Recent studies demonstrate that the aryl hydrocarbon receptor (AhR), a transcription factor, plays an active role in liver homeostasis, and that this process is dysregulated by the environmental toxicant 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). TCDD is a potent AhR agonist responsible for activation and nuclear translocation of AhR from the cytosolic compartment. Previous studies determined that AhR-mediated transcriptional activity requires partnering with the nuclear ARNT protein and DNA binding of this heterodimer to a consensus xenobiotic response element (XRE) linked to target genes. Preliminary evidence examining TCDD induction of plasminogen activator inhibitor-1 (PAI-1), a key protein in liver regeneration, exposed a novel AhR-dependent regulatory mechanism involving DNA binding to a non- consensus XRE (NC-XRE) independent of the ARNT protein. We hypothesize that the AhR regulates PAI- 1 gene expression by a formerly unrecognized mechanism of action. This study will identify the components of the complex bound to the NC-XRE and characterize the AhR protein-protein and protein- DNA interactions at this site.
Specific Aim 1 will employ DNA-affinity chromatography using the NC-XRE binding site characterized by EMSA and functional studies to fractionate the complex from mouse liver nuclear extracts following induction with TCDD. MALDI-TOF/TOF will be used to identify the proteins in the NC-XRE-bound complex.
Specific Aim 2 will verify immunologically that the proteins identified in aim 1 are indeed components of the NC-XRE binding complex using EMSA supershift assays and co- immunoprecipitation studies. Studies in Specific Aim 3 will focus on characterizing the nature of AhR interaction in this complex through a series of in vitro assays designed to examine protein-protein and protein-DNA interactions. Collectively, these studies will explore a new mechanism in the growing repertoire of distinct AhR activities that must be resolved if biologists are to fully understand the physiological role of the receptor or obtain reliable human health risk assessments following exposure to TCDD and related environmental toxicants.
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|Mitchell, Kristen A; Wilson, Shelly R; Elferink, Cornelis J (2010) The activated aryl hydrocarbon receptor synergizes mitogen-induced murine liver hyperplasia. Toxicology 276:103-9|