Shimamura R, Fraizer G C, Trapman J, Saunders G F
Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
Clin Cancer Res. 1997 Dec;3(12 Pt 2):2571-80.
Genital abnormalities associated with Wilms' tumors in the WAGR and Denys-Drash syndromes and the failure of the gonads to develop in Wilms' tumor gene (wt1)-homozygous mutant mice suggest that WT1 may also function in sexual development. To elucidate the mechanism of action of WT1 in embryonal sexual development, we examined how the four isoforms of WT1 regulate the transcription of several genes involved in sexual development using cotransfection assays. SRY (the sex-determining region of the Y chromosome) promoter was strongly activated by the WT1 isoforms without the KTS tripeptide, WT1(-)KTS, but was not activated by the WT1 isoforms with the KTS tripeptide, WT1(+)KTS, in all cells tested. The second alternative splicing site, which inserts the tripeptide KTS, alters the DNA binding capability. The MüAdullerian-inhibiting substance (MIS) promoter was strongly repressed by WT1(-)KTS isoforms and more weakly repressed by the WT1(+)KTS isoforms in Sertoli cells but not in HeLa cells. The androgen receptor (AR) promoter was strongly repressed by the WT1(-)KTS isoforms in all cells tested and was more weakly or not repressed by WT1(+)KTS isoforms depending on cell lines. Electrophoretic mobility shift assays showed strong binding by recombinant WT1(-)KTS protein and weaker or no binding by the WT1(+)KTS protein to DNA probes containing WT1 binding sites from these three promoters. The results of these functional and binding assays suggest that WT1 has an important role in regulation of genes involved in embryonal sexual development and that WT1 can function as a transcriptional activator.
WAGR综合征和Denys-Drash综合征中与威尔姆斯瘤相关的生殖器异常，以及威尔姆斯瘤基因（wt1）纯合突变小鼠性腺发育的失败，表明WT1可能也在性发育中发挥作用。为了阐明WT1在胚胎性发育中的作用机制，我们使用共转染试验研究了WT1的四种异构体如何调节几个参与性发育的基因的转录。在所有测试细胞中，不含KTS三肽的WT1异构体WT1(-)KTS能强烈激活Y染色体性别决定区（SRY）启动子，但含KTS三肽的WT1异构体WT1(+)KTS不能激活。插入三肽KTS 的第二个可变剪接位点改变了DNA结合能力。在支持细胞中，WT1(-)KTS异构体强烈抑制苗勒管抑制物质（MIS）启动子，WT1(+)KTS异构体的抑制作用较弱，而在HeLa细胞中则不然。在所有测试细胞中，WT1(-)KTS异构体强烈抑制雄激素受体（AR）启动子，WT1(+)KTS异构体的抑制作用较弱或不抑制，具体取决于细胞系。电泳迁移率变动分析表明，重组WT1(-)KTS蛋白与包含这三个启动子WT1结合位点的DNA探针有强烈结合，而WT1(+)KTS蛋白与探针的结合较弱或无结合。这些功能和结合试验的结果表明，WT1在调节参与胚胎性发育的基因方面具有重要作用，并且WT1可以作为转录激活因子发挥作用。
