Nucleic Acids Research, Vol 24, Issue 24 4978-4986, Copyright © 1996 by Oxford University Press
DE Sabath, KM Koehler and WQ Yang
The human zeta-globin promoter contains a strong positive regulatory
element in the 5' flanking region, designated the zeta-globin upstream
regulatory element (URE). In this study, we define the minimal sequences
required for URE function and characterize the associated protein-DNA
interactions. Deletion experiments show that the URE spans a 60 bp region
located between 220 and 279 bp 5' to the transcription start site. Further
subdivision of this region shows that multiple cis acting sequences are
present. Electrophoretic mobility shift assays demonstrate that the
erythroid transcription factor GATA-1 binds a site at -230, and Sp1 and an
unidentified factor bind a CCACC site at -240. The unidentified CCACC
factor is distinct from two other CCACC factors, EKLF and BKLF/TEF-2. A
third complex contains a novel DNA-binding activity that interacts with a
site in the -269 to -255 region, designated URE binding factor (URE-BF).
This factor is present in K562 cells that express zeta-globin, but is
absent in the OCIM1 cell line, a human erythroid cell line that does not
express zeta-globin. URE-BF appears to interact with a GATA factor, since
formation of the URE-BF complex can be prevented by the presence of
unlabeled oligonucleotides containing GATA sites. Finally, increasing the
distance from the -230 GATA site to the two upstream sites causes a
progressive decrease in zeta-globin promoter activity. There is no
indication of a requirement for GATA-1 to be on the same side of the DNA
helix as the other upstream factors. These results show that zeta-globin
promoter function is highly dependent on a 60 bp region to which at least
three different factors bind. Two of these factors may represent
DNA-binding proteins not previously identified as important for regulation
of globin gene expression. It is likely that these factors interact
physically to create a functional regulatory unit.
ARTICLES
Structure and function of the zeta-globin upstream regulatory element
Department of Laboratory Medicine, University of Washington School of Medicine, Seattle 98195-7110, USA. dsabath@u.washington.edu
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