Identification and characterization of the human myeloperoxidase promoter.
Myeloperoxidase (MPO) is a microbicidal protein present in the primary granules of myeloid cells. Transcription of the MPO gene is turned on only during the late myeloblast and promyelocyte stages of myeloid maturation. Identification of cis-regulatory elements and transcription factors which regulate the MPO gene should, therefore, shed light on myeloid maturation. We report transfection and in vitro transcription experiments which demonstrate promoter activity in the proximal 5'-flanking region of the human MPO gene. Using a chloramphenicol acetyl transferase (CAT) reporter vector system, and segments of the 5'-flanking MPO DNA, we constructed a series of MPO promoter-CAT expression vectors. By electroporation and lipofectin-mediated transient transfection assays, as well as by in vitro transcription studies, a 594-bp MPO DNA sequence (bp -583 to +11) showed promoter activity in a variety of MPO-expressing and non-MPO-expressing cell lines. Compared with the SV40 early promoter, the MPO promoter had greater relative activity in MPO-expressing than in non-MPO-expressing cell lines, suggesting slight tissue specificity. However, a CAT reporter plasmid containing 1099-bp of 5'-flanking MPO DNA showed greater specificity for MPO expressing cell lines. Analysis of a group of promoter deletion mutants showed that the minimal promoter was contained in a DNA fragment extending from bp-128 to +11. The remainder of the promoter region contained several segments which appeared to enhance the activity of the minimal promoter. One such enhancer sequence was homologous to an enhancer previously described in the human elastase promoter. Activity of the 594-bp MPO promoter in HL-60 was reduced by only approximately 30% following treatment of the cells with chemical inducers of maturation, but the 1099-bp MPO promoter showed 60% reduction in activity after DMSO treatment. A previously described enhancer region in intron 9 of the MPO gene had little or no effect on activity of the 594-bp MPO promoter. The availability of the MPO promoter will facilitate determination of other factors involved in the regulation of this myeloid-specific gene.
Austin
G E
GE
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA.
Zhao
W G
WG
Zhang
W
W
Austin
E D
ED
Findley
H W
HW
Murtagh
J J
JJ
D001483:Base Sequence; D002874:Chromosome Mapping; D004273:DNA, Neoplasm / Q000235:genetics; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology / Q000235:genetics; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009195:Peroxidase / Q000235:genetics*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000201:enzymology / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012680:Sensitivity and Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D009195:Peroxidase; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide