Characterization of the promoter for the human P-selectin gene.
P-selectin, an adhesion receptor for leukocytes, is synthesized selectively by megakaryocytes and endothelial cells. We have cloned the 5'-flanking region of the human P-selectin gene and conducted a preliminary analysis of its features. As determined by primer extension, RNase protection, and anchored polymerase chain reaction cloning, there were multiple transcriptional initiation sites from -95 to -25 nucleotides relative to the start of protein-coding sequence. Transfection of bovine aortic endothelial cells with serially truncated segments of the 5'-flanking region linked to luciferase indicated that the sequence from -249 to -13 was sufficient to promote high level gene expression. Deletions to -197, -147, and -128 gradually reduced expression to basal levels, and further deletion to -100 abolished expression. The sequence from -309 to -13 supported only basal luciferase expression in COS-7, 293, or HeLa cells. Putative regulatory elements in the short 5'-flanking sequence included a CACCC sequence, two inverted repeats similar to binding sites for the ETS and NF-kappa B/rel families, a GATA motif, and a sequence related to the GT-IIC element of the SV40 enhancer. The GATA element was functional, as it bound recombinant GATA-2, and mutations in the core sequence impaired both nuclear protein binding and gene expression. These data suggest that the P-selectin gene is regulated by a combination of cis elements and their cognate transcription factors.
Pan
J
J
W. K. Warren Medical Research Institute, Department of Medicine, University of Oklahoma, Health Sciences Center, Oklahoma City 73104.
McEver
R P
RP
D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000235:genetics*; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005091:Exons; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D008156:Luciferases / Q000096:biosynthesis / Q000378:metabolism; D008533:Megakaryocytes; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D013539:Simian virus 40; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D008156:Luciferases