Tm. Antalis et al., REGULATION OF THE PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-2 GENE IN MONOCYTES - LOCALIZATION OF AN UPSTREAM TRANSCRIPTIONAL SILENCER, Blood, 88(10), 1996, pp. 3686-3697
Transcriptional regulation of the plasminogen activator inhibitor type
-2 (PAI-2) gene appears to be an important factor in the response of m
ononuclear phagocytes to inflammation. We have investigated here the m
olecular basis for PAI-2 synthesis in monocytic cells by reporter gene
deletion analysis. A DNA fragment containing 5.1 kb of 5' flanking re
gion through to the start of the second exon was fused to a chloramphe
nicol acetyl transferase (CAT) reporter gene, transfected into macroph
age and nonmacrophage cells and tested for PAI-2 promoter-directed CAT
activity in the presence and absence of phorbol ester. Deletion analy
sis showed the existence of three major transcription regulatory regio
ns. (1) A positive regulatory region contained in the proximal promote
r mediates basal transcription and 12-phorbol 13-myristate acetate ind
ucibility. (2) A negative regulatory region, or silencer, present betw
een -1977 and -1675, was found to repress PAI-2 promoter activity in a
n orientation- and position-independent manner, but not in a cell-spec
ific manner, (3) A second positive regulatory element, located upstrea
m between approximately -5100 and -3300, appears to overcome inhibitio
n mediated by the silencer in a cell-specific manner, suggesting a mec
hanism for the regulation of this gene. We have localized the motif re
sponsible for silencer activity to a 28-bp DNA sequence containing a u
nique 12-bp palindrome centered at an Xba I restriction enzyme site, C
TCTCTAGAGAG, which is designated the PAI-2-upstream silencer element-1
(PAUSE-1). This element binds a specific PAUSE-1 binding factor as de
termined by mobility shift analysis, We conclude that PAI-2 gene trans
cription is regulated by both positive and negative control mechanisms
that may be important for the regulation of other genes as well. (C)
1996 by The American Society of Hematology.