Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: Role of the proximal promoter GC-rich region

• Published in: Biochemical pharmacology Vol. 84; no. 5; pp. 722 - 735
• Main Authors: Englert, Neal A., Turesky, Robert J., Han, Weiguo, Bessette, Erin E., Spivack, Simon D., Caggana, Michele, Spink, David C., Spink, Barbara C.
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01-09-2012
• Subjects: (GGGGC)n repeat polymorphism; 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; AhR gene; Aryl hydrocarbon receptor; Aryl hydrocarbon receptors; Breast cancer; breast neoplasms; C.D; Carcinogenesis; Carcinogens; Cell Line, Tumor; Chromatin; Chromatin Immunoprecipitation; Chromatography, Liquid; Circular Dichroism; circular dichroism spectroscopy; cytochrome P-450; Cytochrome P-450 CYP1A1 - metabolism; Cytochrome P450; DNA adducts; DNA methylation; electrophoresis; Electrophoretic mobility; Electrophoretic Mobility Shift Assay; Epigenesis, Genetic; Epigenetic; epigenetics; Estrogens; GC Rich Sequence; gene expression; Gene Expression Regulation, Neoplastic; Gene frequency; Guanine-quadruplex; Histones; Humans; Immunoprecipitation; Long-term estrogen exposure; luciferase; Lysine; Metabolism; Methylation; mRNA stability; Mutagenesis, Site-Directed; Neonates; pharmacology; Population genetics; precipitin tests; promoter regions; Promoter Regions, Genetic; Promoters; pyridines; Real-Time Polymerase Chain Reaction; Receptors, Aryl Hydrocarbon - genetics; RNA; RNA processing; Sp Transcription Factors - metabolism; Spectrometry, Mass, Electrospray Ionization; Spectroscopy; Transcription factors; New York; TCDD; Long-term estrogen exposure; LTEE; RNA Pol II; Epigenetic; ChIP; E2; BaP; SE; Guanine-quadruplex; hnRNA; LC-ESI/MS3; EMSA; ODN; PhIP; CYP; CD; EROD; 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; H3K4me2; AIP; AhR; KLF4; ER; Aryl hydrocarbon receptor; TSS; PAH; (GGGGC)n repeat polymorphism; PGR; dG-C8-PhIP; Sp; PCR; H3K27me3
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2012.06.013

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)n repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)n was n=4>5≫6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.