Functional activity of three distinct myeloperoxidase (MPO) promoters in human myeloid cells

Abstract

The enzyme myeloperoxidase (MPO) is synthesized only in myeloid and monocytic cells, making it an important marker of the myeloid lineage. Transcription of the MPO gene is turned on early during the myeloblast stage of myeloid differentiation and is turned off when myeloid precursors are induced to differentiate along any one of a number of pathways. MPO transcripts show heterogeneity in size and sequence due, in part, to differential RNA splicing. We recently reported transfection studies which showed the presence of three distinct MPO promoters in the 5′-flanking region of the human MPO gene, suggesting that MPO transcription may also be regulated through the use of multiple promoters. We now report results of primer extension and RT-PCR experiments designed to determine if transcription of the human MPO gene is initiated at multiple promoter sites in vivo. MPO RNA obtained from myeloid cell lines was analyzed by primer extension using primers located at various sites between bp −1100 and bp +120 of the MPO gene. In addition, RT-PCR experiments were carried out using primer pairs located at intervals between bp −1000 and bp +2500 of the MPO gene. MPO transcripts were found to be initiated at three sites located about bp −920, bp −310, and bp +1 of the MPO gene, corresponding closely to our previously described P3, P2 and P1 promoters, respectively. Transcription initiated at the P1 site gave rise to large transcripts and showed the expected downregulation following induction of differentiation. On the other hand, transcripts initiated at the P3 and P2 sites did not show downregulation following induction of macrophage differentiation by TPA, and most did not appear to extend into the MPO coding region. Northern blot analysis of transcripts initiated at the P3 and P2 sites suggested that transcription at these sites was non-tissue-specific and indicated that many of these transcripts undergo premature termination. These results demonstrate that the MPO gene is transcribed in vivo primarily using the P1 promoter and that the low level of transcription occurring at the P2 and P3 sites is nonspecific and does not contribute significantly to physiologic regulation of MPO gene expression.