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因子，也与多种先天性和获得性人类疾病有关。在胚胎发育过程中，Pax2是肾间质向上皮转化的起始和维持所必需的。它的存在也是激活GDNF和输尿管芽生长所必需的。Pax2的表达随着肾小球、近端和远端小管上皮的成熟而关闭。在小鼠基因敲除中，Pax2表达的缺失导致肾脏缺失，而在人类中，部分缺失（杂合性）导致RenaI-Coloboma综合征。Pax2在人和小鼠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功能的生化机制尚不清楚。为了了解Pax2表达如何指导早期上皮分化和增殖，有必要确定Pax2蛋白激活或抑制的基因。本课题旨在利用细胞培养的差异筛选方法鉴定Pax2的基因靶点。我们已经从胚胎肾间质中获得细胞系，当用表达Pax2的逆转录病毒转化时，这些细胞系表现出基因表达模式的改变。采用差分减法和抑制PCR相结合的方法鉴定了Pax2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各种方法被确认。我们的鉴别筛选中的候选靶克隆将通过全挂载和常规原位杂交来检测发育过程中肾脏特异性表达。将检测Pax2与基因组位点的直接结合，并确定Pax2利用来自候选靶标的上游调控元件反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通过在反活化区假定的磷酸化位点突变的Pax2蛋白来检测。Pax2如何改变或重塑候选靶位点的染色质结构将开始被解决。最初的实验将集中在候选靶基因的dna敏感性和甲基化状态，这两个已知的结构特征可以增强基因表达。进一步的实验将集中在靶基因与核基质的关联上，当染色质变得活跃时，它也提供了从随机到特定锚点的变化。这一建议将为理解肾上皮分化和生长的早期事件提供重要的新数据。因此，它可能为控制上皮正常分化和增殖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.