Mutation of the novel NFY binding element (R) activates the VWF promoter in nonendothelial cells.

A schematic representation of the VWF-HGH plasmids containing the wild-type and mutant VWF promoter fragments is shown in the left. Solid triangles represent the base substitutions of CCG (nucleotides +226 to +228) in the novel NFY binding site. The open triangle represents the base substitution mutation in upstream NF1 binding site. HEK293 (A) and BAE (B) cells were stably transfected with a mixture of each VWF-HGH test plasmid and a plasmid containing the neomycin resistance gene. Transfected cells were continuously grown in presence of neomycin analogue G418 to select for stably transfected cells. Individual colonies of transfected cells were selected and growth hormone expression from each colony was determined and normalized to the cell number. The average level of growth hormone expression from HGH-1 plasmid in BAE and HEK293 cells was 25 ng/mL per 100 000 and 18 ng/mL per 100 000 cells, respectively, and the level of expression from other plasmids is shown as percentages of these levels in each graph. The results shown are the average of 12 to 15 colonies from 2 independent transfections for each plasmid and the error bar represents the standard deviation. The P values were more than .05 for transfections in BAE cells, which demonstrates no statistical significance in level of expressions from various plasmids in this cell type. The P values were less than .01 where indicated by double asterisk for transfection in HEK293. The bracket in graph A represents the plasmid activities that were compared for statistical analysis.