Regulatory sequences and protein-binding sites involved in the expression of the rat plasminogen activator inhibitor-1 gene.
Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor that inhibits both tissue-type and urokinase-type plasminogen activators. Expression of PAI-1 is regulated by growth factors, cytokines, and hormones. To determine the molecular mechanisms involved in the basal expression of the rat PAI-1 gene, we have analyzed the cis-acting sequences and the trans-acting factors involved in the transcription of this gene in the HTC rat hepatoma cell line. DNase I protection analyses revealed eight regions within the first 764 base pairs of 5'-flanking sequence that interact specifically with HTC cell nuclear proteins. The proteins that bind to five of the eight footprinted sites were identified as PEA3-, Sp1-, and CTF/NF-1-like proteins using competition electrophoretic mobility shift assays. The expression of fusion genes containing progressive 5' deletions of the rat PAI-1 promoter linked to the chloramphenicol acetyltransferase reporter gene were analyzed in transient transfection experiments in HTC cells. These studies demonstrated the Sp1 and CTF/NF-1 sites to be important for transcriptional activation. Two of the footprinted sites contain the sequence 5'-TTTGn(n)TCAAT-3' and were shown in competition electrophoretic mobility shift assays to bind the same or related protein(s). Sequences containing these sites, from -764 to -628 base pairs, and from -266 to -188 base pairs, were identified in functional studies as repressor elements of transcription.
Johnson
M R
MR
Department of Human Genetics, University of Michigan Medical School, Ann Arbor 48109-0618.
Bruzdzinski
C J
CJ
Winograd
S S
SS
Gelehrter
T D
TD
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA; D016172:DNA Fingerprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D015849:Plasminogen Inactivators; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase