Molecular analysis of the human myeloperoxidase promoter region

Myeloperoxidase (MPO) is a granule protein, transiently expressed during th e promyelocyte stage of myeloid differentiation. It is transcribed in a sta ge and lineage specific manner. Studies of MPO gene regulation can help to elucidate the mechanism of normal and abnormal myeloid differentiation. Our preliminary data indicated the lack of basal promoter activity in the regi on immediately 5' to the MPO cDNA. Here, we report the results of the detai led molecular studies of the human MPO promoter region. To locate potential promoter elements active in HL60 cells, we made promoter deletion construc ts ranging in size from 200 bp to 4.5 kb of the 5' region of the hMPO gene, cloned into the chloramphenicol acetyl transferase (CAT) reporter vector. Following electroporation of the promoter constructs into HL60 cells, CAT e nzyme production was found only in the construct containing approximately t he 1 kb region upstream of the reported MPO cDNA. A separate set of constru cts was made to look for putative MPO enhancer elements. Several fragments upstream of the 'minimal' MPO promoter showed prominent transactivation of the TK promoter, indicating a possible enhancer. Tissue specificity of MPO promoter fragments was determined in myeloid cells arrested either before i nduction of MPO expression (KG1), during MPO expression (HLGO), or after it had ceased (U937), as well as in non-MPO expressing non-myeloid cells. The construct containing an approximate 1000 bp fragment of the 5' region of M PO was found to direct CAT expression only in HL60 cells. The 3'-truncation s of this promoter region resulted in loss of tissue-specificity, while the promoter activity remained largely unchanged. A negative regulatory elemen t was found upstream of the MPO promoter which repressed heterologous promo ters in all the tested cell lines. Enhancer elements showed ho tissue- or s tage-specificity that were characteristic for native MPO gene. Sequence ana lysis of the putative MPO promoter region showed a number of potential tran scription factor binding sites. Of special interest is the region containin g the purine-rich site that can bind proteins from the Ets-family of transc ription factors and a duplicate GATA-like site. When inserted upstream of a reporter containing the minimal Herpes simplex viral thymidine kinase (HSV -TK) promoter (into pBL2CAT plasmid) this site strongly activated the TK pr omoter in transfected myeloid cells. Further studies showed that oligonucle otides derived from the MPO promoter region bind multiple proteins in a ban d-shift assay. Taken together, our experiments located the regulatory eleme nts important for human MPO gene expression in HL60 promyelocytes.