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. Subclones of a 17-kilobase (kb) murine neutrophil elastase genomic clone were assessed 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 one gene in the myeloid protease cluster. Deletional analysis of the enhancer identified several segments which activated the neutrophil elastase and thymidine kinase promoters 3-6-fold. The most active segment was a 220 base pair region centered at -8.6 kb, which activated transcription 31-fold. This segment contains an Sp1 consensus site, which bound Sp1, flanked by two Ets family consensus sequences, which bound PU.1, GABP, and an Ets factor present in myeloid cell extracts. Mutation of the Sp1-binding site reduced enhancer 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.