The role of the 5′-flanking region in the cell-specific transcription of the human von Willebrand factor gene

Abstract

Transcriptional regulation of the human von Willebrand factor (vWF) gene was investigated in calf pulmonary artery endothelial (CPAE), HeLa, COS 7 and Hep G2 cells. Various lengths of flanking sequences extending up to 2123 bp 5' of the transcription initiation site and containing 19 bp of the first exon, were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and these constructs were assayed in transient transfection assays. Sequences up to 89 bp upstream of the cap site showed transcriptional activity in all cell types. Sequences between -147 and -419 bp markedly reduced CAT activity in CPAE cells and abolished it in other cell lines. A domain from -592 to -810 bp generated low levels of expression only in CPAE cells. This transcriptional activity was repressed with constructs containing 1041 to 1240 bp upstream of the cap site. Endothelial cell-specific transcription was restored by a construct that contained 1286 bp upstream of the cap site. The additional 46 bp upstream of the negative regulatory domain were within the 5' end of an inverse human Alu-family DNA repeat. RNAase-protection assays confirmed the correct transcriptional initiation. The sequence between -89 and -420 contained at least one negative regulatory element able to repress the CAT gene expression controlled by the heterologous thymidine kinase promoter in all cell types. A construct that included the sequence from -89 to -1286 bp increased the transcriptional activity directed by the thymidine kinase promoter only in CPAE cells. These results indicate that negative and positive elements in the 5'-flanking region interact to regulate vWF gene expression.

Ets transcription factors bind and transactivate the core promoter of the von Willebrand factor gene

“…Previous results obtained in two laboratories including ours (Ferreira et al, 1993;Jahroudi and Lynch, 1994) indicated that a fragment spanning nt789 to +19 of the vWF gene promoter was active in cell types of endothelial and non-endothelial origins. In this study we have identi®ed the minimal sequences required for core promoter activity in HUVEC, CPAE, COS and HeLa cells.…”

“…Previous studies of the human vWF gene promoter have identi®ed a promoter spanning nt 789 to +19 which is active in all endothelial and non-endothelial cell-types which have been analysed (Ferreira et al, 1993;Jahroudi and Lynch, 1994). A survey of the sequence between nt 789 and 730 revealed that this region contains two consensus binding sites in the reverse orientation for Ets transcription factors at positions 749 to 756 (Ets binding site 1, EBS1) and 736 to 743 (EBS2).…”

“…As shown in Figure 2, the antisense RNA probe complementary to the pGvWCAT constructs protects a fragment migrating at the level of a 292 nt marker, corresponding to an RNA initiated at the correct cap site (Ferreira et al, 1993).…”

“…This promoter is not cell-type speci®c and an upstream negative regulatory region between nt 7500 and 789 represses its activity in all cell types analysed (Ferreira et al, 1993;Jahroudi and Lynch, 1994). Recent results showed that an NF1-like protein functions as a repressor of the human vWF promoter fragment 7487/+155 in bovine smooth muscle cells and in bovine aorta endothelial (BAE) cells through interaction with the sequence from 7440 to 7470 (Jahroudi et al, 1996).…”

A strategy for enhancing the transcriptional activity of weak cell type-specific promoters

“…[4][5][6]15 Frequently, however, specific promoters are inefficient activators of transcription which severely limits their applicability. A typical example is the von Willebrand factor (vWF) promoter, which exhibits a particularly high degree of endothelial cell (EC) specificity compared with other EC promoters, but is a poor activator of transcription [16][17][18][19] (see also Results below). Other EC promoters are stronger, but less specific, such as the promoters for PECAM-1/CD31 20,21 or flk-1/KDR.…”