YY1 Transcription Factor Down-regulates Expression of CCR5, a
Major Coreceptor for HIV-1*
Masako
Moriuchi
and
Hiroyuki
Moriuchi
§¶
From the
Division of Medical Virology, Department of
Molecular Microbiology and Immunology, Nagasaki University Graduate
School of Biomedical Sciences, Nagasaki 852-8523, Japan and the
§ Department of Pediatrics, Nagasaki University Hospital,
Nagasaki 852-8501, Japan
Received for publication, May 21, 2002, and in revised form, January 3, 2003
 |
ABSTRACT |
Expression of CCR5, a major coreceptor for human
immunodeficiency virus type 1 (HIV-1), is regulated by a number of
transcription factors. Here we report that the YY1 transcription
factor down-regulates CCR5 promoter activity and that overexpression of
YY1 reduces cell surface CCR5 expression and infectibility by
R5-HIV-1. Because YY1 also down-regulates promoter activities of
CXCR4, another major coreceptor for HIV-1 and HIV-1 long terminal
repeat, this transcription factor may play a critical role in the
pathogenesis of HIV-1 disease.
 |
INTRODUCTION |
YY1 is a ubiquitous transcription factor that regulates a
number of cellular and viral promoters, depending on the gene as well
as the cell type in question (reviewed in Ref. 1). This transcription
factor down-regulates expression from the human immunodeficiency virus
type 1 (HIV-1)1 long terminal
repeat (2) as well as the promoter for CXCR4, a major coreceptor for
X4-HIV-1 (3). Furthermore, YY1 may influence maturation and
uncoating of the HIV-1 virions through its interaction with cyclophilin
A (4). Thus, the YY1 transcription factor may play a critical role in
the pathogenesis of HIV-1 disease.
CCR5, a receptor for the CC chemokines regulated upon activation,
normal T cell-expressed and -secreted, macrophage inflammatory protein-1
and -1
is essential for cellular entry of R5-HIV-1 (reviewed in Ref. 5). Levels of its expression appear to be critical
for infectibility by R5 HIV-1 (6) or rate of disease progression in
infected individuals (7). We have cloned and characterized the promoter
region of CCR5 and identified several transcription factors that
transactivate it (8-10).
In this study, we demonstrate that YY1 down-regulates CCR5 expression,
and overexpression of YY1 reduces cell surface CCR5 expression and
infectibility by R5-HIV-1, further emphasizing its importance in the
pathogenesis of HIV-1 disease.
 |
EXPERIMENTAL PROCEDURES |
Plasmids--
Plasmids pGL-CCR5 and pGL-CCR5(
417) were
described previously (8). Plasmid pGL-CCR5 (YY1-WT) contains the CCR5
promoter sequence spanning
607 to +61 relative to the major
transcription start site (TSS) (8), followed by the luciferase reporter
gene in plasmid pGL3-basic (Promega Corp., Madison, WI). Plasmid
pGL-CCR5 (YY1-MT) has mutations (shown in bold letters below) at
594
and
593 relative to the TSS on the YY1 motif (indicated by underline) (AAAAAGATGGGAAA
AAAAAGATCCGAAA).
Plasmids pCMV-YY1 and phPES2(
327/+59), a luciferase reporter driven
by human cyclooxygenase-2 gene promoter, are generous gifts from T. Shenk (Princeton University, Princeton, NJ) (11) and H. Inoue (National
Cardiovascular Center Research Institute, Osaka, Japan), respectively
(12). To construct plasmid pcDNA/GFP-YY1, the YY1 coding
region was amplified by polymerase chain reaction using forward primer
5'-ATGGCCTCGGGCGACACCCTCTAC-3' and reverse primer
5'-TCACTGGTTGTTTTTGGCCTTAGC-3', and cloned into
pcDNA3.1/ NT-GFP-TOPO (Invitrogen). The resultant plasmid expresses green fluorescence protein GFP·YY1 fusion protein under the
control of the human cytomegalovirus major immediate early promoter. To
construct pcDNA/GFP, pcDNA3.1/NT-GFP-TOPO was digested with
KpnI, treated with T4 DNA polymerase, and re-ligated with T4
DNA ligase.
Cells--
Peripheral blood mononuclear cells (PBMC) were
obtained from healthy volunteers (Red Cross Blood Center in Nagasaki
Prefecture, Japan), and CD4+ T cell-enriched PBMC were
propagated as described previously (13). Where indicated,
CD4+ T cell-enriched PBMC were stimulated with recombinant
human interleukin-2 (IL-2) (100 units/ml) for 7 days.
Transfection and Transient Expression Assays--
Transfections
of PBMC were performed using electroporation as described previously
(14) or the Human T Cell NucleofectorTM kit (Amaxa Biosystems). In
brief, 5 × 106 PBMC were resuspended in 100 µl of
Human T Cell Nucleofector solution, mixed with a total of 5 µg of
plasmid DNA, and pulsed using the Nucleofector program U-14. Luciferase
assays for transient expression assays were performed as described
previously (14). 293 cells were transfected as described
previously (13).
Gel Mobility Shift Assays--
Nuclear extracts were prepared
from PBMC or 293 cells as described previously (15), and gel mobility
shift assays were performed as described previously (15). Anti-YY1
rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Oligonucleotides used for competition studies are
listed in Table I.
Flow Cytometric Analysis--
Expression of cell surface CCR5 or
CD4 was determined by staining cells with monoclonal anti-CCR5 Ab 2D7
phycoerythrin (PE)-conjugate or monoclonal anti-CD4 Ab RPA-T4
PE-conjugate (BD PharMingen), respectively, and analyzing in FACScan
(Becton-Dickinson Immunocytometry Systems, San Jose, CA). GFP
expression from pcDNA/GFP or pcDNA/GFP-YY1 was also
analyzed in FACScan.
Single-round Viral Infection and Flow Cytometric Analysis for
Intracellular Expression of p24 Ag--
The recombinant,
replication-incompetent virus NL4-3luc-R
E
that had been complemented with R5 JR-FL Env was designated
NL4-3luc/JR-FL here and propagated as described previously (13).
Approximately 5 × 106 PBMC were stimulated with hIL-2
(100 units/ml) for 7 days, transfected with 5 µg of pcDNA/GFP or
pcDNA/GFP-YY1, and mock-infected or infected with NL4-3luc/JR-FL
at 16 h after transfection.
For intracellular p24 Ag staining, the transfected and (mock-)infected
cells were washed once in phosphate-buffered saline containing 5% male
AB human serum (Sigma) and 0.04% sodium azide. The cells were fixed
and permeabilized using the Cytofix/Cytoperm kit (BD PharMingen),
washed twice with the wash buffer provided by the manufacturer, and
resuspended in 50 µl of a 1:150 dilution of mouse anti-HIV-1 p24
monoclonal Ab PE conjugate (KC57; Coulter, Inc., Miami, FL). After a
30-min incubation in dark on ice, the cells were washed twice and
analyzed in FACScan for GFP and p24 expression.
 |
RESULTS |
Overexpression of YY1 Down-regulates CCR5 Promoter--
Because we
and others have demonstrated that the YY1 transcription factor mediates
a variety of effects on HIV-1 infection, we wanted to investigate
whether this transcription factor plays a role in CCR5 expression on
CD4+ T cells, a target for HIV-1. First, we transfected
CD4+ T cell-enriched PBMC or 293 cells with pGL-CCR5 along
with pCMV-YY1 or control plasmid. Gel mobility shift assay using YY1-C
oligonucleotide (Table I) containing the
YY1 consensus binding site demonstrated that the endogenous level of
YY1 expression in PBMC or 293 cells was increased by transfection with
pCMV-YY1 (Fig.
1A).
Overexpression of YY1 down-regulated CCR5 promoter activity in a
dose-dependent manner (Fig. 1B); however,
truncation of the CCR5 promoter region down to
417 relative to the
TSS resulted in much reduced responsiveness to YY1 (Fig.
1C), suggesting that a region upstream from
417 relative
to the TSS contains a YY1-responsive element(s). In contrast, YY1 had
little, if any, effect on cox-2 promoter activity (Fig. 1C).
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Table I
Oligonucleotides used for gel mobility shift assays
Sequences corresponding to cis-acting elements are underlined.
Mutated nucleotides are shown in bold letters.
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Fig. 1.
YY1 down-regulates CCR5 promoter.
A, nuclear extracts were prepared from 40 × 106 PBMC that had been transfected with pcDNA3.1
(lane 2) or pCMV-YY1 (lanes 3-6), or from 293 cells that had been transfected with pcDNA3.1 (lane 8)
or pCMV-YY1 (lane 9). Gel mobility shift assay was performed
with those nuclear extracts and YY1-C as a probe. Lanes 1 and 7 represent probe alone. Where indicated, anti-YY1
rabbit polyclonal antibody (lane 5) or control rabbit serum
(lane 6) was added to the reaction. FP indicates
free probe. An arrow indicates the YY1 complex. A
bracket indicates nonspecific complexes that appeared
variably among experiments (data not shown) and were not disrupted by
anti-YY1 antibody (lane 5). B, five million
CD4+ T cell-enriched PBMC were transfected with 2.5 µg of
pGL-CCR5 along with the indicated amount of pCMV-YY1 and/or
pcDNA3.1. Luciferase assays in the transfected cell lysates were
performed 24 h after transfection. Reporter activity was shown as
luciferase activity relative to that without pCMV-YY1 co-transfection.
Results are reported as means ± S.E. from three independent
experiments. C, five million CD4+ T
cell-enriched PBMC were transfected with 2.5 µg of pGL-CCR5, pGL-CCR5
( 417), or phPES2( 327/+59) along with 2.5 µg of pCMV-YY1 or
pcDNA3.1. Luciferase activity was shown as arbitrary light units.
Results are reported as means ± S.E. from three independent
experiments.
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Identification of a YY1 Binding Element on the CCR5
Promoter--
By scanning the DNA sequence, we found two candidate
sites for YY1 binding on the CCR5 promoter region upstream from
417 relative to the TSS. Gel mobility shift assays demonstrated that one of
the two, spanning from
607 to
584 relative to the TSS, can form a
DNA-protein complex that was disrupted by the YY1 consensus oligonucleotide (Fig. 2A,
lanes 7 and 8). Unlabeled R5(
607/
584) oligonucleotide, but not R5(
607/
584)-MT in which the putative YY1
site was mutated (Table I), also disrupted the DNA-protein complex
formation (Fig. 2A, lanes 3-6). Anti-YY1
antibody specifically disrupted DNA-protein complex formation (Fig.
2B, lane 3), indicating that this complex
contains YY1. On the contrary, R5(
786/
763) probe, spanning from
786 to
763 relative to the TSS, could not form the YY1 complex
(data not shown).

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Fig. 2.
YY1 binds to CCR5 promoter.
A, gel mobility shift assays and competition studies.
R5( 607/ 584) probe was incubated with PBMC nuclear extracts in the
presence of a 50- (lanes 3, 5, 7, and
9) or 500-fold (lanes 4, 6,
8, and 10) molar excess of non-labeled
oligonucleotides (see Table I), indicated above the figure as
competitors. Lane 1 represents probe alone. Lanes
2-10 represent reactions in the presence of PBMC nuclear
extracts. FP indicates free probe. An arrow
indicates the YY1 complex. B, gel-shift interference
analysis with anti-YY1 antibody. The reaction mixture for binding
between R5( 607/ 584) oligonucleotide and PBMC extracts was incubated
with either normal rabbit serum (lane 2) or anti-YY1 rabbit
polyclonal antibody (lane 3). The solid arrow
indicates the YY1 complex, which was disrupted by anti-YY1 antibody
(lane 3), but not control serum (lane 2).
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Mutation on the YY1 Binding Site Markedly Reduces YY1-mediated
Down-regulation of CCR5 Promoter--
To demonstrate that the
aforementioned YY1 binding site is functional, YY1 effects on pGL-CCR5
(YY1-WT) or pGL-CCR5 (YY1-MT) in which the YY1 site is mutated were
compared. Mutations on the YY1 site had minimal effect on basal
activity of the CCR5 promoter but significantly inhibited CCR5 promoter
activity induced by IL-2 stimulation (Fig.
3A) or phorbol myristate
acetate plus ionomycin (data not shown). Thus, endogenous YY1 may play
an important role in down-regulating the CCR5 promoter in cells that
were stimulated to induce CCR5 expression, but not in unstimulated
cells. YY1 potently down-regulated reporter activity of pGL-CCR5
(YY1-WT) by more than 10-fold; however, mutations on the YY1 site
markedly reduced YY1 suppression of reporter activity (less than 3-fold reduction) (Fig. 3B). These results suggest that the YY1
binding site is functional and plays a critical role in YY1-mediated
down-regulation of the CCR5 promoter. Although residual responsiveness
of pGL-CCR5 (YY1-MT) to YY1 may imply the presence of other YY1 binding
site(s) on the promoter, we could not find any sequence resembling the YY1 motif.

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Fig. 3.
YY1 down-regulates CCR5 promoter.
A, effect of YY1 site mutation on CCR5 promoter activity.
CD4+ T cell-enriched PBMC were unstimulated or stimulated
with IL-2 (100 units/ml) for 7 days and transfected with 2.5 µg of
pGL-CCR5(YY1-WT) or pGL-CCR5(YY1-MT). Reporter
activities are shown as arbitrary light units. Results are reported as
means ± S.E. from three independent experiments. B,
the YY1 motif on the CCR5 promoter mediates YY1-mediated
transrepression. Five million CD4+ T cell-enriched PBMC
were transfected with 2.5 µg of pGL-CCR5(YY1-WT) or
pGL-CCR5(YY1-MT) along with 2.5 µg of pCMV-YY1 or
pcDNA3.1. Fold reduction indicates luciferase activity
relative to basal promoter activity (pcDNA3.1 co-transfection).
Results are reported as means ± S.E. from three independent
experiments.
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Overexpression of YY1 Down-regulates Cell Surface Expression of
CCR5--
As shown above, the YY1 transcription factor can
down-regulate the CCR5 promoter; however, it is well known that YY1 can
mediate totally different effects, depending upon promoter constructs and cell types to be tested. To demonstrate whether cell surface expression of CCR5 is actually down-regulated by YY1, we overexpressed YY1 by transfecting CD4+ T cell-enriched PBMC with
pcDNA/GFP-YY1 and determined cell surface expression of CCR5 or CD4
by flow cytometry. Like pCMV-YY1, pcDNA/GFP-YY1 down-regulated
reporter activity from pGL-CCR5 when co-transfected (data not shown).
After a 7-day stimulation with IL-2, more than 30% of
CD4+ T cell-enriched PBMC expressed CCR5 (Fig.
4, lower panels; data not
shown). GFP-positive cells expressed CCR5 at levels comparable with
GFP-negative cells after pcDNA/GFP transfection (Fig. 4, left
panels). However, significantly fewer GFP-positive cells expressed CCR5 after pcDNA/GFP-YY1 transfection (Fig. 4,
right panels). Thus, overexpression of YY1 appears to
reduce cell surface expression of CCR5. On the contrary, CD4 expression
on GFP-positive cells after pcDNA/GFP transfection (94%; mean
fluorescence intensity 36.9) was comparable with that on
GFP-positive cells after pcDNA/GFP-YY1 transfection (93%; mean
fluorescence intensity 34.0).

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Fig. 4.
Overexpression of YY1 reduces cell surface
expression of CCR5. Five million CD4+ T cell-enriched
PBMC were stimulated with IL-2 for 7 days and transfected with 5 µg
of pcDNA/GFP (left panels) or pcDNA/GFP-YY1
(right panels), stained with IgG2a PE (upper
panels) or anti-CCR5 PE (lower panels), and analyzed in
FACScan. The dot blot shows green fluorescence intensity
(GFP or GFP-YY1 expression) in x-axis
and PE color intensity (CCR5 expression) in
y-axis. Results are representative of three independent
experiments.
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Overexpression of YY1 Decreased Infectibility by
R5-HIV-1--
Having had the aforementioned results, we wanted to
determine whether overexpression of YY1 could inhibit HIV-1 infection. Because YY1 has variable effects depending upon cell type and because
continuous and strong expression of YY1 is toxic to cells (data not
shown), YY1 was transiently overexpressed in IL-2-stimulated, CD4+ T cell-enriched PBMC by transfection with
pcDNA/GFP-YY1. The transfected cells were then mock-infected or
infected with NL4-3luc/JR-FL that is competent only for a single-round
infection. HIV-1 infection was demonstrated by intracellular p24 Ag
staining. As shown in Fig. 5,
GFP-positive cells expressed p24 at levels comparable with GFP-negative
cells after pcDNA/GFP transfection (Fig. 5, left
panels). In contrast, many fewer GFP-positive cells expressed p24
after pcDNA/GFP-YY1 transfection (Fig. 5, right panels).
Thus, overexpression of YY1 appeared to render CD4+ T cells
less infectible by R5-HIV-1.

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Fig. 5.
Overexpression of YY1 reduces infectibility
of cells by R5-HIV-1. CD4+ T cell-enriched PBMC were
stimulated with IL-2 for 7 days and transfected with pcDNA/GFP
(left panels) or pcDNA/GFP-YY1 (right panels)
and, 16 h later, mock-infected or infected with NL4-3luc/JR-FL.
The cells were stained with anti-HIV-1 p24 Ab-PE or IgG1 PE (data not
shown). The dot blot shows green fluorescence intensity
(GFP-YY1 expression) in x-axis and PE color
intensity (p24 expression) in y-axis. Results are
representative of three independent experiments.
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|
 |
DISCUSSION |
In this study we have demonstrated that the YY1 transcription
factor can down-regulate expression of CCR5 at the promoter level.
Levels of CCR5 expression appear to correlate well with infectibility
of CD4+ T cells by R5 HIV-1 (6) and rate of disease
progression (7). Expression of CCR5 appears to be highly regulated by a
number of cytokines, cell activation, or differentiation (16-20). At
the promoter level, several transcription factors have been
demonstrated to up-regulate CCR5 expression, including p65 (RelA) (21),
C/EBP-
(22), GATA-1 (9), and Octamer (10). The present study has extended our understanding of the molecular mechanism of regulation of
CCR5 promoter activity by adding YY1 as the first transcriptional repressor of the promoter. We further demonstrated that overexpression of YY1 actually down-regulates cell surface CCR5 expression and infectibility by R5-HIV-1.
We have previously demonstrated that YY1 down-regulates the promoter
for CXCR4, another major co-receptor for HIV-1 (3). YY1 is also known
to down-regulate the HIV-1 long terminal repeat promoter (2).
Furthermore, YY1 associates with cyclophilin A, which may be critical
for maturation and uncoating of HIV-1 virions through its interaction
with Gag (4). Taken together with the present study, it is reasonable
to consider that the YY1 transcription factor plays important roles in
the pathogenesis of HIV-1 disease (Fig.
6). Further investigations to delineate the molecular and cellular mechanisms that regulate expression of HIV
coreceptors and to develop therapeutic interventions using anti-HIV-1
host factor(s) such as YY1 are warranted.
 |
ACKNOWLEDGEMENTS |
We thank T. Shenk, H. Inoue, and N. Landau
for reagents, S. Chiyoda, H. Okuda, and K. Deguchi (Nagasaki Red Cross
Blood Center) for blood samples, and M. Yokoyama for excellent
technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by a grant from the Research
for the Future Program (JSPS-RFTF97L00705) of the Japan Society for the
Promotion of Science and a grant-in-aid for Scientific Research on
Priority Areas from the Ministry of Education, Science, Sports, and
Culture, Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Pediatrics, Nagasaki University Hospital, 1-7-1 Sakamoto, Nagasaki
852-8501, Japan. Tel.: 81-95-849-7297; Fax: 81-95-849-7301; E-mail:
hiromori@net.nagasaki-u.ac.jp.
Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M204980200
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ABBREVIATIONS |
The abbreviations used are:
HIV, human
immunodeficiency virus;
GFP, green fluorescent protein;
PBMC, peripheral blood mononuclear cells;
IL-2, interleukin-2;
PE, phycoerythrin;
TSS, transcription start site.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.