Crosstalk Between Nephrogenesis and Ahr Signaling

描述（由申请人提供）：Ramos实验室最近表明，环境碳氢化合物激活Ahr信号调节wt1剪接并破坏肾发生。本申请中提出的研究的总体目标是评估Ahr和wt1之间的分子相互作用，以及它们在碳氢化合物诱导的肾形成缺陷中的作用。研究将验证以下假设：1)Ahr激活剂苯并（a)芘（BaP）激活Ahr和氧化还原信号，介导剪接体功能的改变，这些改变与肾母细胞分化和肾形成的缺陷有关；2）BaP激活Ahr信号，在肾形成过程中调节WT1的下游效应物，并诱导聚集在间质表型或“吸引子”中的全局mRNA表达的变化。从11.5个C57BL/6J胚胎中建立后肾培养物，用DMSO或3mum BaP攻毒4天。通过RT-PCR和Western分析检测Cyp1a1和1b1 mRNA和蛋白水平。为了解决bap诱导的DNA内聚或突变在wt1剪接调节中的作用，将进行突变分析和32P后标记实验。评估Ahr与剪接机制直接相互作用能力的第一种方法将依赖于酵母双杂交方法。为了确定Ahr和U2AF65是否形成蛋白复合物，我们将在两个杂交实验之后进行拉下实验和共免疫沉淀实验。Ahr激活后剪接因子的转录调控将通过实时或RT-PCR实验进行检测。微阵列分析将使用从Ahr+/+和Ahr-/- metahephroi中分离的RNA在Ahr配体存在和不存在的情况下进行，以确定WT1的关键靶点和协同调节的功能网络。如上所述的基因表达谱将使用多重二进制表达系统进行评估。布尔遗传网络的规则将使我们能够识别在肾发育的不同阶段，即胚细胞从间充质表型向上皮表型转变时表达的特定基因，以及这些变化发生的转录背景网络。

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.