Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq analysis reveals endogenous aryl hydrocarbon receptor regulation is highly associated with eicosanoid synthesis and tumor necrosis factor activity in MCF-7 cancer cells

ABSTRACT: Background: The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is activated by xenobiotic chemicals that function as AHR ligands. The response to xenobiotic AHR ligands is toxicity and the induction of drug metabolizing enzymes. The impact of AHR knockdown on gene expression and pathways in human breast cancer cells in absence of xenobiotic AHR ligands has not been investigated on a genome-wide scale. Methods: MCF-7 cells were used as a model of human breast cancer. The AHR was silenced with short interfering RNA against AHR (siAHR). RNA-sequencing coupled with Ingenuity pathway analysis (IPA) was used to determine the impact of AHR knockdown on gene expression and pathways in the absence of xenobiotic AHR ligands. Western blot analysis and recombinant tumor necrosis factor (TNF) was used to investigate the impact of AHR knockdown on TNF induction of MNSOD expression. Results: We found that the AHR is transcriptionally active in MCF-7 breast cancer cells in the absence of xenobiotic AHR ligands. In total, the expression of 634 genes was significantly changed in AHR knockdown cells compared to controls at a false discovery rate of < 10%. The analysis confirmed that drug metabolizing enzymes were AHR targets; however, we found that AHR also promoted the expression of genes that were not directly related to the metabolism of xenobiotics, such as those involved in lipid and eicosanoid synthesis. Gene pathway analysis of the AHR regulated gene dataset predicted TNF activity to be reduced in AHR knockdown cells. Our finding that AHR knockdown inhibited TNF-stimulated increases in MNSOD expression confirmed this IPA prediction. Conclusions: This is the first gene expression profiling study of AHR knockdown breast cancer cells. Several known and novel AHR targets were identified. The results suggest that endogenous AHR regulation impacts eicosanoid synthesis by regulating gene expression. The IPA prediction of reduced TNF activity in AHR knockdown cells was confirmed by showing that TNF-induced increases in MNSOD expression was inhibited in AHR knockdown cells. The requirement of AHR for MNSOD activation is novel and provides a new link by which AHR may impact reactive oxidative species (ROS) signaling. Expression profiles by mRNA sequencing were generated for human MCF-7 cells transfected with cRNAi (6 replicates) or AHR-siRNA (5 replicates) for 36hr. Sequencing was performed on an Illumina HiSeq 1000 using a 2x100 base paired-end strategy.

ORGANISM(S): Homo sapiens

SUBMITTER: James Denvir

PROVIDER: E-GEOD-52036 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Endogenous aryl hydrocarbon receptor promotes basal and inducible expression of tumor necrosis factor target genes in MCF-7 cancer cells.

Salisbury Travis B TB Tomblin Justin K JK Primerano Donald A DA Boskovic Goran G Fan Jun J Mehmi Inderjit I Fletcher Jackie J Santanam Nalini N Hurn Estil E Morris Gary Z GZ Denvir James J

Biochemical pharmacology 20140624 3

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that upon activation by the toxicant 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) stimulates gene expression and toxicity. AHR is also important for normal mouse physiology and may play a role in cancer progression in the absence of environmental toxicants. The objective of this report was to identify AHR-dependent genes (ADGs) whose expression is regulated by AHR in the absence of toxicants. RNA-Seq analysis revealed t ...[more]

PMID: 24971714

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Project description:Background: The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is activated by xenobiotic chemicals that function as AHR ligands. The response to xenobiotic AHR ligands is toxicity and the induction of drug metabolizing enzymes. The impact of AHR knockdown on gene expression and pathways in human breast cancer cells in absence of xenobiotic AHR ligands has not been investigated on a genome-wide scale. Methods: MCF-7 cells were used as a model of human breast cancer. The AHR was silenced with short interfering RNA against AHR (siAHR). RNA-sequencing coupled with Ingenuity pathway analysis (IPA) was used to determine the impact of AHR knockdown on gene expression and pathways in the absence of xenobiotic AHR ligands. Western blot analysis and recombinant tumor necrosis factor (TNF) was used to investigate the impact of AHR knockdown on TNF induction of MNSOD expression. Results: We found that the AHR is transcriptionally active in MCF-7 breast cancer cells in the absence of xenobiotic AHR ligands. In total, the expression of 634 genes was significantly changed in AHR knockdown cells compared to controls at a false discovery rate of < 10%. The analysis confirmed that drug metabolizing enzymes were AHR targets; however, we found that AHR also promoted the expression of genes that were not directly related to the metabolism of xenobiotics, such as those involved in lipid and eicosanoid synthesis. Gene pathway analysis of the AHR regulated gene dataset predicted TNF activity to be reduced in AHR knockdown cells. Our finding that AHR knockdown inhibited TNF-stimulated increases in MNSOD expression confirmed this IPA prediction. Conclusions: This is the first gene expression profiling study of AHR knockdown breast cancer cells. Several known and novel AHR targets were identified. The results suggest that endogenous AHR regulation impacts eicosanoid synthesis by regulating gene expression. The IPA prediction of reduced TNF activity in AHR knockdown cells was confirmed by showing that TNF-induced increases in MNSOD expression was inhibited in AHR knockdown cells. The requirement of AHR for MNSOD activation is novel and provides a new link by which AHR may impact reactive oxidative species (ROS) signaling.

Project description:The Aryl hydrocarbon Receptor (AhR) is a signal regulated transcription factor, which is canonically activated by the direct binding of xenobiotics. In addition, switching cells from adherent to suspension culture also activates the AhR, representing a non-xenobiotic, physiological activation of AhR signaling. Here, we show that the AhR is recruited to target gene enhancers in both ligand (YH439) treated and suspension cells, suggesting a common mechanism of target gene induction between these two routes of AhR activation. However, gene expression profiles critically differ between xenobiotic and suspension activated AhR signaling. Por, and Cldnd1 were regulated predominately by ligand treatments, while in contrast, ApoER2 and Ganc were regulated predominately by the suspension condition. Classic xenobiotic metabolizing AhR targets such as Cyp1a1, Cyp1b1, and Nqo1 were regulated by both ligand and suspension conditions. Temporal expression patterns of AhR target genes were also found to vary, with examples of transient activation, transient repression, or sustained alterations in expression. Furthermore, sequence analysis coupled with ChIP assays and reporter gene analysis identified a functional XRE (xenobiotic response element) within the mouse Tiparp gene that features a concatemer of 4 XRE cores (GCGTG) residing in the first intron ~1.2kb downstream of the Tiparp transcription start site. Our data suggest that this XRE concatemer site concurrently regulates the expression of both Tiparp gene and its cis anti-sense non-coding RNA following ligand or suspension induced AhR activation. This work lends novel insights into how AhR signaling drives different transcriptional programs via the ligand versus suspension modes of activation. Reference: Murray IA et al. (2005) Evidence that ligand binding is a key determinant of Ah receptor-mediated transcriptional activity. Arch Biochem Biophys 442(1):59-71. Reference: Monk SA et al. (2001) Transient expression of CYP1A1 in rat epithelial cells cultured in suspension. Arch Biochem Biophys 393(1):154-162. Reference: Chua SW et atl. (2006) A novel normalization method for effective removal of systematic variation in microarray data. Nucleic acids research 34(5):e38. 18 microarray samples consisting of AhR null and reconstituted hepatocytes subjected to 10M-NM-<M YH439, 0.1% DMSO carrier control or grown in suspension culture for 8 hours in 3 biologial replicates.

Project description:The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates a broad range of target genes involved in the xenobiotic response, cell cycle control and circadian rhythm. AhR is constitutively expressed in macrophages, acting as key regulator of cytokine production. While proinflammatory cytokines, i.e., IL-1β, IL-6, IL-12, are suppressed through AhR activation, anti-inflammatory IL-10 is induced. However, the underlying mechanisms of those effects and the importance of the specific ligand structure are not yet completely understood. Therefore, we have compared the global gene expression pattern in activated murine bone marrow-derived macrophages (BMMs) subsequently to exposure with either benzo[a]pyrene (BaP) or indole-3-carbinol (I3C), representing xenobiotic vs. nutritional AhR ligands, respectively, by means of mRNA sequencing. AhR dependency of observed effects was proved using BMMs from AhR-knockout mice. In total, more than 1,000 differentially expressed genes (DEGs) could be mapped, covering a plethora of AhR-modulated effects on basal cellular processes, i.e., transcription and translation, but also immune functions, i.e., antigen presentation, cytokine production, and phagocytosis. Among DEGs were genes that are already known to be regulated by AhR, i.e., Irf1, Ido2, and Cd84. However, we identified DEGs not yet described to be AhR-regulated in macrophages so far, i.e., Slpi, Il12rb1, and Il21r. All six genes likely contribute to shifting the macrophage phenotype from proinflammatory to anti-inflammatory. The majority of DEGs were specific for BaP but not affected through I3C exposure, probably due to higher AhR affinity of BaP in comparison to I3C. Mapping of known aryl hydrocarbon response element (AHRE) sequence motifs in identified DEGs revealed more than 200 genes not possessing any AHRE, and therefore being not eligible for canonical regulation. Bioinformatic approaches modeled a central role of type I and type II interferons in the regulation of those genes. Additionally, RT-qPCR and ELISA confirmed a BaP- and AhR-dependent expressional induction and AhR-dependent secretion of IFN-γ, suggesting a potentially novel auto- or paracrine activation pathway of macrophages.

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.

Dataset Information

RNA-seq analysis reveals endogenous aryl hydrocarbon receptor regulation is highly associated with eicosanoid synthesis and tumor necrosis factor activity in MCF-7 cancer cells

Publications

Endogenous aryl hydrocarbon receptor promotes basal and inducible expression of tumor necrosis factor target genes in MCF-7 cancer cells.

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