REGULATION OF CD34 EXPRESSION IN DIFFERENTIATING M1 CELLS

CD34 is a cell surface glycoprotein expressed on hematopoietic stem an d progenitor cells, but not on mature blood cells. In the present stud y we found that CD34 downregulation during hematopoiesis occured at th e level of transcriptional initiation. Two transcription initiation si tes (TISs) were identified in each of three different CD34(+) cell lin es: these TISs were located at 120 and 80 bp 5' of the translation sta rt site, respectively. The promoter lacks TATA elements and, like othe r 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. Transien t transfection assays using CD34 promoter-luciferase reporter construc ts, containing sequences up to 3 kb upstream and inclusive of the TLS, indicate that this promoter drives transcription in hematopoietic CD3 4(+) cells but not CD34(+) nonhematopoietic cells. Both cell type-spec ific expression and full promoter activity are maintained in construct s that contain as little as 454 bp upstream of the TISs. Optimal promo ter activity requires the 5' untranslated region of exon I, which cont ains a 51-bp element that has the potential to form an extensive secon dary structure. In the plasmid DNA, however, this secondary structure was not detectable by P1 nuclease digestion. At least three proteins p resent in uninduced M1 nuclear extracts bind to this element. Two of t he 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 factor s that interact with regulatory elements within the first 230 bp of th e promoter and that optimal expression requires a 60-bp segment of the 5' untranslated region.