TY - JOUR
T1 - A nuclear factor Y (NFY) site positively regulates the human CD34 stem cell gene
AU - Radomska, Hanna S.
AU - Satterthwaite, Anne B.
AU - Taranenko, Natalie
AU - Narravula, Sailaja
AU - Krause, Diane S.
AU - Tenen, Daniel G.
PY - 1999/12/1
Y1 - 1999/12/1
N2 - Proper regulation of the human CD34 gene requires a combinatorial action of multiple proximal and long-range, cis elements. This report shows that, like the murine CD34 5' untranslated region (UTR), the corresponding region of the human CD34 gene is necessary for optimal promoter activity. We localized the most critical element of this region to base pairs +48/+75. Through oligonucleotide competition and antibody supershift experiments in electrophoretic mobility shift assays, we found that this sequence contains a binding site (CCAAT box) for the transcription factor NFY (nuclear factor Y), a factor mediating cell type-specific and cell-cycle regulated expression of genes. Mutating this site led to a 5-fold decrease in CD34 promoter activity in transient transfection experiments. Interestingly, NFY binds adjacently to the earlier identified c-myb binding site. Here we show that both binding sites are important for CD34 promoter function: mutating either site alone decreased CD34 promoter-driven reporter gene activity 4-fold. We also show that the integrity of the c-myb binding site is necessary for stabilization 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, NFY did not bind to this region. Thus, our results indicate that the functional similarities between the human and murine CD34 5' UTRs are achieved through different molecular mechanism(s).
AB - Proper regulation of the human CD34 gene requires a combinatorial action of multiple proximal and long-range, cis elements. This report shows that, like the murine CD34 5' untranslated region (UTR), the corresponding region of the human CD34 gene is necessary for optimal promoter activity. We localized the most critical element of this region to base pairs +48/+75. Through oligonucleotide competition and antibody supershift experiments in electrophoretic mobility shift assays, we found that this sequence contains a binding site (CCAAT box) for the transcription factor NFY (nuclear factor Y), a factor mediating cell type-specific and cell-cycle regulated expression of genes. Mutating this site led to a 5-fold decrease in CD34 promoter activity in transient transfection experiments. Interestingly, NFY binds adjacently to the earlier identified c-myb binding site. Here we show that both binding sites are important for CD34 promoter function: mutating either site alone decreased CD34 promoter-driven reporter gene activity 4-fold. We also show that the integrity of the c-myb binding site is necessary for stabilization 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, NFY did not bind to this region. Thus, our results indicate that the functional similarities between the human and murine CD34 5' UTRs are achieved through different molecular mechanism(s).
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U2 - 10.1182/blood.v94.11.3772
DO - 10.1182/blood.v94.11.3772
M3 - Article
C2 - 10572091
AN - SCOPUS:0033485858
SN - 0006-4971
VL - 94
SP - 3772
EP - 3780
JO - Blood
JF - Blood
IS - 11
ER -