This study further elucidates the role of the AhR in mediating the hepatic effects of TCDD in C57BL6 mice. Recent studies have mapped AhR binding using promoter-focused ChIP-chip arrays and found that ~50% of the AhR enriched regions were devoid of the DRE core [32–34]. The lack of a DRE core in regions of AhR enrichment was also reported in a AhR genome-wide ChIP-chip study performed in mouse CH12.LX cells . ChIP-seq experiments for other TFs have also demonstrated enrichment in remote genome regions, which may serve important regulatory roles [10, 11, 14, 17]. Collectively these data suggest the AhR uses different mechanisms to regulate gene expression. Moreover, the integration of genome-wide in silico DRE search, with de novo motif analysis and TCDD-elicited hepatic temporal gene expression data has further elucidated the hepatic AhR gene regulatory network.
ChIP-chip analysis identified 14,446 TCDD-induced AhR regions at 2 hrs and 974 regions at 24 hrs, consistent with the rapid nuclear export and subsequent degradation of the AhR following TCDD activation . Approximately half of these regions were within intragenic regions (10 kb upstream of a TSS to the end of the 3' UTR). Furthermore, 25% of these enriched regions at 2 hrs and 19% at 24 hrs were within 2 kb of a TSS, indicating that a large subset of AhR enrichment occurs adjacent to a TSS. Unlike other studies that report a normal distribution of TF binding centered around the TSS [15, 58–60], the AhR density profile exhibited a cleft immediately adjacent to the TSS, possibly to accommodate recruited transcriptional machinery.
Although most AhR enrichment regions are intragenic, a significant number are located in distal intergenic regions (i.e. 4,163 of 14,446 at 2 hrs and 344 of 974 at 24 hrs). Studies with the ER, p53 and forkhead box protein A1 [10, 11, 14, 17] suggest distal TF binding may have distinct regulatory roles. Binding proximal to the TSS is presumed to stabilize the general transcriptional machinery, while distal binding regulates transcription by a looping mechanism or by altering chromatin structure [9, 61, 62]. Consequently, AhR binding outside of the proximal promoter region may have important regulatory roles that remain largely uninvestigated.
Comparing AhR enriched regions with DRE cores revealed that their intergenic, intragenic and genic (10 kb upstream, UTRs, and CDS) density distributions were similar. The greatest density of AhR enrichment associated with a DRE core occurred within the proximal promoter. Both exhibited comparable distribution profiles except for the cleft in enrichment at the TSS. The decrease in AhR enrichment at the TSS coincides with RNA polymerase II binding at the TSSs  of transcriptionally responsive genes. Although TCDD-elicited differential gene expression is thought to be mediated by the substitution intolerant DRE core sequence (5'-GCGTG-3'), only ~50% of the AhR enriched regions contained a DRE core, consistent with findings in other promoter targeted AhR ChIP-chip studies [33, 35] (Lo et al., in submission). Moreover, relatively few alternative AhR response elements (5'-CATGN6C[T|A]TG-3') [40, 41] were identified in AhR enriched regions lacking a DRE core sequence. Enrichment in regions lacking DRE cores provides additional evidence of AhR-DNA interactions that don not involve the basic bHLH domain , such as tethering to other DNA interacting TFs and/or tertiary interactions with looping DNA.
Integration of gene expression, ChIP-chip, and DRE distribution data suggests that ~35% of all differentially expressed hepatic genes are mediated by direct AhR binding to a DRE. Consequently, 65% of the gene expression responses elicited by TCDD do not involve direct AhR binding to a DRE. However, TF binding analyses based on tiling arrays is limited by the extent of probe coverage (Figure 1). Genomic regions lacking probe coverage may falsely inflate the number of DRE-absent AhR enriched regions, thus underestimating the number of AhR regulated genes involving a DRE. Furthermore, the analyses may not be exhaustive due to the technical limitations of ChIP-chip assay coupled with the conservative FDR threshold used to identify statistically significant signals, which may have excluded some positive signals. These limitations of the technology could be addressed in ChIP-seq experiments, which have greater resolution and sensitivity [64, 65]. The shorter sequence reads would improve resolution, but may also identify fewer regions containing a DRE. The higher sensitivity of ChIP-seq could also identify additional regions of AhR enrichment. ChIP-seq studies could also confirm AhR binding in these genomic regions in either a DRE-dependent or -independent manner.
TCDD induces hepatic vacuolization and lipid accumulation with differential gene expression associated with fatty acid metabolism and transport [25, 53]. Independent functional annotation analysis of differentially expressed genes with significant AhR enrichment using DAVID and IPA identified over-represented processes related to fatty acid and lipid metabolism. Computational analysis also identified over-represented binding motifs for TFs involved in the regulation of lipid and cholesterol metabolism, including sites for HNF4, LXR, PXR, PPAR and COUP-TF. COUP-TF is a potent repressor that antagonizes transcriptional responses mediated by other nuclear receptors including HNF4, PPAR, ER, RAR and VDR . For example, COUP-TF antagonizes HNF4α-mediated responses by binding HNF4α response elements [67–71]. Furthermore, AhR interactions with COUP-TF repress ER-mediated gene expression responses . Therefore, AhR interactions with COUP-TF may regulate lipid and fatty acid metabolism by blocking HNF4α target gene expression (Figure 10A). Coincidentally, the HNF4 binding motif is over represented within AhR enriched regions lacking a DRE core.
Consistent with this proposed mechanism, several HNF4α regulated genes, including Cyp7a1 and Gck, exhibited AhR enrichment and were repressed by TCDD. Cyp7a1 is the rate-limiting enzyme in the bile acid biosynthetic pathway that converts cholesterol into bile acids. Transgenic mice over-expressing Cyp7a1 are protected from high-fat diet induced obesity, fatty liver and insulin resistance . Moreover, a genetic deficiency of Cyp7a1 in humans results in hyperlipidemia . Gck phosphorylates glucose in the initial step of glycolysis. Mutations in Gck that reduce kinase activity are associated with insulin resistance and maturity onset diabetes of young 2 (MODY2) in humans [74–76]. Furthermore, mice over-expressing Gck are resistant to MODY2 . The down-regulation of Cyp7a1 and Gck, possibly due to AhR - COUP-TF interactions at HNF4α response elements, is consistent TCDD-induced hepatic lipid accumulation in mice. Interestingly, TCDD exposure has been linked to diabetes and metabolic syndrome in humans [78–84]. Studies examining AhR-COUP-TF interactions and their effects on HNF4 target gene expression are being investigated further.