A nuclear factor Y (NFY) site positively regulates the human CD34 stem cell gene

Proper regulation of the human CD34 gene requires a combinatorial action of multiple proximal and long-range, cis elements. This report shows that, li ke the murine CD34 5' untranslated region (UTR), the corresponding region o f the human CD34 gene is necessary for optimal promoter activity, We locali zed the most critical element of this region to base pairs +48/+75. Through oligonucleotide competition and antibody supershift: experiments in electr ophoretic mobility shift assays, we found that this sequence contains a bin ding site (CCAAT box) for the transcription factor NN (nuclear factor Y), a factor mediating cell type-specific end cell-cycle regulated expression of genes. Mutating this site led to a 5-fold decrease in CD34 promoter activi ty in transient transfection experiments, Interestingly, NFY binds adjacent ly to the earlier identified c-myb binding site. Here we show that both bin ding sites are important for CD34 promoter function: mutating either site a lone decreased CD34 promoter-driven reporter gene activity 4-fold. We also show that the integrity of the c-myb binding site is necessary for stabiliz ation of NFY binding to its site. Such cooperation between c-myb, which is expressed in early hematopoietic cells, and NFY, which is expressed in many cell types, might contribute to specific activation of CD34 in stem cells. The CCAAT box motif was also noted in the 5' UTR of the murine CD34 gene, however, NN did not bind to this region. Thus, our results indicate that th e functional similarities between the human and murine CD34 5' UTRs are ach ieved through different molecular mechanism(s). (C) 1999 by The American So ciety of Hematology.