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. [RNA-Seq]

Abstract: Inhibitory crosstalk between estrogen receptor alpha (ERalpha) and aryl hydrocarbon receptor (AHR) regulates 17ß-estradiol (E2)-dependent breast cancer cell signalling. ERalpha and AHR are transcription factors activated by E2 and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) respectively. Dietary ligands resveratrol (RES) and 3,3´diindolylmethane (DIM) also activate ERalpha while only DIM activates AHR and RES represses it. DIM and RES are reported to have anti-cancer and anti-inflammatory properties. Studies with genome wide targets and AHR and ERalpha regulated genes after DIM and RES are unknown. We used chromatin immunoprecipitation with high-throughput sequencing and transcriptomics to study ERalpha as well as AHR coregulation in MCF-7 human breast cancer cells treated with DIM, RES, E2 or TCDD alone or E2+TCDD for 1 and 6 hours respectively. ERalpha bound sites after DIM enriched for the AHR motif but not after E2 or RES while AHR bound sites after DIM and E2+TCDD enriched for the ERE motif but not after TCDD. More than 90% of the differentially expressed genes closest to an AHR binding site after DIM or E2+TCDD also had an ERalpha site and 60% of coregulated genes between DIM and E2+TCDD were common. Collectively our data shows that RES and DIM differentially regulate multiple transcriptomic targets via ERalpha and ERalpha/AHR coactivity respectively, which need to be considered to properly interpret their cellular and biological responses. These novel data also suggest that when both receptors are activated, ERalpha dominates with preferential recruitment of AHR to ERalpha target genes. Overall design: The MCF-7 cells were treated with 10 nM or 10uM of various ligands for 6 hours or DMSO in triplicate. Total RNA was isolated with the Aurum™ total RNA mini kit (BioRad, Hercules, CA, USA), following the manufacturer's protocol. The RNA yield was assessed with NanoDrop™ 1000 from Thermo Fisher Scientific. The RNA quality was evaluated using the Agilent 2100 bioanalyzer from Agilent (Palo Alto, California, USA) with the RNA 6000 Nano LabChip kit and subjected to RNA sequencing. They were multiplexed and sequenced using strand specific TruSeq RNA sequencing library prep as single end reads on a NextSeq 500 machine using 75 base reads.