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.
Bibliographical noteFunding Information:
This work was supported by National Institutes of Health grants R01 CA081243 (to DCS), R01 CA122320 (to RJT), R01 CA106186 (to SDS) and R21 CA104812 (to SDS). The authors gratefully acknowledge use of the Wadsworth Center's Tissue Culture Facility and the Biochemistry and Applied Genomic Technologies Core Facilities. We thank our Wadsworth Center colleagues for the following DNA samples: from PANC-1 and HEK293 cells, Dr. Erasmus Schneider; from A549 cells, Dr. Xinxin Ding; and from NCI-H23 and NCI-H292 lung cancer cell lines, and four leukemia cell lines, Dr. Noel Espina. DNA from A2780 and HOSE-1 cells was a kind gift of Dr. Gareth Owen of the Pontifical Catholic University of Chile (Santiago, Chile). The AHR promoter-luciferase reporter plasmid pGL3-hAhRP was a kind gift from Drs. Sandra Wolff and Josef Abel of the Medical Institute of Environmental Hygiene at the Heinrich-Heine-University, Düsseldorf, Germany.
- (GGGGC) repeat polymorphism
- 2-Amino-1-methyl-6- phenylimidazo[4,5-b]pyridine
- Aryl hydrocarbon receptor
- Long-term estrogen exposure