Molecular analysis of the human myeloperoxidase promoter region.

Myeloperoxidase (MPO) is a granule protein, transiently expressed during the promyelocyte stage of myeloid differentiation. It is transcribed in a stage 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 region immediately 5' to the MPO cDNA. Here, we report the results of the detailed molecular studies of the human MPO promoter region. To locate potential promoter elements active in HL60 cells, we made promoter deletion constructs 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 enzyme production was found only in the construct containing approximately the 1 kb region upstream of the reported MPO cDNA. A separate set of constructs was made to look for putative MPO enhancer elements. Several fragments upstream of the 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 induction of MPO expression (KG1), during MPO expression (HL60), 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 MPO was found to direct CAT expression only in HL60 cells. The 3'-truncations of this promoter region resulted in loss of tissue-specificity, while the promoter activity remained largely unchanged. A negative regulatory element was found upstream of the MPO promoter which repressed heterologous promoters in all the tested cell lines. Enhancer elements showed no tissue- or stage-specificity that were characteristic for native MPO gene. Sequence analysis of the putative MPO promoter region showed a number of potential transcription factor binding sites. Of special interest is the region containing the purine-rich site that can bind proteins from the ets-family of transcription 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 promoter in transfected myeloid cells. Further studies showed that oligonucleotides derived from the MPO promoter region bind multiple proteins in a band-shift assay. Taken together, our experiments located the regulatory elements important for human MPO gene expression in HL60 promyelocytes.

Localization of the c-ets-2 transactivation domain.

The human ets-2 proto-oncogene is one of the homologs of the v-ets gene, found in avian acutely transforming retrovirus E26 (D. Leprince, A. Gegonne, J. Call, C. de Taisne, A. Schneeberger, C. Lagrou, and D. Stehelin, Nature [London] 306:395-397, 1983; M. F. Nunn, P. H. Seeburg, C. Moscovici, and P. H. Duesberg, Nature [London] 306:391-395, 1983), which causes leukemia in chickens. We used the DNA-binding domain of yeast transcriptional activator GAL4 to locate the transactivation region of human ets-2. The transactivation domain of ets-2 was found in the N-terminal part of the protein, which is homologous to ets-1, and can be disrupted by deletion of a stretch of acidic amino acid residues. A transactivation-deficient mutant of ets-2 failed to transform Rat-1 cells and suppressed the transforming activity of coexpressed wild-type ets-2. A mutation in the putative DNA-binding region of ets-2 abolished transforming activity. We show that the motif crucial for ets-2 transactivation capability is necessary for transforming activity in Rat-1 cells. Mutant ets-2 protein that lacks the transactivation domain has a dominant negative effect on transformation by wild-type ets-2. We were unable to detect ets-2-dependent transcriptional regulation of several enhancers containing ets-binding motifs.