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.
Antalis
T M
TM
Queensland Cancer Fund Experimental Oncology Unit, Queensland Institute of Medical Research, Brisbane, Australia.
Costelloe
E
E
Muddiman
J
J
Ogbourne
S
S
Donnan
K
K
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