Dietary ligands diindolylmethane and resveratrol result in diverse ERa signaling not seen after E2 and a subset of diindolylmethane mediated signaling needs concurrent AHR activation. [ChIP-Seq]

Background: Estrogen receptor (ERa) and aryl hydrocarbon receptor (AHR) are two nuclear receptors involved in regulating gene expression. ERa and AHR are regulated by estradiol(E2) and TCDD respectively. They are also regulated by dietary ligands including 3,3´diindolylmethane (DIM) and resveratrol (RES). DIM is an ERa and AHR agonist, while RES is an ERa agonist and AHR antagonist. Few studies have investigated the impact of RES and DIM on ERa and AHR signaling at a genome-wide level. This study assessed ERa and AHR binding and associated gene expression changes after treatment of MCF-7 human breast cancer cells with DIM, RES, E2, TCDD, and E2+TCDD for 1 hour and 6 hours before ChIP and RNA sequencing respectively. Results: 88% and 86% of the ERa bound sites after RES and DIM overlapped with E2 ERa sites. RES and DIM resulted in 577 and 446 differentially expressed genes (DEGs) respectively compared to 866 after E2. 68% and 62.3% of the DEGs after RES and DIM were closest to an ERa binding site. Motif analysis indicated enrichment for AHRE motif among DIM ERa sites but not among E2 or RES ERa sites. Both DIM and E2+TCDD resulted in greater genome wide binding of AHR than TCDD. DIM and E2+TCDD resulted in 10546 and 8904 AHR and ERa co-occupied sites respectively. While co-occupied sites for both enriched for the AHR and ERE motif among others, DIM mediated co-occupied sites enriched for Tcfcp2l1, Tgif1, Gata3 motifs while E2+TCDD mediated ones enriched for the NF1 and Ppara motifs. Overlap of coregulated DEGs after DIM and E2+TCDD indicated 123 were the same among both with 81 and 85 unique coregulated DEGs respectively. Enrichment analysis indicated that more enrichment terms were different than similar for coregulated genes after E2+TCDD and DIM. Conclusions: AHR activation is responsible for DIM mediated reduced regulation of gene expression by ERa relative to E2 and only a subset of the DEG's after DIM and RES are ERa targets indicating future studies into other transcription factors regulated by DIM and RES are needed for insights into the regulation of gene expression by these ligands. Overall design: MCF-7 human breast cancer cells (HTB-22, ATCC) were cultured in low-glucose (1 g/L) DMEM (Dulbecco's modified Eagle's media) supplemented with 1% (v/v) penicillin/streptomycin (P/S) either 10% (v/v) fetal bovine serum (FBS) or 5% dextran coated charcoal (DCC)-treated FBS. They were then treated with activated charcoal and subject to various ligands or DMSO for 1 hour and the ChIP sequencing experiment was done in triplicates.