ABSTRACT Exposure to byproducts of industrial combustion, such as dioxins and polycyclic aromatic hydrocarbons (PAH), are one of the most important, and arguably best appreciated, environmental exposures. Their adverse impact on health is clear, ranging from thymic atrophy to tumorigenesis. Dioxins and PAHs exert their toxic effects by activating aryl hydrocarbon receptors (AHR), ligand-dependent transcription factors that regulate gene expression. AHR forms a complex with aryl hydrocarbon receptor nuclear translocator proteins (ARNT), and similar to other transcription factors, binds transcriptional coregulators and recognizes a consensus DNA sequence termed the xenobiotic response element (XRE). While this fundamental biology is well-explored, important questions related to AHR signaling still remain. These include how the choice of ligand determines AHR activity, and the possibility that AHRs also regulate gene expression via non-consensus DNA sequences. Answering these questions is of importance, as they hold the potential to reveal new pathways by which known and yet-to-be-identified AHR ligands may exert their toxic effects. The choice of ligand influences which transcriptional cofactors interact with AHRs, and this transcriptional cofactor complex in turn determines which genes respond to AHR signaling. While the field has assumed that a consensus XRE is necessary for AHR regulation of gene expression, emerging evidence suggests AHRs recognize DNA sequences other than XREs. What determines the activity of the AHR at XRE vs non-consensus regulatory elements is not well understood. Our overarching hypothesis is that AHR binding to non-consensus DNA sequences directs AHR activity and determines the choice of AHR interacting proteins. In cultured cells and mouse livers, AHR can bind a non- consensus DNA sequence (NC-XRE) and recruit the transcription factor KLF6 following exposure to 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). In contrast, when TCDD-AHR binds a consensus XRE DNA sequence, no KLF6 is recruited. This suggests that the DNA sequence to which AHR binds influences which proteins bind AHR. We will identify interactions between AHR, protein coregulators and DNA sequence in mouse liver following exposure to multiple AHR ligands (Aim 1) and determine whether AHR binds NC-XRE DNA to regulate target gene expression in vivo (Aim 2). While dogma holds that AHR must dimerize with ARNT1 to bind DNA and regulate transcription, recent data suggests AHR can bind DNA in the absence of ARNT1. Whether this binding occurs in vivo is not known, as global ARNT1 or ARNT2 mutant mice are not viable. We overcame this limitation by developing ARNT1/2 mutant zebrafish, which we will use to explore the role of ARNT1 and ARNT2 in AHR signaling at non-consensus DNA sequence (Aim 3). By combining genetic and biochemical approaches in multiple model organisms, we will determine how ligands, DNA sequence and protein coregulators influence AHR target gene expres...