Dioxin Increases the Interaction Between Aryl Hydrocarbon Receptor and Estrogen Receptor Alpha at Human Promoters

Abstract

Recent studies have shown that activated aryl hydrocarbon receptor (AHR) induced the recruitment of estrogen receptor-alpha (ERalpha) to AHR-regulated genes and that AHR is recruited to ERalpha-regulated genes. However, these findings were limited to a small number of well-characterized AHR- or ERalpha-responsive genes with little knowledge of what was occurring at other genomic regions. In this study, we showed using chromatin immunoprecipitation followed by hybridization to promoter focused microarrays (ChIP-chip) that 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment significantly increased the overlap of genomic regions bound by both AHR and ERalpha. Conventional and sequential ChIPs confirmed the recruitment of AHR and ERalpha to many of the identified regions. Transcription factor binding site analysis revealed an overrepresentation of aryl hydrocarbon receptor response elements in regions bound by both AHR and ERalpha, suggesting that AHR was the important factor determining the recruitment of ERalpha to these regions. RNA interference-mediated knockdown of AHR confirmed its requirement for the recruitment of ERalpha to some, but not all, of the shared regions. Our findings demonstrate not only that dioxin induces the recruitment of ERalpha to AHR target genes but also that AHR is recruited to estrogen-responsive regions in a gene-specific manner, suggesting that AHR utilizes both of these mechanisms to modulate estrogen-dependent signaling.

FOXA1 Is Essential for Aryl Hydrocarbon Receptor–Dependent Regulation of Cyclin G2

“…Wellstudied AHR target genes include the phase I and II drug-metabolizing enzymes, such as cytochrome P450 1A1 (CYP1A1), CYP1B1, and glutathione S-transferases. Chromatin immunoprecipitation combined with microarrays (ChIP-chip) and gene expression microarray experiments revealed that AHR regulates numerous genes involved in diverse cellular pathways, including metabolism and cellcycle regulation (8)(9)(10). In support of these findings, previous studies have shown that TCDD inhibits cell proliferation (11)(12)(13).…”

“…After 72 hours, cells were treated with DMSO (final concentration, 0.1%), 10 nmol/L TCDD, 10 nmol/L E2, or E2 þ TCDD (10 nmol/L) for 0.75 hours. ChIP and sequential ChIP assays were conducted as previously described (8). Enrichment levels (relative to 100% total input) were determined by quantitative real-time PCR (qPCR) using the following primers: CCNG2 AHRE2 (enhancer): 5 0 -TGGGTTACCAAGGACCAA-GAA-3 0 5 0 -CCAGAGGTTGTAGTGCTGTTGTTT-3 0 ; CCNG2 AHRE1: 5 0 -AACTCTCCCGTGGCTGAAAA and 5 0 -CGCGGCGCTTCTCCTAA-3 0 CCNG2 TATA: 5 0 -GGGAGGCCGCGAGAGA-3 0 and 5 0 -TCCTCCACG-GACTTTAAAAAAGAG-3 0 .…”

“…Cells were treated with either 10 nmol/L TCDD or pretreatment for 1 hour with 1 mmol/L CH223191 for 0.75, 1.5, 3, 6, or 24 hours. RNA was isolated using illustra RNAspin mini columns (GE Healthcare) and reverse-transcribed as previously described (8). All target gene transcripts were normalized to ribosomal 18S RNA and fold inductions were calculated using timematched DMSO-treated samples and the comparative C T (DDC T ) method was used for data analysis.…”

“…All target gene transcripts were normalized to ribosomal 18S RNA and fold inductions were calculated using timematched DMSO-treated samples and the comparative C T (DDC T ) method was used for data analysis. qPCR primers used in the study have been described elsewhere (8) with exception of the following primers: FOXA1 mRNA 5 0 -GAAGATGGAAGGGCATGAAA-3 0 and 5 0 -GCCTGA-GTTCATGTTGCTGA-3 0 .…”

“…Our previous ChIP-chip studies identified an AHR-bound AHRE-containing sequence in the upstream regulatory region of CCNG2. In contrast to estrogen, the AHR agonists, TCDD and 3MC, induced AHR recruitment to CCNG2 causing an increase in CCNG2 mRNA levels (8,9). However, the molecular mechanisms and characterization of the AHR-dependent regulation of CCNG2 remain elusive.…”