Regulation of CD34 expression in differentiating M1 cells.

CD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells, but not on mature blood cells. In the present study we found that CD34 downregulation during hematopoiesis occured at the level of transcriptional initiation. Two transcription initiation sites (TISs) were identified in each of three different CD34+ cell lines; these TISs were located at 120 and 80 bp 5' of the translation start site, respectively. The promoter lacks TATA elements and, like other TATA-less promoters, the TISs conform to the consensus sequence for an INR (PyPyCAPyPyPyPy). An additional 3000 bp of upstream genomic DNA were sequenced and found to contain consensus sites for transcription factors, suggesting their potential role in gene regulation. Transient transfection assays using CD34 promoter-luciferase reporter constructs, containing sequences up to 3 kb upstream and inclusive of the TIS, indicate that this promoter drives transcription in hematopoietic CD34+ cells but not CD34+ nonhematopoietic cells. Both cell type specific expression and full promoter activity are maintained in constructs that contain as little as 454 bp upstream of the TISs. Optimal promoter activity requires the 5' untranslated region of exon 1, which contains a 51-bp element that has the potential to form an extensive secondary structure. In the plasmid DNA, however, this secondary structure was not detectable by P1 nuclease digestion. At least three proteins present in uninduced M1 nuclear extracts bind to this element. Two of the three proteins were identified as Sp 1 and Sp 3 based on supershift experiments. These data suggest that CD34 expression by hematopoietic stem and progenitor cells involves hematopoietic cell-specific factors that interact with regulatory elements within the first 230 bp of the promoter and that optimal expression requires a 60-bp segment of the 5' untranslated region.