Regulation of CD34 transcription by Sp 1 requires sites upstream and downstream of the transcription start site

Objective. CD34 is a cell surface glycoprotein expressed on hematopoietic s tem and progenitor cells, but not on fully differentiated cells in the peri pheral blood. To better understand the molecular regulation of early hemato poiesis, we are elucidating the mechanisms of CD34 transcriptional regulati on. Materials nod Methods. By deletion analysis we identify a 39-bp element in the proximal region of murine CD34 promoter that is critical for promoter a ctivity. Electromobility shift assays indicate that nuclear proteins of hem atopoietic cells bind to this domain; however, the presence of this binding activity does not correlate directly with CD34 expression, Results, Using methylation interference, the DNA binding site for this acti vity was localized to four guanine residues within the GGGGTCGG sequence fr om -48 to -54 bp, When the four contact guanines were mutated, both protein binding and promoter activity were abolished, Although this sequence does not contain a standard consensus for Spl, this transcription factor binds s pecifically to the 39-bp region and stimulates promoter activity in both he matopoietic cells and in Spl null Drosophila S2 cells. In addition, Ku bind s to this domain in a sequence-specific manner. Activation of the CD34 prom oter by Spt requires the presence of a binding domain at -48 bp as well as the 5' untranslated region, which also binds Spl, Conclusion. A functional interaction between regulatory regions upstream an d downstream of the transcription start site is required for CD34 gene expr ession, (C) 2000 International Society for Experimental Hematology. Publish ed by Elsevier Science Inc.