In both tissue sections and cell culture, the endothelial nature of a cell is most commonly determined by demonstration of its expression of von Willebrand factor (vWf) protein and/or mRNA. Thus, the mechanism of cell-type-specific transcriptional regulation of the vWf gene is central to studying the basis of endothelialcell-specific gene expression. In this study, deletion analyses were carried out to identify the region of the vWf gene which regulates its endothelial-cell-specific expression. A 734-bp fragment which spans the sequence from -487 to +247 relative to the transcription start site was identified as the cell-type-specific promoter. It consists of a minimal core promoter located between -90 and +22, a strong negative regulatory element located upstream of the core promoter (ca. -500 to -300), and a positive regulatory region located downstream of the core promoter in the first exon. The activity of the core promoter is not cell type specific, and the negative regulatory region is required to inhibit its activity in all cell types. The positive regulatory region relieves this inhibition only in endothelial cells and results in endothelial-cell-specific gene expression. The positive regulatory region contains sequences predicting possible SP1, GATA, and octamer binding sites. Mutations in either the SP1 or octamer sequence have no effect on transcriptional activity, while mutation in the GATA binding element totally abolishes the promoter activity. Evidence that a GATA factor is involved in this interaction is presented. Thus, the positive regulatory region with an intact GATA binding site is required to overcome the inhibitory effect of the negative regulatory element and activate vWf gene expression in an endothelial-cell-specific manner.
Severe von Willebrand disease is characterized by undetectable or trace quantities of von Willebrand factor in plasma and tissue stores. We have studied the genomic DNA of 10 affected individuals from six families with this disorder using probes from the 5' and 3' ends of the vWF cDNA and with a probe extending from the 5' end into the central region. Southern blots of restriction endonuclease digests and gene dosage analysis measurements carried out with quantitative slot blots of undigested genomic DNA separated these patients into three groups. The first group consisted of a family with complete homozygous deletions of the vWF gene in the four probands. Gene dosage analysis was consistent with heterozygous deletions in both of the asymptomatic parents and four asymptomatic siblings of this kindred (P less than 0.01). The second group was comprised of a family in which there was a complete heterozygous deletion of the vWF gene in the proband and one asymptomatic parent, suggesting that a different type of genetic abnormality was inherited from the other parent. Thus, the patient appeared to be doubly heterozygous for interacting genetic abnormalities affecting vWF expression. In the third group, no gene deletions could be detected. Alloantibodies developed only in the kindred with homozygous deletions. These techniques should prove useful in identifying carriers of severe von Willebrand disease and also in defining patients predictably at risk of developing alloantibodies to vWF.
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