PAX2 gene targets, binding sites and function

描述(由申请人提供)：Pax2基因是早期肾脏发育的重要调节基因，也与多种人类先天性和获得性疾病有关。在胚胎发育过程中，Pax2是启动和维持肾间充质细胞向上皮细胞转化所必需的。它的存在也是激活GDNF和输尿管芽生长所必需的。随着肾小球、近端和远端肾小管上皮的成熟，Pax2的表达被关闭。在基因敲除的小鼠中，Pax2的表达缺失导致肾脏缺失，而在人类中，部分缺失(杂合性)导致肾癌-缺陷症候群。Pax2在人和小鼠的PKD、发育不良和囊性疾病中持续表达，也与肾细胞癌和肾母细胞瘤有关。虽然Pax2被认为是一种DNA结合转录因子，但Pax2功能的生化机制仍不清楚。为了了解Pax2的表达如何指导早期的上皮分化和增殖，识别Pax2蛋白激活或抑制的基因是至关重要的。这项建议旨在利用细胞培养中的差异筛选方法来确定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，这些细胞系的基因表达模式发生了变化。采用差异消减和抑制性聚合酶链式反应相结合的方法鉴定对Pax2有反应的激活基因。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各种方法得到确认。在我们的差异筛选中的候选目标克隆将通过整体安装和常规的原位杂交来检测发育过程中肾脏的特异性表达。将检测Pax2与基因组位点的直接结合，并将确定Pax2使用候选靶标的上游调控元件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假定的磷酸化位点突变的Pax2蛋白来检验。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目标基因的DNAsel敏感性和甲基化状态，这两个已知的结构特征会增强基因的表达。进一步的实验将集中在靶基因与核基质的联系上，这也是已知的，当染色质变得活跃时，这也提供了从随机到特定锚定点的变化。这一建议将为理解肾上皮细胞分化和生长的早期事件提供关键的新数据。因此，它可能为控制上皮细胞正常分化和增殖的机制以及参与先天性和获得性人类疾病过程的机制提供新的见解。

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.