Southwestern blot mapping of potential regulatory proteins binding to the DNA encoding plasminogen activator inhibitor type 2

Abstract

In order to investigate the molecular basis for the regulated expression of plasminogen activator inhibitor type 2 (PAI-2), we sought to identify monocyte-derived nuclear factors which interact with the PAI-2 gene. We have explored the application of Southwestern blot mapping as an approach for identifying specific DNA-protein interactions and targeting potential regulatory DNA elements. The procedure involves an initial global screening of a crude preparation of nuclear proteins with radiolabelled DNA fragments (200–300 bp) derived from a large region (8.8 kb) of PAI-2. The bound DNA fragments are eluted and their location within PAI-2 mapped by Southern blot hybridization analysis. We have used this procedure to examine the differential binding of nuclear factors from the U937 monocytic cell in the absence and in the presence of the differentiating agent, 12-phorbol 13-myristate acetate (PMA), in order to identify proteins that bind specifically to the 5' flanking promoter region and first intron of PAI-2. Eleven DNA-binding proteins ranging in molecular mass from 27 to 92 kDa were identified, and the results define three regions of the gene which contain DNA-binding sites which may be involved in the transcriptional regulation of PAI-2. Deletion analysis using a series of 5' deletion mutants spanning PAI-2 fused to a chloramphenicol acetyltransferase-encoding reporter gene ( cat) demonstrates that two of the regions identified by Southwestern blot mapping contain elements which can function to modulate PAI-2 expression in transient transfections of U937 cells.