AN NF1-LIKE PROTEIN FUNCTIONS AS A REPRESSOR OF THE VON-WILLEBRAND-FACTOR PROMOTER

The expression of the von Willebrand factor (vWf) gene is restricted t o endothelial cells and megakaryocytes, We have previously reported th e identification of a region of the vWf gene that regulates its cell-t ype-specific expression in cell culture, This region (spanning nucleot ides -487 to +247) consists of a core promoter (spanning nucleotides - 90 to +22), a positive regulatory region (spanning nucleotides +155 to +247), and a negative regulatory region spanning nucleotides -312 to -487. To identify the trans-acting factor(s) that interacts with the n egative regulatory region, we carried out gel mobility and DNase1 foot print analyses of sequences -312 to -487, These analyses demonstrated that an NF1-like protein interacts with DNA sequences spanning -440 to -470 nucleotides in the negative regulatory region of the vWf promote r, Base substitution mutations of the NF1 binding site abolished the N F1-DNA interaction, Furthermore, mutation of the NF1 binding site in t he promoter fragment (-487 to +155) that contained the core and the ne gative regulatory region resulted in activation of the mutant promoter in both endothelial and nonendothelial cells, The wild type promoter fragment (-487 to +155) was not activated in either cell type. These r esults demonstrate that an NF1-like protein functions as a repressor o f vWf promoter activity, In contrast, the mutation of the same NF1 bin ding site, but now in the context of the larger 734-base pair endothel ial cell-specific promoter fragment (-487 to +247), did not result in promoter activation in nonendothelial cells, The data indicate that th ere are additional repressor elements within the vWf promoter region s uppressing its activity specifically, in nonendothelial cells, and sug gest that there is a secondary repressor element(s) that is located in the terminal region of the first exon of this gene.