| The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that mediates biological responses to environmental pollutants like dioxins. In an effort to characterize the regulatory mechanism of AHR signaling, in chapter II we describe the development of a high throughput approach that allowed the use of a powerful yeast deletion array to identify sixty-six novel modifiers of the AHR pathway. To characterize these modifiers of AHR signaling, an integrated approach was established. This approach combines multiple bioinformatic and experimental techniques. By overlapping the data from these methods, we are able to classify the sixty-six modifiers into five functional groups. These groups can be described as those that regulate receptor folding, nuclear translocation, transcriptional activation, receptor level, and one unknown function related to the PAS domain. Based on these results, an expanded model of AHR signaling has been proposed. This model suggests that receptor folding involves Hsp90 cochaperones Sti1p, Cpr7p, and that transactivation requires the SAGA/PCAF complex subunits Gcn5p, Spt3p, and Spt8p, as well as the SWI/SNF complex subunits Snf12p and Swi3p. In chapter III we describe EMS mutagenesis and transposon insertion mutagenesis that were employed in early phase of this study. Approximately 150 AHR signaling mutants were generated by these experiments in a yeast model system. These random mutagenesis methods generate a spectrum of subtle mutations, and are complementary to the deletion strategy in building up a complete picture of AHR signaling. In chapter IV we describe the study on homotypic modifiers of AHR signaling. In this experiment, we employed mammalian cell culture system to examine cross-talk between the AHR and hypoxia inducible factor 1α (HIF1α) signaling pathways. Our data suggests that inhibitory and additive cross-talk occurs between these two pathways via dioxin/hypoxia responsive elements. |
