title: Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites.
abstract: 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.
authors.affiliation: Department of Haematology, Imperial College London-Faculty of Medicine, Hammersmith Hospital Campus, London, United Kingdom. j.rance@imperial.ac.uk
authors.forename: James B
authors.initials: JB
authors.lastname: Rance
authors.forename: George A
authors.initials: GA
authors.lastname: Follows
authors.forename: Peter N
authors.initials: PN
authors.lastname: Cockerill
authors.forename: Constanze
authors.initials: C
authors.lastname: Bonifer
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Simmonds
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D001779:Blood Coagulation Factors / Q000235:genetics*; D003850:Deoxyribonuclease I; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011956:Receptors, Cell Surface / Q000235:genetics*; D015183:Restriction Mapping; D016415:Sequence Alignment; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D001779:Blood Coagulation Factors; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D016329:Sp1 Transcription Factor; C091963:activated protein C receptor; D003850:Deoxyribonuclease I