The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic effects of many environmental contaminants, including dioxin-like compounds (DLCs) such as 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). DLC effects are wide-ranging, targeting multiple organ systems. Among these is the thyroid hormone (TH) system, which is important for neurodevelopment and metabolic regulation. TH effects are mediated by the thyroid receptors (TRs). Like AHR, TRs are nuclear receptors. This AREA project will support an undergraduate lab group to define a novel molecular mechanism by which DLCs disrupt the thyroid system. The overall objective is to test the hypothesis that AHR activation disrupts tightly regulated gene expression patterns established by thyroid receptors, resulting in elevated and/or mistimed expression of TH target genes. The project will utilize an important model of thyroid system function and disruption during development: Xenopus tadpole metamorphosis. Metamorphosis of a tadpole into a frog involves dramatic tissue remodeling, including tail resorption, limb growth, and development of lungs to enable air breathing. It is initiated by TH, which activates TR? and induces expression of transcription factor klf9. klf9 is a key regulator of subsequent metamorphic gene expression changes, and its expression is a molecular biomarker of TH exposure and an excellent endpoint for assessing TR activity. Preliminary studies in tadpoles and a frog cell line demonstrate that klf9 and other TR target genes are upregulated not only by TH but also by TCDD, suggesting a functional interaction between the TR and AHR signaling pathways. Such ?cross-talk? may contribute to thyroid- related DLC toxicity, representing a novel mechanism of thyroid disruption. This project will investigate the mechanisms of TR:AHR cross-talk through two specific aims that use klf9 induction as a model endpoint. Studies under Aim 1 will use transactivation assays and chromatin immunoprecipitation to determine the molecular basis klf9 regulation, identifying functional enhancer elements through which AHR acts.
Aim 1 studies will also test the hypothesis that klf9 induction by TCDD is tissue-specific, underlying different TCDD effects in tadpole tails and hindlimbs during metamorphosis.
Aim 2 seeks to characterize a novel molecular mechanism of transcription regulation by AHR, testing the hypothesis that klf9-associated enhancer RNA (eRNA) is necessary for AHR- mediated disruption of klf9 expression. These studies use antisense and genome editing technologies to reduce expression of a non-coding RNA associated with the ?klf9 synergy module? (KSM), a cluster of transcription factor binding sites, examining the effect on induction of klf9 by TCDD. This AREA project will provide new insight into the molecular mechanisms of AHR-mediated thyroid disruption, a potential fetal basis of adult disease. It will provide mechanistic underpinning to a widely used model of thyroid function, the Xenopus tadpole, crucial information for the valid use of US EPA?s Amphibian Metamorphosis Assay (AMA; a Tier 1 endocrine disruption screen) in toxicological risk assessment for humans.
Dioxins are widespread and persistent chemical pollutants that nearly everyone is exposed to through the diet. They have many toxic effects, including disruption of the thyroid hormone system. This research project uses developing tadpoles as a model for studying how thyroid-related genes and proteins are disrupted by dioxin and ultimately how dioxin exposure during development causes thyroid disease in people.
