BackgroundThe aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor (TF) that mediates responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Integration of TCDD-induced genome-wide AhR enrichment, differential gene expression and computational dioxin response element (DRE) analyses further elucidate the hepatic AhR regulatory network.ResultsGlobal ChIP-chip and gene expression analyses were performed on hepatic tissue from immature ovariectomized mice orally gavaged with 30 μg/kg TCDD. ChIP-chip analysis identified 14,446 and 974 AhR enriched regions (1% false discovery rate) at 2 and 24 hrs, respectively. Enrichment density was greatest in the proximal promoter, and more specifically, within ± 1.5 kb of a transcriptional start site (TSS). AhR enrichment also occurred distal to a TSS (e.g. intergenic DNA and 3' UTR), extending the potential gene expression regulatory roles of the AhR. Although TF binding site analyses identified over-represented DRE sequences within enriched regions, approximately 50% of all AhR enriched regions lacked a DRE core (5'-GCGTG-3'). Microarray analysis identified 1,896 number of TCDD-responsive genes (|fold change| ≥ 1.5, P1(t) > 0.999). Integrating this gene expression data with our ChIP-chip and DRE analyses only identified 625 differentially expressed genes that involved an AhR interaction at a DRE. Functional annotation analysis of differentially regulated genes associated with AhR enrichment identified overrepresented processes related to fatty acid and lipid metabolism and transport, and xenobiotic metabolism, which are consistent with TCDD-elicited steatosis in the mouse liver.ConclusionsDetails of the AhR regulatory network have been expanded to include AhR-DNA interactions within intragenic and intergenic genomic regions. Moreover, the AhR can interact with DNA independent of a DRE core suggesting there are alternative mechanisms of AhR-mediated gene regulation.

The aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) activated complex regulates genes in response to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR has also emerged as a potential therapeutic target for the treatment of human diseases and different cancers, including breast cancer. To better understand AHR and ARNT signaling in breast cancer cells, we used chromatin immunoprecipitation linked to high-throughput sequencing to identify AHR- and ARNT-binding sites across the genome in TCDD-treated MCF-7 cells. We identified 2594 AHR-bound, 1352 ARNT-bound, and 882 AHR/ARNT cobound regions. No significant differences in the genomic distribution of AHR and ARNT were observed. Approximately 60% of the cobound regions contained at least one core an aryl hydrocarbon response element (AHRE), 5'-GCGTG-3'. AHR/ARNT peak density was the highest within 1 kb of transcription start sites (TSS); however, a number of AHR/ARNT cobound regions were located as far as 100 kb from TSS. De novo motif discovery identified a symmetrical variation of the AHRE (5'-GTGCGTG-3'), as well as FOXA1 and SP1 binding motifs. Microarray analysis identified 104 TCDD-responsive genes where 98 genes were upregulated by TCDD. Of the 104 regulated genes, 69 (66.3%) were associated with an AHR- or ARNT-bound region within 100 kb of their TSS. Overall our study identified AHR/ARNT cobound regions across the genome, revealed the importance but not absolute requirement for an AHRE in AHR/ARNT interactions with DNA, and identified a modified AHRE motif, thereby increasing our understanding of AHR/ARNT signaling pathway.

Estrogen receptor α (ERα) mediates the biological actions of estrogens and also contributes to the development and progression of breast cancer. To gain a more comprehensive understanding of ERα-mediated transcription, we used chromatin immunoprecipitation and promoter focused microarrays (ChIP-chip) to identify ERα binding sites in T-47D human breast cancer cells. Transcription factor binding site analysis revealed that the estrogen response element (ERE) was significantly over-represented and was found in 50% of the 243 ERα-bound regions identified. Interestingly, multiple ERα-bound regions were detected in the upstream regulatory sequences of the CYP2B gene cluster. Because ERα has been reported to regulate the expression of other cytochrome P450 enzymes and CYP2B6 is highly expressed in ERα-positive breast tumors, we focused on characterizing the ERα-dependent regulation of CYP2B6. Reporter gene assays revealed that ERα and ERβ increased CYP2B6-regulated gene expression through a functional ERE located at −1669 to −1657 in the upstream regulatory region of CYP2B6. E2 increased ERα and nuclear receptor coactivator 3 (NCoA3) recruitment to the 5′-flanking region of CYP2B6, and increased CYP2B6 mRNA levels in T-47D but not in MCF-7 human breast cancer cells. RNAimediated knockdown of ERα in the T-47D cells resulted in a significant decrease in CYP2B6 mRNA levels. Taken together, our study provides evidence for cell-type specific transcriptional regulation of the CYP2B6 gene by ERs.

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Epigenetic regulation is facilitated by a battery of proteins that act as 'writers' or 'erasers' to add or remove biochemical modifications to DNA and histone proteins. DNA modifications, histone modifications and long noncoding RNAs function interdependently through reciprocal crosstalk. This review will focus on DNA methylation and DNA methyltransferases with emphasis on how biological and biochemical factors such as histone modifications and noncoding RNAs might play a role in modulating DNA methylation. Other physiological and biochemical factors that can modify DNA methylation marks will also be discussed including chemical exposures and inflammation.

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