Hofmann W, Royer H D, Drechsler M, Schneider S, Royer-Pokora B
Institute of Human Genetics, University of Heidelberg, Germany.
Oncogene. 1993 Nov;8(11):3123-32.
Specific control of the expression of the Wilms tumor gene WT1 is important for normal development of the kidney. In order to characterise the transcriptional control region of the WT1 gene we have isolated genomic clones spanning the upstream region, the first WT1 exon and the 5' end of the Wit1 gene. DNA sequencing revealed that the WT1 promoter lacks a TATA box or CCAAT motif and has a GC content of 71%. Four transcriptional start sites are clustered within a 32 bp region. GC-boxes are present at nucleotide positions -413, -160, +84 and +158. DNAase I protection assays with purified Sp1 protein revealed the existence of 11 different binding sites in the WT1 promoter. WT1 and Wit1 promoter activities were tested in COS-7 cells with luciferase reporter gene constructs either containing or lacking an SV40 enhancer. WT1 promoter activity was found in a fragment extending from 449 bp upstream to 201 bp downstream of the WT1 start site. It was 26 fold lower in the absence of the SV40 enhancer than in the presence. Cotransfection with a Sp1 expression vector stimulated both constructs 3-4 fold. Wit1 promoter activity was identified in a DNA fragment extending from 200 bp upstream of the putative Wit1 TATA box to 130 bp downstream. Several potential recognition sites for WT1/EGR, Pax-8, and GAGA-like transcription factors are present in the WT1 promoter.
肾母细胞瘤基因WT1表达的特异性调控对肾脏的正常发育至关重要。为了表征WT1基因的转录调控区域，我们分离了跨越上游区域、首个WT1外显子和Wit1基因5'端的基因组克隆。DNA测序显示WT1启动子缺乏TATA盒或CCAAT基序，GC含量为71%。四个转录起始位点聚集在一个32 bp的区域内。GC盒位于核苷酸位置-413、-160、+84和+158处。用纯化的Sp1蛋白进行的DNA酶I保护试验揭示了WT1启动子中存在11个不同的结合位点。用含有或缺乏SV40增强子的荧光素酶报告基因构建体在COS-7细胞中测试了WT1和Wit1启动子活性。在从WT1起始位点上游449 bp延伸至下游201 bp的片段中发现了WT1启动子活性。在没有SV40增强子的情况下，其活性比有增强子时低26倍。与Sp1表达载体共转染可使两种构建体的活性提高3-4倍。在从假定的Wit1 TATA盒上游200 bp延伸至下游130 bp的DNA片段中鉴定出了Wit1启动子活性。WT1启动子中存在几个WT1/EGR、Pax-8和GAGA样转录因子的潜在识别位点。
