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Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1433-1435
CORRESPONDENCE
GATA-1 Transcription Factor Transactivates the Promoter for CCR5,
a Coreceptor for Human Immunodeficiency Virus Type 1 Entry
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LETTER |
To the Editor:
Infection of host cells by human immunodeficiency virus type 1 (HIV-1)
is determined by binding of the viral envelope to the CD4
molecule as well as to a coreceptor such as CCR5 or
CXCR4.1 Expression of CCR5 is apparently restricted to
certain cell types (ie, lymphocytes, monocyte/macrophages, or
CD34+ progenitor cells),2-4 which are
susceptible to macrophage (M)-tropic (or R5) strains of HIV-1.
Because most primary isolates of HIV-1 use CCR5 to enter
cells and levels of CCR5 expression correlate well with the
infectability with M-tropic HIV-1 in vitro,2 it is
important to delineate the cellular and molecular mechanisms whereby
expression of CCR5 is regulated. The present study demonstrates that the transcription factor GATA-1 can upregulate CCR5 promoter activity.
The CCR5 promoter region contains at least three GATA-binding elements
(Fig 1A). We have previously identified one
of those elements as a GATA-1 binding site4 and have
designated it as GATA#1. Characterization of the further upstream
region demonstrated another GATA binding site (GATA#2) between  734
and 739 relative to the transcription start site
(TSS4; data not shown). Further characterization of the
promoter showed that one of the DNase-protected areas
(FP-5)4 contains a nonconsensus GATA element (Fig 1B), designated as GATA#3. Therefore, we tested whether transcription factors of GATA family can modulate CCR5 promoter activity in transient expression assays. Plasmid pGL-CCR5(WT) contains the CCR5
promoter region between 770 and +61 relative to the TSS, followed by
the luciferase gene, and plasmids pGL-CCR5 GATA#1, pGL-CCR5GATA#2,
pGL-CCR5GATA#3, and pGL-CCR5GATA#1+2+3 have mutations on these
GATA-binding sites individually or in combination (see Fig 1A for
mutated nucleotides). The indicated CCR5 promoter-luciferase reporter
construct was cotransfected with either a GATA-1 expression vector
(pMT-GATA1; kindly provided by S.H. Orkin, Harvard University, Boston,
MA5) or its parent plasmid (pMT2T) into
PM1 cells (CCR5-positive,2,4 CD4+
T-lymphoid cell line) or THP-1 cells (CCR5-positive,4
monocytoid cell line). Expression of GATA1, but not GATA-2 or GATA-3
(data not shown), upregulated CCR5 promoter activity up to 15-fold; however, mutation on GATA#1 markedly reduced GATA-1-mediated
transactivation of the CCR5 promoter (Fig
2). Whereas mutation on GATA#2 or GATA#3 alone had only modest effects on the promoter activity, mutation on all
three GATA elements further diminished GATA-1 responsiveness compared
with mutation on GATA#1 alone (Fig 2). These results indicate that
GATA-1 transcription factor upregulates CCR5 promoter activity through
interaction with several GATA elements on the promoter.
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| Fig 1.
Identification of GATA elements on the CCR5 promoter
region. (A) Map and sequence of the CCR5 promoter region. Location and
sequences of three GATA elements (GATA#1, GATA#2, and GATA#3) are
shown. The sequence shown for GATA#3 was also used as an
oligonucleotide probe (F5) for gel-mobility shift assays (see B).
Nuleotides shown in upward arrows indicate mutated nucleotides in
plasmids pGL-CCR5 GATA#1, pGL-CCR5GATA#2, pGL-CCR5GATA#3, and
pGL-CCR5GATA#1+2+3. Brackets indicate GATA elements. (B)
Identification of GATA#3 as a binding site for GATA-1 by gel mobility
shift assays. A radiolabeled oligonucleotide F5 corresponding to FP-5
(a DNase-protected area in footprinting assays4) was
incubated with nuclear extracts from PM1 cells. Lanes 1 and 9 represent
probe alone. Where indicated, a 50- or 500-fold molar excess of
oligonucleotide indicated above the figure was added as a competitor.
F5-m has mutation on the GATA element as in plasmid pGL-CCR5GATA#3.
For GATA or AP1 consensus oligonucleotide, see Moriuchi et
al.4 Gel shift disruption experiments (lanes 9 through 14)
were performed by adding either control Ab or monoclonal antibodies to
either GATA-1, GATA-2, or GATA-3 (Santa Cruz Biotechnology, Inc, Santa
Cruz, CA), as indicated above the figure. The arrow indicates the
GATA-1 complex. NS, nonspecific bands; FP, free probe. The experiments
were repeated twice with similar results.
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| Fig 2.
GATA-1 upregulates CCR5 promoter activity through
interaction with GATA elements on the promoter. Twenty million PM1
cells were transfected with 10 µg of the indicated CCR5
promoter-luciferase construct along with 10 µg of either pMT2T or
pMT-GATA-1, and luciferase assays were performed 40 hours after
transfection, as described previously.4 Results are
reported as means ± SD from four independent experiments. Similar
results were obtained for THP-1 cells (data not shown).
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GATA-1 is critical for gene expression in hematopoietic progenitor
cells5 or eosinophils,6,7 which have been shown to be infectable with M-tropic HIV-13,8; therefore, GATA-1 may play a role in the regulation of CCR5 expression and thereby in the
susceptibility of these cells to infection with M-tropic HIV-1.
Masako Moriuchi
Hiroyuki Moriuchi
Anthony S. Fauci
Laboratory of Immunoregulation
National Institute of Allergy and
Infectious Diseases
National Institutes of Health
Bethesda,
MD
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