Genomic regions with significant AhR enrichment were mapped to intragenic and non coding intergenic regions. Most regions were enriched 5. 7 fold with values ranging from 1. 7 to 111. 4 fold. Enriched regions varied done in width from 108 to 6,990 bp with 90. 5% spanning 1,500 bp. There was no correlation between fold enrichment and region width. Of the 974 significantly enriched regions at 24 h 899 of them overlapped with a 2 hr enriched region, consistent with reports of constant shuttling of the AhR between the nucleus and cytoplasm, and AhR promoter occupancy of targeted genes in untreated cells. Relaxing the FDR to 0. 05 increased the overlap to 906, while reducing the number of 24 hr specific enriched regions to 68.

Comparable overlaps were identified in promoter specific ChIP chip studies of TCDD induced AhR enrichment at 2 and 24 hrs in the livers of intact C57BL 6 mice, which identified 1,397 number of genes with 403 overlap. Further analysis of the 899 enriched regions found that the fold enrichment values from both time points were positively correlated. Although only 40% of the mouse genome consists of intragenic DNA, 71. 8% and 64. 7% of all sites with signif icant AhR enrichment at 2 hrs and 24 hrs, respectively, were within this region. The density of AhR enrichment was calculated across the entire genome in order to consider the cumulative intergenic and intragenic DNA region lengths. Genome and chromosomal analyses revealed increased enrichment within intragenic regions compared to non coding intergenic regions further illustrating a bias for gene encoding regions.

However, these values may be inflated due to incom plete probe coverage in the intergenic regions and sequence gaps in the genome. Specific analysis of the 10 kb upstream, 5 and 3 UTRs and CDS regions revealed the highest density of AhR enrichment was proximal to the TSS. AhR enrichment density was greatest within 1. 5 kb at 2 and 24 hrs, coinciding with proximal promoter DRE core densities and RNA polymerase II binding at the TSSs. Interestingly, there is a nota ble cleft in AhR enrichment 200 bp directly upstream and downstream of the TSSs, possibly to accommodate general transcription machinery. Both global and proximal promoter density analyses illustrate TCDD induced AhR enrichments are more prominent in regions directly associated with a gene.

Nevertheless, there are a significant number of distally located enrich ment sites that may also be functionally relevant. Confirmation of AhR ChIP chip Enrichment Analysis Selected regions of AhR enrichment identified by ChIP chip analysis at 2 hrs were confirmed by ChIP PCR. Three representative ChIP chip enrichments from each genomic region were selected Dacomitinib to vali date AhR enrichments with and without a DRE core at different positions relative to the TSS.