A CELL PROLIFERATION-DEPENDENT MULTIPROTEIN COMPLEX NC-3A POSITIVELY REGULATES THE CD34 PROMOTER VIA A TCATTT-COUTAINING ELEMENT

The CD34 cell surface antigen is a glycoprotein expressed by hematopoi etic stem and progenitor cells and also by certain nonhematopoietic ce ll types. Because CD34 expression is regulated both at the transcripti onal and the posttranscriptional level, we attempted to identify facto rs that, by interacting with the 5' flanking region of the human CD34 gene, may regulate its promoter activity in proliferating hematopoieti c cells. By electrophoretic mobility shift assay, UV cross-linking and DNase I footprinting analyses, we identified a multiprotein complex, designated NC-3A, that specifically interacts with the CD34 promoter r egion from nucleotides -375 to -351. Sequence analysis of this region revealed the presence of a distinct motif, TCATTT. Chloramphenicol ace tyl-transferase assays used to assess promoter activity in transiently transfected cells showed that this TCATTT-containing element, which i s conserved in both the human and the murine CD34 genes, mediates posi tive regulatory activity in hematopoietic and nonhematopoietic cells, and acts as an enhancer when placed upstream of a heterologous promote r. Moreover, loss of CD34 promoter activity was caused by mutation of the TCATTT motif. In addition, the interaction of the nuclear multipro tein complex NC-3A with this enhancer element is prolife ration-depend ent. These data indicate that, although not cell-type specific, the fo rmation of a multiprotein complex NC-OA interacting with the region fr om nucleotides -375 to -351 plays an important role in controlling CD3 4 promoter activity in proliferating hematopoietic cells. (C) 1996 by The American Society of Hematology.