PAX2 gene targets, binding sites and function

PAX2 基因靶标、结合位点和功能

• 批准号: 7570464
• 负责人: PATRICK D BROPHY
• 金额: $ 9.02万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7570464
• 关键词: Address; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biological Assay; Candidate Disease Gene; Cell Differentiation process; Cell Line; Chromatin; Chromatin Structure; Coloboma; Cultured Cells; Cystic Kidney Diseases; DNA Binding; DNA-Binding Proteins; DNA-Protein Interaction; Data; Development; Differentiation and Growth; Disease; Distal; Dysplasia; Embryo; Embryonic Development; Epithelial; Epithelial Cells; Epithelium; Event; Exhibits; GDNF gene; Gene Activation; Gene Dosage; Gene Expression; Gene Targeting; Genes; Genetic Programming; Genetic Transcription; Genomics; Human; In Situ Hybridization; Kidney; Kidney Diseases; Knockout Mice; Localized; Loss of Heterozygosity; Maintenance; Mammals; Mesenchymal; Mesenchyme; Metanephric Diverticulum; Methods; Methylation; Mus; Mutate; Nephroblastoma; Northern Blotting; Nuclear Matrix; Pattern; Phosphorylation Site; Polymerase Chain Reaction; Process; Proteins; Regulatory Element; Renal Cell Carcinoma; Reporter Genes; Retroviridae; Screening procedure; Structure; Syndrome; Techniques; Testing; Transactivation; Transcriptional Regulation; Transformed Cell Line; Tubular formation; design; human disease; insight; kidney cell; mutant; novel; renal epithelium; research study; response; transcription factor

DESCRIPTION (provided by applicant): The Pax2 gene is an essential regulator of early renal development and has also been implicated in a variety of human diseases both congenital and acquired. During embryonic development, Pax2 is required for the initiation and maintenance of the conversion of the renal mesenchyme to epithelium. Its presence is also required to activate GDNF and thus ureteric bud outgrowth. Pax2 expression is turned off as the glomerular, proximal, and distal tubular epithelium matures. Loss of Pax2 expression leads to an absence of kidneys in the mouse knockout, while partial loss (heterozygosity) leads to the RenaI-Coloboma syndrome in humans. Persistent expression of Pax2 is found in human and mouse PKD, dysplastic and cystic disease and also is associated with renal cell carcinoma and the Wilms Tumor. Although Pax2 is presumed to be a DNA binding transcription factor, the biochemical mechanisms of Pax2 function remain obscure. In order to understand how Pax2 expression directs early epithelial differentiation and proliferation, it is essential to identify the genes activated or repressed by the Pax2 protein. This proposal aims to identify gene targets of Pax2 using a differential screening method in cell culture. We have derived cell lines from embryonic kidney mesenchyme that exhibit altered gene expression patterns when transformed with Pax2 expressing retroviruses. Genes activated in response to Pax2 were identified using a combination differential subtraction and suppressive PCR strategy. One clone has been confirmed as a kidney specific target of Pax2 and several others will be confirmed by a variety of methods. Candidate target clones in our differential screen will be assayed for kidney specific expression during development by whole mount and conventional in situ hybridization. Direct binding of Pax2 to genomic loci will be assayed and the ability of Pax2 to transactivate transcription of reporter genes, using upstream regulatory elements from candidate targets, will be determined. The effect of Pax2 phophorylation status on target gene activation will also be examined utilizing Pax2 proteins mutated in presumed phosphorylation sites in the transactivation region. How Pax2 can alter or remodel the chromatin structure of candidate target loci will begin to be addressed. Initial experiments will focus on the DNAsel sensitivity of candidate target genes and the methylation state, two structural features known to potentiate gene expression. Further experiments will focus on target gene association with nuclear matrix, which also is known to provide a change from random to specific anchorage points when chromatin becomes active. This proposal will provide critical new data for understanding the early events underlying renal epithelial differentiation and growth. As such, it may provide novel insight into the mechanisms controlling the normal differentiation and proliferation of epithelium as well as those involved in both congenital and acquired human disease processes.

描述（由申请人提供）：Pax2基因是早期肾脏发育的重要调节因子，也与多种先天性和获得性人类疾病有关。在胚胎发育期间，Pax2是启动和维持肾间充质向上皮转化所必需的。它的存在也需要激活GDNF，从而输尿管芽生长。Pax2表达随着肾小球、近端和远端肾小管上皮成熟而关闭。Pax2表达缺失导致敲除小鼠肾脏缺失，而部分缺失（杂合性）导致人类肾脏缺损综合征。在人类和小鼠PKD、发育异常和囊性疾病中发现Pax2的持续表达，并且还与肾细胞癌和Wilms肿瘤相关。虽然Pax2被认为是一种DNA结合转录因子，但其功能的生化机制仍不清楚。为了了解Pax2表达如何指导早期上皮细胞分化和增殖，必须确定由Pax2蛋白激活或抑制的基因。该建议旨在使用细胞培养中的差异筛选方法来鉴定Pax2的基因靶点。我们已经从胚胎肾间充质中获得了细胞系，当用表达Pax2的逆转录病毒转化时，这些细胞系表现出改变的基因表达模式。使用差异减法和抑制性PCR策略的组合来鉴定响应Pax2而激活的基因。一个克隆已被确认为Pax2的肾脏特异性靶点，其他几个克隆将通过多种方法确认。在我们的差异筛选中，候选靶克隆将在发育过程中通过整体安装和常规原位杂交测定肾脏特异性表达。将测定Pax2与基因组基因座的直接结合，并使用来自候选靶标的上游调控元件测定Pax2反式激活报告基因转录的能力。Pax2磷酸化状态对靶基因激活的影响也将利用在反式激活区域中的假定磷酸化位点突变的Pax2蛋白进行检查。Pax2如何改变或重塑候选靶基因座的染色质结构将开始得到解决。最初的实验将集中在候选靶基因的DNAsel敏感性和甲基化状态，这两个结构特征已知可以增强基因表达。进一步的实验将集中在靶基因与核基质的关联上，这也是已知的，当染色质变得活跃时，它提供了从随机到特定锚定点的变化。这一建议将为了解肾上皮分化和生长的早期事件提供关键的新数据。因此，它可以提供新的洞察控制上皮细胞的正常分化和增殖的机制，以及参与先天性和后天性人类疾病过程。