Regulation of the plasminogen activator inhibitor type-2 gene in monocytes: localization of an upstream transcriptional silencer.
Transcriptional regulation of the plasminogen activator inhibitor type-2 (PAI-2) gene appears to be an important factor in the response of mononuclear phagocytes to inflammation. We have investigated here the molecular basis for PAI-2 synthesis in monocytic cells by reporter gene deletion analysis. A DNA fragment containing 5.1 kb of 5' flanking region through to the start of the second exon was fused to a chloramphenicol acetyl transferase (CAT) reporter gene, transfected into macrophage and nonmacrophage cells and tested for PAI-2 promoter-directed CAT activity in the presence and absence of phorbol ester. Deletion analysis showed the existence of three major transcription regulatory regions. (1) A positive regulatory region contained in the proximal promoter mediates basal transcription and 12-phorbol 13-myristate acetate inducibility. (2) A negative regulatory region, or silencer, present between -1977 and -1675, was found to repress PAI-2 promoter activity in an orientation- and position-independent manner, but not in a cell-specific manner. (3) A second positive regulatory element, located upstream between approximately -5100 and -3300, appears to overcome inhibition mediated by the silencer in a cell-specific manner, suggesting a mechanism for the regulation of this gene. We have localized the motif responsible for silencer activity to a 28-bp DNA sequence containing a unique 12-bp palindrome centered at an Xba I restriction enzyme site, CTCTCTAGAGAG, which is designated the PAI-2-upstream silencer element-1 (PAUSE-1). This element binds a specific PAUSE-1 binding factor as determined by mobility shift analysis. We conclude that PAI-2 gene transcription is regulated by both positive and negative control mechanisms that may be important for the regulation of other genes as well.
Queensland Cancer Fund Experimental Oncology Unit, Queensland Institute of Medical Research, Brisbane, Australia.
T M
TM
Antalis
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Costelloe
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Muddiman
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S
Ogbourne
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K
Donnan
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D005354:Fibrosarcoma / Q000473:pathology; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007938:Leukemia / Q000473:pathology; D016403:Lymphoma, Large B-Cell, Diffuse / Q000473:pathology; D008264:Macrophages / Q000378:metabolism*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D017396:Plasminogen Activator Inhibitor 2 / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D017396:Plasminogen Activator Inhibitor 2; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate