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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3734-3741
Sp1 and C/EBP are necessary to activate the lactoferrin gene
promoter during myeloid differentiation
Arati Khanna-Gupta,
Theresa Zibello,
Carl Simkevich,
Alan G. Rosmarin, and
Nancy Berliner
From the Section of Hematology, Yale University School of Medicine,
New Haven, CT; the Division of Hematology, Brown University and the
Department of Medicine and the Division of Hematology/Oncology, The
Miriam Hospital, Providence, RI.
In this study, we sought to identify factors responsible for the
positive modulation of lactoferrin (LF), a neutrophil-specific, secondary-granule protein gene. Initial reporter gene transfection assays indicated that the first 89 base pairs of the LF promoter are
capable of directing myeloid-specific LF gene expression. The presence
of a C/EBP site flanked by 2 Sp1 sites within this segment of the LF
promoter prompted us to investigate the possible role of these sites in
LF expression. Cotransfection studies of LF-89luc plasmid with
increasing concentrations of a C/EBP expression vector in myeloid
cells resulted in a linear transactivation of luciferase reporter
activity. Electrophoretic mobility shift assays found that the C/EBP
site is recognized by C/EBP and that both LF Sp1 binding sites bind
the Sp1 transcription factor specifically in myeloid cells. Mutation of
either Sp1 site markedly reduced activity of the LF-89luc plasmid in
myeloid cells, and neither Sp1 mutant plasmid was transactivated by a
C/EBP expression plasmid to the same extent as wild-type LF-89luc.
We also transfected LF-89luc into Drosophila Schneider cells,
which do not express endogenous Sp1, and demonstrated up-regulation of
luciferase activity in response to a cotransfected Sp1 expression
plasmid, as well as to a C/EBP expression plasmid. Furthermore,
cotransfection of LF-89luc plasmid simultaneously with C/EBP and
Sp1 expression plasmids resulted in an increase in luciferase activity
greater than that induced by either factor alone. Taken together, these observations indicate a functional interaction between C/EBP and Sp1 in
mediating LF expression.
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