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

Abstract

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.

Analysis of the AHR gene proximal promoter GGGGC-repeat polymorphism in lung, breast, and colon cancer

“…Although transcription of heterologous luciferase constructs that are composed of “naked” DNA differs from that of gDNA associated with proteins and with possible epigenetic changes, these results suggest that in vivo transcription of the AHR allele with only 2 polymorphic repeats may be reduced. Indeed, we found in our previous study (Englert et al, 2012) that oligonucleotides containing the polymorphic repeats formed G-quadruplexes as measured by circular dichroism, and those with higher numbers of repeats displayed increased peak intensities. These spectra also suggested that the G-quadruplex formed with oligonucleotides containing the 2-repeat was of a qualitatively different conformation from the G-quadruplexes formed with oligonucleotides containing higher numbers of repeats.…”

“…Our data indicate that the most prominent allele is (GGGGC) 4, followed in incidence by (GGGGC) 5 ; (GGGGC) 2 and (GGGGC) 6 were found to be rare alleles, and the (GGGGC) 1 and (GGGGC) 3 alleles were not observed. In our previous study of New York State neonates (Englert et al, 2012), we found that the individuals of Asian descent also showed (GGGGC) 4 as the most prominent allele, followed in incidence by (GGGGC) 5 . The reasons for the discrepancies among our results and those of Fukushima-Uesaka et al (2004) and Li et al (2013) are not apparent.…”

“…The resulting construct, referred to as AhRΔ(-120) 4 , in which the subscript refers to the number of GGGGC-repeats, encompasses 120 bp of the proximal AHR promoter region and 159 bp of the region corresponding to the mRNA. To prepare a construct containing (GGGGC) 2 , two repeats, or 10 bp, were removed from the AhRΔ(-120) 4 construct using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, Agilent Technologies, Santa Clara, CA) as described (Englert et al, 2012), and is referred to as AhRΔ(-120) 2 . This strategy was also used to prepare a construct containing the single nucleotide polymorphism (SNP) C/A: (GGGGC) 2 GGGG A GGGGC, in the AhRΔ(-120) 4 construct, which is referred to as AhRΔ(-120) C/A .…”

“…Surplus DNA derived from bloodspots of New York State neonates was obtained from the Newborn Screening Program of New York State Department of Health and represents an expanded data set from our previous study (Englert et al, 2012). Frozen non-tumor lung tissue from lung cancer patients and formalin-fixed, paraffin-embedded (FFPE) tumor tissue from breast and colon cancer patients were obtained from the National Cancer Institute’s Cooperative Human Tissue Network (CHTN).…”

“…Based on our previous studies (Englert et al, 2012), we contend that one of the most important genetic polymorphisms of the human AHR gene is the (GGGGC) n repeat (with n = 2, 4, 5 or 6) in the proximal promoter region, which has essentially been overlooked for technical reasons. The TATA-less promoter of the human AHR gene is dependent on specificity protein (Sp) transcription factors for promoter activation.…”