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Myb binding sites mediate negative regulation of c-myb expression in T-
cell lines
J Guerra, DA Withers and LM Boxer
Center for Molecular Biology in Medicine, Palo Alto Veterans Affairs
Medical Center, CA, USA.
In hematopoietic cell development, the c-myb transcription factor plays an
important role. c-myb mRNA is expressed at high levels in immature
proliferating cells and in leukemic cells. We have investigated the
regulatory role of Myb protein binding to the human c-myb promoter. Three
Myb binding sites have been described at approximately 600 bp upstream of
the cap site. By transient transfection assays in hematopoietic cell lines,
we found that deletion of the previously defined most 5' Myb binding site
had no effect on activity, whereas deletion of the region containing the
remaining two Myb binding sites resulted in an increase in activity in both
a T-cell line and a myeloid cell line. To specifically test the importance
of these two Myb binding sites, the activity of three-point mutation
constructs was measured. Mutation of either Myb binding site resulted in an
increase in activity compared with the wild-type promoter in T cells.
Mutation of both sites produced even higher activity. Transfection of the
Myb site mutants into the myeloid cell line resulted in no change in
activity compared with the wild type construct. Results from gel shift
analysis, UV cross- linking, and Western blots showed that both c-Myb and
B-Myb bound to the Myb I and II sites. We conclude that the Myb family
proteins negatively regulate c-myb expression in T-cell lines in contrast
to the positive regulation via these sites, which has been shown in
fibroblasts. In addition, in a myeloid cell line, the Myb binding sites are
nonfunctional.
Volume 86,
Issue 5,
pp. 1873-1880,
09/01/1995
Copyright © 1995 by The American Society of Hematology
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