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Dietary ligands diindolylmethane and resveratrol result in diverse ERα signaling not seen after E2 and a subset of diindolylmethane mediated signaling needs concurrent AHR activation.

ABSTRACT: Inhibitory crosstalk between estrogen receptor alpha (ER alpha ) and aryl hydrocarbon receptor (AHR) regulates 17-estradiol (E2)-dependent breast cancer cell signaling. ER alpha and AHR are transcription factors activated by E2 and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively. Dietary ligands resveratrol (RES) and 3,30diindolylmethane (DIM) also activate ER alpha while only DIM activates AHR and RES represses it. DIM and RES are reported to have anti-cancer and antiinflammatory properties. Studies with genome-wide targets and AHR- and ER alpha-regulated genes after DIM and RES are unknown. We used chromatin immunoprecipitation with high-throughput sequencing and transcriptomics to study ER alpha 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 h, respectively. ER alpha bound sites after being DIM enriched for the AHR motif but not after E2 or RES while AHR bound sites after being 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 ER alpha site, and 60% of the coregulated genes between DIM and E2+TCDD were common. Collectively, our data show that RES and DIM differentially regulate multiple transcriptomic targets via ER alpha and ER alpha /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, ER alpha dominates with preferential recruitment of AHR to ER alpha target genes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE232235 | GEO | 2023/10/16

REPOSITORIES: GEO

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Project description:Background: Estrogen receptor (ERα) and aryl hydrocarbon receptor (AHR) are two nuclear receptors involved in regulating gene expression. ERα 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 ERα and AHR agonist, while RES is an ERα agonist and AHR antagonist. Few studies have investigated the impact of RES and DIM on ERα and AHR signaling at a genome-wide level. This study assessed ERα 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 ERα bound sites after RES and DIM overlapped with E2 ERα 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 ERα binding site. Motif analysis indicated enrichment for AHRE motif among DIM ERα sites but not among E2 or RES ERα 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 ERα 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 ERα relative to E2 and only a subset of the DEG’s after DIM and RES are ERα 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.

Project description:To increase the utility of the zebrafish gene expression bioassay for assessing EDC effects on multiple gene targets and tissue types, and to expand our understanding of genetic overlap between estrogen receptor (ER) and arylhydrocarbon receptor (AhR) mediated signaling pathways, we conducted microarray analysis of zebrafish embryos exposed to estradiol and dioxin alone or in combination. Of >16,000 probe sets on the array, 34 were regulated by estradiol (E2), 86 by dioxin (TCDD) and 109 by E2+TCDD (as chosen by >2-fold change and p<0.1). Of 62 genes selected for verification by QPCR, 14 genes, or 22% were reproducibly up- or down-regulated, offering potential additional target genes for screening of estrogen- and dioxin-like EDC. The majority of these successful hits, 11, were TCDD-responsive. In addition, all of the target genes routinely evaluated in this laboratory (AroB, Vtg1, and Esr1: E2-responsive; Cyp1a: TCDD- responsive; Vtg1: E2+TCDD-responsive) were verified with these arrays, testifying to the power of microarray analysis in finding reliable responsive genes. However, over two-thirds of the novel up- or down- regulated probes were not annotated as zebrafish genes, and many of the identified genes were changed only 2- to 3-fold, an effect often not reproduced by QPCR. Additional responsive genes were identified for each treatment condition, and while low levels of expression and low magnitude fold changes make those for E2 responsiveness or interaction between E2 and TCDD response unlikely to serve as robust biomarkers, their response to EDCs may assist in understanding the regulation of existing biomarkers and zebrafish endocrine systems as a whole. In addition to a source for potential EDC screening biomarkers, these studies provide an entry point to further study physiological effects of ER and AHR ligands and cross-talk between these signaling pathways, in a physiologically relevant in vivo model. Hybridizations of three biological replicates (n=30 zebrafish embryos/larvae pooled per treatment group) were performed, in two independent batches, for each of four treatment groups: 0.1% DMSO (control), 0.1µM E2, 1nM TCDD, and 0.1µM E2 + 1nM TCDD

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Dietary ligands diindolylmethane and resveratrol result in diverse ERα signaling not seen after E2 and a subset of diindolylmethane mediated signaling needs concurrent AHR activation.

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