275:6770–6776. 2000.PubMed/NCBI View Article : Google Scholar
24	Tang YM, Wo YY, Stewart J, Hawkins AL, Griffin CA, Sutter TR and Greenlee WF: Isolation and characterization of the human cytochrome P450 CYP1B1 gene. J Biol Chem. 271:28324–28330. 1996.PubMed/NCBI View Article : Google Scholar
25	Shen ES and Whitlock JP Jr: The potential role of DNA methylation in the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Biol Chem. 264:17754–17758. 1989.PubMed/NCBI
26	Takahashi Y, Suzuki C and Kamataki T: Silencing of CYP1A1 expression in rabbits by DNA methylation. Biochem Biophys Res Commun. 247:383–386. 1998.PubMed/NCBI View Article : Google Scholar
27	Wolffe AP: Transcriptional control: Imprinting insulation. Curr Biol. 10:R463–R465. 2000.PubMed/NCBI View Article : Google Scholar
28	Siegfried Z and Cedar H: DNA methylation: A molecular lock. Curr Biol. 7:R305–R307. 1997.PubMed/NCBI View Article : Google Scholar
29	Chang H, Chang LW, Cheng YH, Tsai WT, Tsai MX and Lin P: Preferential induction of CYP1A1 and CYP1B1 in CCSP-positive cells. Toxicol Sci. 89:205–213. 2006.PubMed/NCBI View Article : Google Scholar