Crosstalk Between Nephrogenesis and Ahr Signaling

描述（由申请人提供）：拉莫斯实验室最近表明，环境碳氢化合物激活Ahr信号传导可调节wt 1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Wt 1之间的分子相互作用，以及它们在烃诱导的肾发生缺陷中的作用。将进行研究以检验以下假设：1）苯并（a）芘（BaP）（一种Ahr激活剂）激活Ahr和氧化还原信号传导，介导剪接体功能的改变，所述剪接体功能的改变与成肾细胞分化和肾发生中的缺陷相关联，以及2）BaP激活Ahr信号传导，在肾发生过程中调节WT 1的下游效应子，并诱导聚集在间充质表型或“吸引子”内的整体mRNA表达的变化。将从11.5个C57 BL/6 J胚胎建立后肾培养物，并用媒介物（DMSO）或3 μ M BaP攻击4天。将通过RT-PCR和Western分析检查Cyp 1a 1和1b 1 mRNA和蛋白质水平。为了解决BaP诱导的DNA加合或突变在调节wt 1剪接中的作用，将进行突变分析和32 P后标记实验。第一种评估Ahr直接与剪接机制的组分相互作用的能力的方法将依赖于酵母双杂交方法。为了确定Ahr和U2 AF 65是否形成蛋白复合物，我们将在两个杂交实验后进行下拉测定和共免疫沉淀实验。通过真实的时间或RT-PCR实验检查Ahr激活后剪接因子的转录调节。在存在和不存在Ahr配体的情况下，将使用从Ahr+/+和Ahr-/-偏肝中分离的RNA进行微阵列分析，以鉴定WT 1的关键靶标和共调节的功能网络。将使用多个二元表达系统评价如上所述的基因表达谱。布尔遗传网络的规则将使我们能够确定特定的基因表达在不同阶段的肾发育的胚细胞从间充质上皮表型的转变，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.