NF1 regulatory element functions as a repressor of tissue plasminogen activator expression.
OBJECTIVE
Analysis of the distribution of endothelial cell tissue plasminogen activator (tPA) in the vasculature of rodents and primates demonstrated that tPA is constitutively expressed predominantly in small artery endothelial cells of brain and lung. The regulatory elements responsible for the highly selective expression of arterial endothelial cell tissue plasminogen activator were sought.


RESULTS
Transcription factor binding sites were defined by electrophoretic mobility-shift assay (EMSA) analysis using rat lung and brain nuclear extracts and the tPA promoter sequence from -609 to +37 bp. Protein binding to the promoter was found to be mediated by an NF1 site between -158 and -145 bp upstream from the transcriptional start site. Specific binding was confirmed through mutational analysis and competition binding studies. Infection of endothelial cells with a tPA promoter-green fluorescent protein (GFP) (-609 to +37 bp) reporter construct resulted in expression of the GFP, whereas no expression was found in smooth muscle cells. Mutation of the NF1 site increased the GFP expression indicating that the element acts as a repressor.


CONCLUSIONS
These results suggest that the 600 bp of the tPA promoter upstream of the transcription start site conveys cell specificity to tPA expression and that an NF1 site within this region acts as a repressor.
Pham
Nhat-Long
NL
Division of Vascular Biology, La Jolla Institute for Molecular Medicine, San Diego Calif 92121, USA.
Franzen
Amy
A
Levin
Eugene G
EG
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D020868:Gene Silencing; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D051839:NFI Transcription Factors; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D051839:NFI Transcription Factors; D014157:Transcription Factors; C080333:transcription factor nuclear factor 1; D049452:Green Fluorescent Proteins; D010959:Tissue Plasminogen Activator