MYB BINDING-SITES MEDIATE NEGATIVE REGULATION OF C-MYB EXPRESSION IN T-CELL LINES

In hematopoietic cell development, the c-myb transcription factor play s an important role, c-myb mRNA is expressed at high levels in immatur e proliferating cells and in leukemic cells. We have investigated the regulatory role of Myb protein binding to the human c-myb promoter. Th ree Myb binding sites have been described at approximately 600 bp upst ream of the cap site. By transient transfection assays in hematopoieti c cell lines, we found that deletion of the previously defined most 5' Myb binding site had no effect on activity, whereas deletion of the r egion containing the remaining two Myb binding sites resulted in an in crease in activity in both a T-cell line and a myeloid cell line. To s pecifically test the importance of these two Myb binding sites, the ac tivity of three-point mutation constructs was measured. Mutation of ei ther Myb binding site resulted in an increase in activity compared wit h the wild-type promoter in T cells. Mutation of both sites produced e ven higher activity. Transfection of the Myb site mutants into the mye loid cell line resulted in no change in activity compared with the wil d type construct. Results from gel shift analysis, UV crosslinking, an d Western blots showed that both c-Myb and B-Myb bound to the Myb I an d II sites. We conclude that the Myb family proteins negatively regula te c-myb expression in T-cell lines in contrast to the positive regula tion via these sites, which has been shown in fibroblasts. In addition , in a myeloid cell line, the Myb binding sites are nonfunctional. (C) 1995 by The American Society of Hematology.