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J Thromb Haemost. 2009 Aug;7(8):1384-92. doi: 10.1111/j.1538-7836.2009.03501.x. Epub 2009 May 30.

A +220 GATA motif mediates basal but not endotoxin-repressible expression of the von Willebrand factor promoter in Hprt-targeted transgenic mice.

Author information

1
The Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA.

Abstract

BACKGROUND:

The von Willebrand factor (VWF) gene is a marker for spatial and temporal heterogeneity of the endothelium. A GATA motif at +220 has been implicated in basal VWF expression in vitro. Other studies have shown that GATA3 and VWF are transcriptionally downregulated in response to inflammatory mediators.

OBJECTIVES:

Our goal was to determine the importance of the +220 GATA motif in mediating expression of VWF promoter in vivo, and to elucidate whether the GATA element plays a role in spatial and/or temporal regulation of VWF expression.

METHODS:

ChIP and electrophoretic mobility shift assays were carried out in human umbilical vein endothelial cells (HUVEC). Reporter gene constructs containing 3.6 kb of the human VWF promoter with and without a mutation of the +220 GATA element were transfected into cultured endothelial cells or targeted to the Hprt locus of mice. The Hprt-targeted mice were subjected to endotoxemia.

RESULTS:

In protein-DNA binding assays, the +220 GATA motif bound GATA-2, -3 and -6. Mutation of the GATA site resulted in reduced basal promoter activity in HUVEC. When targeted to the Hprt locus of mice, the GATA mutation resulted in a significant, proportionate reduction of promoter activity in LacZ expressing vascular beds. Systemic administration of lipopolysaccharide (LPS) resulted in a widespread reduction in VWF mRNA expression and promoter activity. LPS-mediated repression of the VWF promoter was unaffected by the GATA mutation.

CONCLUSIONS:

A region of the VWF promoter between -2182 and the end of the first intron contains information for LPS-mediated gene repression. The +220 GATA motif is important for basal, but not LPS-repressible expression of the VWF gene.

PMID:
19496923
PMCID:
PMC5303625
DOI:
10.1111/j.1538-7836.2009.03501.x
[Indexed for MEDLINE]
Free PMC Article
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