An enhancer located between the neutrophil elastase and proteinase 3 promoters is activated by Sp1 and an Ets factor

The adjacent neutrophil elastase, proteinase 3, and azurocidin genes encode serine proteases expressed specifically in immature myeloid cells. Subclon es of a 17-kilobase (kb) murine neutrophil elastase genomic clone were asse ssed for their ability to stimulate the neutrophil elastase promoter in 32D cl3 myeloid cells. Region -9.3 to -7.3 kb stimulated transcription 7-fold, whereas other genomic segments were inactive. This enhancer is located in the second intron of the proteinase-3 gene and so may regulate more than on e gene in the myeloid protease cluster. Deletional analysis of the enhancer identified several segments which activated the neutrophil elastase and th ymidine kinase promoters 3-6-fold. The most active segment was a 220 base p air region centered at -8.6 kb, which activated transcription 31-fold. This segment contains an Sp1 consensus site, which bound Sp1, flanked by two Et s family consensus sequences, which bound PU.1, GABP, and an Ets factor pre sent in myeloid cell extracts. Mutation of the Sp1-binding site reduced enh ancer activity 8-fold in 32D cl3 cells, and mutation of either or both Ets- binding sites reduced activity 3-4-fold. Sp1 activated the distal enhancer 5-fold, GABP 8-fold, and the combination 8-fold in Schneider cells.