YY1 Transcription Factor Down-regulates Expression of CCR5, a Major Coreceptor for HIV-1*

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.

Transfection and Transient Expression Assays-Transfections of PBMC were performed using electroporation as described previously (14) or the Human T Cell Nucleofector™ kit (Amaxa Biosystems). In brief, 5 ϫ 10 6 PBMC were resuspended in 100 l of Human T Cell Nucleofector solution, mixed with a total of 5 g 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.
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 l 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 ϩ T cells, a target for HIV-1. First, we transfected CD4 ϩ 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 Ϫ417 relative to the TSS resulted in much reduced responsiveness to YY1 (Fig. 1C), suggesting that a region upstream from Ϫ417 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).
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 Ϫ417 relative to the TSS. Gel mobility shift assays demonstrated that one of the two, spanning from Ϫ607 to Ϫ584 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(Ϫ607/Ϫ584) oligonucleotide, but not R5(Ϫ607/Ϫ584)-MT in which the putative YY1 site was mutated (Table I), also disrupted the DNA-protein complex forma- . 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 ϩ T cell-enriched PBMC were transfected with 2.5 g 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 cotransfection. Results are reported as means Ϯ S.E. from three independent experiments. C, five million CD4 ϩ T cellenriched PBMC were transfected with 2.5 g of pGL-CCR5, pGL-CCR5 (Ϫ417), or phPES2(Ϫ327/ϩ59) along with 2.5 g of pCMV-YY1 or pcDNA3.1. Luciferase activity was shown as arbitrary light units. Results are reported as means Ϯ S.E. from three independent experiments.
Mutation on the YY1 Binding Site Markedly Reduces YY1mediated 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  5, 7, and 9) or 500-fold (lanes 4, 6, 8, and 10) molar excess of non-labeled oligonucleotides (see Table I 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 downregulation 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.
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 ϩ 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 ϩ 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).
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 ϩ T cell-enriched PBMC by transfection with pcDNA/GFP-YY1. The transfected cells were then mockinfected or infected with NL4 -3luc/JR-FL that is competent only for a single-round infection. HIV-1 infection was demon-strated 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 ϩ T cells less infectible by R5-HIV-1.

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 ϩ 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 transcrip-

YY1 Transcription Factor Down-regulates Expression of CCR5
tional 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.