Crosstalk Between Nephrogenesis and Ahr Signaling

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DESCRIPTION (provided by applicant): The Ramos laboratory has recently shown that activation of Ahr signaling by environmental hydrocarbons modulates wt1 splicing and disrupts nephrogenesis. The overall goal of the research proposed in this application is to evaluate molecular interactions between Ahr and wt1, and their role in hydrocarbon-induced deficits in nephrogenesis. Studies will be conducted to test the hypotheses that 1) activation of Ahr and redox signaling by benzo(a)pyrene (BaP), an Ahr activator, mediates alterations in spliceosome function that are coupled to deficits in nephroblast differentiation and nephrogenesis and, 2) that activation of Ahr signaling by BaP, modulates downstream effectors of WT1 during nephrogenesis and induces variations in global mRNA expression that cluster within mesenchymal phenotypes or "attractors". Metanephroi cultures will be established from 11.5 C57BL/6J embryos and challenged with vehicle (DMSO) or 3 muM BaP for 4 days. Cyp1a1 and 1b1 mRNA and protein levels will be examined by RT-PCR and Western analysis. To address the role of BaP-induced DNA adduction or mutation in modulation of wt1 splicing, mutational analysis and 32P post-labeling experiments will be conducted. The first approach to evaluate the ability of Ahr to interact directly with components of the splicing machinery will rely on yeast two-hybrid methodology. To determine if Ahr and U2AF65 form protein complexes, we will perform pull down assays and co-immunopercipitation experiments following two hybrid experiments. Transcriptional regulation of splicing factors following Ahr activation will be examined by real time or RT-PCR experiments. Microarray analysis will be performed using RNA isolated from both Ahr+/+ and Ahr-/- metahephroi in the presence and absence of Ahr ligands to identify critical targets of WT1 and functional networks of co-regulation. Gene expression profiles as outlined above will be evaluated using a multiple binary expression system. The rules of Boolean genetic networks will enable us to identify specific genes expressed in different stages of renal development as blastemal cells transition from mesenchymal to epithelial phenotypes, and the transcriptional contextual network in which these changes occur.