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Further characterization of cis-acting regulatory sequences in the genomic
locus of the murine erythropoietin receptor: evidence for stage- specific
regulation
H Youssoufian
Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115.
Expression of the murine erythropoietin receptor (EpoR) gene was
investigated in progenitor cell lines representing distinct stages of
hematopoietic differentiation. In murine erythroid cell lines, the EpoR
mRNA level was fivefold higher in the more mature murine erythroleukemia
(MEL) cells than in CB-5 cells and very low in granulocyte/macrophage-like
FDC-P1 cells. GATA-1 mRNA was present in equivalent levels in both
erythroid cell lines, but at a low level in FDC-P1 cells. To account for
the elevated levels of EpoR mRNA, the activity of the promoter and
expression of DNase I hypersensitive sites were assessed as markers of
transcriptional activity in various cell lines. Among a series of 5'
flanking restriction fragments linked to a reporter gene, a 83-bp fragment
that includes binding sites for the transcription factors GATA-1 and Sp-1
gave low levels of erythroid- specific activity, and a 256-bp fragment that
includes, in addition, two sites for the putative CACCC-binding protein
gave the highest level of erythroid-specific transcription. DNase I
footprinting showed binding of a constitutive factor to the proximal
CACCC-binding site, and deletion or mutation of this site significantly
reduced the overall expression while maintaining tissue-specificity. Three
DNase I hypersensitive sites were detected in the 5' flanking region of the
EpoR gene, two of which were unique to MEL cells. These sites were situated
over the promoter region and approximately 0.5 kb and 2.4 kb upstream of
the transcriptional initiation sites. A 0.8-kb restriction fragment
spanning the distal site caused approximately a four-fold rise in
transcription from the endogenous or a heterologous promoter in MEL cells
independent of its orientation and up to 1.5-fold rise in CB-5 cells, but
it was inactive in COS-1 cells that were cotransfected with an expression
plasmid encoding GATA-1. These results show that (1) basal activity as well
as tissue specificity of the EpoR promoter can be accounted for by its
interaction with GATA-1, and (2) upstream sites regulate the strength of
the promoter. Expression of the distal DNase I hypersensitive site and the
corresponding enhancer activity in MEL cells suggests a role for this
element in stage-specific transcriptional control.
Volume 83,
Issue 5,
pp. 1428-1435,
03/01/1994
Copyright © 1994 by The American Society of Hematology
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