Sequences in Intron 51 of the VWF Gene Target Promoter Activation to a Subset of Lung Endothelial Cells in Transgenic Mice.

Abstract The VWF gene, located on chromosome 12, is 178 kb long and contains 52 exons. VWF is synthesized exclusively by endothelial cells and megakaryocytes. We have previously characterized a region of the VWF gene spanning sequences −487 to +247 that functions as an endothelial specific promoter in vitro. Subsequently a number of transacting factors including, NF1, Oct 1, Ets, GATA6, NFY and Ebp4 that positively and negatively regulate the activity of this promoter were identified by us and others. However, in vivo analysis of the promoter demonstrated that this promoter fragment (sequences −487 to +247) targets activation of fused heterologous transgenes (LacZ and amyloid precursor proteins) specifically and exclusively to brain vascular endothelial cells of transgenic mice. A longer VWF promoter fragment, including 2182 bp of the 5′ flanking sequences, the first exon and the first intron was reported by Aird et al to activate LacZ transgene expression in endothelial cells of the heart and muscle as well as brain of transgenic mice. Considering that endogenous VWF expression is observed in almost all endothelial cells, these results suggested that additional VWF gene sequences were required for transcriptional activation of the VWF promoter in vascular endothelial cells of multiple other organs in vivo; and that distinct regions of the VWF gene are required to achieve promoter activity in endothelial cells of distinct organs. To identify additional cis acting elements within the VWF gene that may participate in its transcriptional regulation generally and/or in distinct organs, we explored the possibility that such sequences may be located in VWF chromatin regions that show hypersensitivity to DNase I. We have now identified a region within intron 51 of the VWF gene that is DNase I hypersensitive (HSS) specifically in non-endothelial cells. This region was shown to interact with YY1 transcription factor in a manner that forms endothelial and non-endothelial specific complexes. In vitro transfection analyses demonstrated that HSS sequences containing this YY1 binding site significantly increased a heterologous SV40 promoter activity specifically in endothelial cell and that this increase was dependent on the presence of an intact YY1 binding site. In contrast, the HSS sequences significantly decreased the SV40 promoter activity in non-endothelial cells. These results suggested that the HSS sequences may participate in activation of gene expression in an YY1 dependent manner in endothelial cells, while repress gene expression in non-endothelial cells. Nevertheless the HSS sequences did not significantly affect the homologous VWF promoter activity that was analyzed by in vitro transfection analyses. However, in vivo analyses demonstrated that addition of these sequences to the VWF promoter (−487 to +247) results in promoter activation in lung and brain vascular endothelial cells. These results demonstrate that the HSS sequences in intron 51 of the VWF gene participate in organ specific regulation of VWF gene expression, an observation that could not be determined by in vitro analysis. These analyses suggest that the HSS sequences contain cis-acting elements that are specifically necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo.

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