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Prepublished online as a Blood First Edition Paper on January 30, 2003; DOI 10.1182/blood-2002-05-1570. This Article Full Text (PDF) All Versions of this Article: 2002-05-1570v1 101/11/4393    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Rance, J. B Articles by Simmonds, R. E Search for Related Content PubMed PubMed Citation Articles by Rance, J. B Articles by Simmonds, R. E Social Bookmarking                   What's this? Submitted May 28, 2002 Accepted January 21, 2003 Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites James B Rance*, George A Follows, Peter N Cockerill, Constanze Bonifer, David A Lane, and Rachel E Simmonds Department of Haematology, Imperial College London, Faculty of Medicine, London, United Kingdom Department of Molecular Medicine, University of Leeds, Leeds, United Kingdom * Corresponding author; email: j.rance{at}imperial.ac.uk. The human endothelial cell protein C receptor (hEPCR) is normally expressed by the endothelium of large blood vessels but the molecular basis for its in vivo specificity is uncertain. In this study, DNaseI hypersensitive site mapping demonstrated the presence of a hypersensitive site in the 5' flanking region of the hEPCR gene in endothelial cells and certain transformed cells (HeLa and U937), known to express hEPCR in vitro. Conversely, this site was only weakly hypersensitive in HepG2 cells, cells which do not express hEPCR mRNA. Functional analysis of this 5' flanking region by in vivo dimethylsulfate footprinting in cultured endothelial cells identified multiple regions, containing high and low homology consensus Sp1 binding sequences, that were protected from methylation in endothelial cells. These sequences were not protected in HepG2 cells. Reporter gene analysis of this region in endothelial cells demonstrated the presence of promoter activity conferred by the proximal 572 bp but failed to identify a functional TATA-box. This promoter was inactive in HepG2 cells. Electrophoresis mobility shift assays using endothelial cell nuclear extracts identified Sp1 family proteins binding to sites that were protected during footprinting. Sp1 sites were identified in regions at -368, -232, -226, -201, -146 and -102 bp relative to the translation start site. With the exception of the site at -102 bp, each identified Sp1 binding site made a positive contribution to reporter gene expression, although no individual site was critically important. We conclude that transcription factor binding to Sp1 binding sites in the 5' flanking region is critical for normal hEPCR gene expression in endothelial cells. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: L. R. Mollica, J. T. B. Crawley, K. Liu, J. B. Rance, P. N. Cockerill, G. A. Follows, J.-R. Landry, D. J. Wells, and D. A. Lane Role of a 5'-enhancer in the transcriptional regulation of the human endothelial cell protein C receptor gene Blood, August 15, 2006; 108(4): 1251 - 1259. [Abstract] [Full Text] [PDF] B. Saposnik, J.-L. Reny, P. Gaussem, J. Emmerich, M. Aiach, and S. Gandrille A haplotype of the EPCR gene is associated with increased plasma levels of sEPCR and is a candidate risk factor for thrombosis Blood, February 15, 2004; 103(4): 1311 - 1318. [Abstract] [Full Text] [PDF]

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