St-Arnaud R, Moir J M
Genetics Unit, Shriners Hospital for Crippled Children, Québec, Canada.
Mol Cell Biol. 1993 Mar;13(3):1590-8. doi: 10.1128/mcb.13.3.1590-1598.1993.
The Wnt-1 proto-oncogene is essential for proper development of the midbrain and is expressed in a spatially and temporally restricted manner during central nervous system development in mice. In vitro, the gene is specifically transcribed during the retinoic acid (RA)-induced neuroectodermal differentiation of the P19 line of embryonal carcinoma cells. The P19 cells differentiate into neurons, astrocytes, and fibroblast-like cells when treated with RA. Treatment of the cells with dimethyl sulfoxide leads to differentiation along mesodermal lineages, including skeletal and cardiac muscle. We have used the P19 cell line to study the Wnt-1 promoter and identify and characterize the transcription factor(s) that regulates the differentiation-specific transcription of Wnt-1 in RA-treated P19 cultures. Transient-transfection assays have revealed that a 230-bp region comprising positions -278 to -47 of the 5' upstream Wnt-1 sequence was sufficient to direct RA-specific transcription. This promoter fragment was shown to contain a binding site for a nuclear factor that was not detected in undifferentiated P19 stem cells or their dimethyl sulfoxide-treated derivatives but was induced in differentiating RA-treated cells. This factor was termed Wnt-1-inducing factor-1 (WiF-1). DNase I footprinting analysis has identified the G/C-rich WiF-1 binding site, and UV cross-linking studies have shown that WiF-1 is a protein with an M(r) of 65,000. WiF-1 binding activity was also detected in postpubertal mouse testis, the only tissue that expresses Wnt-1 in adults. Site-directed mutations that inhibited WiF-1 binding to the Wnt-1 promoter concomitantly abolished the activity of the promoter in RA-treated P19 cells. The active WiF-1 protein was purified by DNA affinity chromatography. Our data suggest that WiF-1 is a novel G/C box-binding transcription factor and support a physiological role for WiF-1 in the developmentally regulated expression of Wnt-1.
Wnt-1原癌基因对中脑的正常发育至关重要，且在小鼠中枢神经系统发育过程中以空间和时间受限的方式表达。在体外，该基因在维甲酸（RA）诱导的胚胎癌细胞P19系神经外胚层分化过程中特异性转录。用RA处理P19细胞时，这些细胞会分化为神经元、星形胶质细胞和成纤维细胞样细胞。用二甲基亚砜处理这些细胞会导致沿中胚层谱系分化，包括骨骼肌和心肌。我们利用P19细胞系研究Wnt-1启动子，并鉴定和表征在RA处理的P19培养物中调节Wnt-1分化特异性转录的转录因子。瞬时转染分析表明，包含5'上游Wnt-1序列-278至-47位的230bp区域足以指导RA特异性转录。该启动子片段显示含有一个核因子结合位点，该位点在未分化的P19干细胞或其经二甲基亚砜处理的衍生物中未检测到，但在分化的RA处理细胞中被诱导。该因子被称为Wnt-1诱导因子-1（WiF-1）。DNase I足迹分析确定了富含G/C的WiF-1结合位点，紫外线交联研究表明WiF-1是一种分子量为65000的蛋白质。在青春期后小鼠睾丸中也检测到了WiF-1结合活性，睾丸是成年期唯一表达Wnt-1的组织。抑制WiF-1与Wnt-1启动子结合的定点突变同时消除了RA处理的P19细胞中启动子的活性。活性WiF-1蛋白通过DNA亲和层析纯化。我们的数据表明WiF-1是一种新型的G/C盒结合转录因子，并支持WiF-1在Wnt-1发育调控表达中的生理作用。
