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The Journal of Immunology, 2008, 180, 402 -408
Copyright © 2008 by The American Association of Immunologists, Inc.
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Pro-IL-16 Recruits Histone Deacetylase 3 to the Skp2 Core Promoter through Interaction with Transcription Factor GABP1

Yujun Zhang2,*, Marina Tuzova*, Zhi-Xiong J. Xiao{dagger}, William W. Cruikshank* and David M. Center*

* Pulmonary Center and {dagger} Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Pro-IL-16 is a PDZ domain-containing protein expressed in T cells. Our previous work showed that upon activation of normal T cells, pro-IL-16 mRNA and protein are diminished in close correlation to the down-regulation of p27KIP1 protein. In addition, we showed that pro-IL-16 regulates the transcription of Skp2, the mechanism of which, however, remains elusive. In this study, we identified GA binding protein β1 subunit (GABPβ1) and histone deacetylase 3 (HDAC3) as binding partners of pro-IL-16. Interestingly, both GABPβ1 and HDAC3 have canonical PDZ-binding motifs and specifically bind to the first and second PDZ domain of pro-IL-16, respectively. Heat shock cognate protein 70 (HSC70) also copurified with the GST-PDZ1-containing fragment but lacks a C-terminal PDZ binding motif, suggesting that it binds through a different mechanism. We further showed that pro-IL-16 is located in a GABP transcriptional complex bound to the Skp2 promoter. In addition, we demonstrated that HDAC activity is critical for pro-IL-16-induced cell cycle arrest. Taken altogether, these data suggest that pro-IL-16 forms a complex with GABPβ1 and HDAC3 in suppressing the transcription of Skp2. Thus, this study has revealed a novel mechanism with which pro-IL-16 regulates T cell growth through the Skp2-p27KIP1 pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In cells of hemopoietic origin, pro-IL-16 is a 631-amino acid protein that acts as the precursor for the secreted cytokine IL-16 (1, 2). Following T cell activation it is cleaved by caspase 3, resulting in liberation of the C-terminal 121 amino acids that comprise mature IL-16 (3, 4). The structure of pro-IL-16 is unusual for a cytokine precursor. It is highly conserved among species (5, 6, 7) and contains a phosphorylation-regulated nuclear localization signal (8) and three PDZ domains (9). Taken altogether, these characteristics suggest that it might have a nuclear function independent of its role as a cytokine precursor. Along these lines, ectopic nuclear expression of pro-IL-16 in pro-IL-16-negative Jurkat cells results in G0/G1 cell cycle arrest, which is associated with elevated levels of the cyclin-dependent kinase inhibitor p27KIP1 (10). In this circumstance, p27KIP1 protein levels are stabilized by transcriptional repression of Skp2, a key component of the SCFSkp2 ubiquitin E3 ligase complex that is responsible for p27KIP1 degradation during the transition from G1 phase to S phase.

Consistent with its repression of Skp2 transcription, it has been observed that the levels of nuclear pro-IL-16 are high in resting T cells and low in actively dividing T cells (11). In fact, pro-IL-16 is rapidly diminished upon T cell activation, and T cell proliferation is associated with its loss from the nucleus (11). It is re-expressed coincident with T cell quiescence, which is inversely correlated with the expression of Skp2 and therefore directly correlated with p27KIP1 levels.

There are no predicted enzymatic or DNA binding motifs in pro-IL-16 and, as a result, there is no direct explanation for the effect of nuclear pro-IL-16 on Skp2 transcription. The nuclear localization and the three PDZ domains suggested to us that it might act as a nuclear scaffold for a transcriptional repression. Along those lines, PDZ 2 has been shown to bind to three closely related myosin phosphatase-targeting subunits, MYPT1, MYPT2, and MBS85, all of which reside in the cytoplasm as part of actin-myosin fibers in COS-7 cells that do not naturally express pro-IL-16 (9). This association is thought to be related to cell motility. The neuronal form of pro-IL-16 has two additional internal PDZ domains that interact with neuronal ion channels, but these are not present in T cell pro-IL-16 (12). Of interest, the nuclear magnetic resonance structure of the PDZ 3 domain of T cell pro-IL-16 has been resolved and is predicted to have an occluded GLGF protein binding cleft that would eliminate binding to other proteins (13). However, functional binding to T cell proteins has never been tested for any of the three PDZ domains present in T cell-derived pro-IL-16. In this study, we have identified three interacting proteins of pro-IL-16 from human peripheral blood T cells by using conventional GST pull-down and immunoprecipitation followed by immunoblotting. We demonstrate that the β1 subunit of GA-binding protein (GABP)3 and heat shock cognate protein 70 (HSC70) copurify with a fragment of pro-IL-16 containing the first PDZ domain fused to GST, while histone deacetylase 3 (HDAC3) binds to a fragment of pro-IL-16 containing the second PDZ domain fused to GST in the extracts of resting human T cells. We show that HDAC activity is essential for the effects of nuclear pro-IL-16 on the cell cycle progression and Skp2 levels, and identify pro-IL-16 in GABP{alpha}2/β2-Skp2 promoter complex by chromatin immunoprecipitation (ChIP). Taken altogether, our results suggest that in mature resting T cells, pro-IL-16 represses Skp2 gene transcription by recruiting HDAC3 to the Skp2 promoter through the interaction with GABP transcription factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and Abs

The Jurkat T cell leukemia cell line was obtained from American Type Culture Collection and maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 25 mM HEPES, and 100 U/ml penicillin/streptomycin. Nylon wool nonadherent human T cells were prepared as described (14). The mAb (clone 14.1) to bioactive IL-16 (C terminus of pro-IL-16) and the rabbit polyclonal Ab to pro-IL-16 have been described previously (15, 16). Goat polyclonal anti-IL-16 Ab was purchased from R&D Systems; Abs to p27KIP1, Skp2, HDAC3, HSC70, and actin were purchased from Santa Cruz Biotechnology; and the Ab to tubulin was obtained from Sigma-Aldrich. Rabbit polyclonal Abs to GABP{alpha} and GABPβ1 were kindly provided by Dr. W. J. Leonard (Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institute of Health, Bethesda, MD). All studies involving the use of human T cells have been approved by the Institutional Review Board at the Boston University Medical Center (Boston, MA).

GST pull-down assay

DNA sequences from three individual PDZ domains of Pro-IL-16 were cloned into the GST vector (pGEX-6P-1). Escherichia coli BL21 transformed with the GST fusion vectors were induced with 0.1 mM isopropyl-1-thio-D-galactopyranoside for 4 h, and fusion proteins were purified from glutathione-Sepharose columns by affinity chromatography per the manufacturer’s instructions (Pharmacia). For in vitro binding interactions, purified GST-PDZ domain fusion proteins were incubated with whole cell lysates of normal human peripheral blood T cells for 4 h at 4°C, respectively. Following incubation, the GST-PDZ domain fusion proteins with their interacting proteins were bound to glutathione-Sepharose columns and unbound proteins were removed by extensive washing. Subsequently, the bound GST-PDZ domain fusion proteins and any proteins that specifically interacted were eluted using a solution containing 10 mM reduced glutathione in 50 mM Tris-HCl (pH 8.0). Eluted proteins were concentrated using Centricon-20 (Amicon), and the reduced glutathione was removed from eluted proteins by washing with PBS several times during concentration. The eluted proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad), and visualized by Ponceau S staining. Bands representing proteins bound to GST-PDZ domain fusion proteins were cut out and sequenced (Beckman LF3000 sequencer).

Coimmunoprecipitation and immunoblotting

For coprecipitation of GABPβ1 or HDAC3 with FLAG-tagged pro-IL-16 derivatives, COS-7 cells were transiently transfected by electroporation using Gene Pulser (Bio-Rad) with 5 µg of each corresponding plasmid. After incubation for 48 h, the cells were dissolved in cell lysis buffer (25 mM Tris (pH 7.9), 0.5% Nonidet P-40, 250 mM NaCl, 1.5 mM EDTA, and a protease inhibitor mixture), and amounts of cell extracts equivalent to 500 µg of protein were incubated with 50 µl of M2 anti-FLAG agarose (Sigma-Aldrich) for 2 h. The agarose beads were precipitated, washed three times with lysis buffer, and resuspended in 2x SDS-gel loading buffer. All of the incubations mentioned above were conducted at 4°C and with constant movement using a head-over-tail rotor. The precipitates were analyzed by SDS-PAGE and Western blotting using the rabbit anti-GABPβ1 antiserum and a rabbit anti-HDAC3 Ab (Santa Cruz Biotechnology).

Far-Western blot

FLAG-tagged pro-IL-16 was expressed in COS cells, immunoprecipitated with anti-FLAG Ab, separated on SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were washed with far-Western buffer (20 mM Tris HCl (pH 7.5), 0.1 mM EDTA, 5% glycerol, 0.04% Nonidet P-40, and 40 mM NaCl) for 10 min, and then blocked with 5% dry milk for 1 h at 4°C. Membranes were then incubated with 10 ml of 5 mg/ml GST-GABPβ1 overnight at 4°C. After washing with far-Western buffer for 10 min at 4°C, membranes were probed with mouse anti-GST Ab (Santa Cruz Biotechnology) for 2 h at 4°C. After three washes with far-Western buffer (5 min each), membranes were incubated with goat anti-mouse IgG secondary Ab. Binding of GST-GABPβ1 to immunoprecipitated FLAG-pro-IL-16 was visualized by autoradiography following the incubation of the membranes with SuperSignal West Dura reagent (Pierce).

Preparation of nuclear extract and EMSA analysis

Nuclear extracts of resting human peripheral blood T cells were prepared and stored at –80°C for EMSA analysis as described previously (10). For "supershift" analysis, 1 µg of Abs to GABP{alpha}, GABPβ1, or pro-IL-16 (R&D Systems) were added to each reaction mixture, respectively. The oligonucleotides –174/–152(+) (5'-GAGCTCCACCCACTTCCGCCTGC-3') and –174/–152(–) (5'-GCAGGCGGAAGTGGGTGGAGC TC-3') and the nonspecific oligonucleotides –152/–126(+) (5'-GGTGTCCTCCTCCTCCTCCTCCTCCTCCT-3') and –152/–126(–) (5'-GGAGGAGGAGGAGGAGGAGGAGGAGGACA-3'), were chemically synthesized; the corresponding (+) and (–) oligonucleotides were annealed and labeled by filling in the guanine-containing 5' overhangs with the use of the Klenow fragment (New England Biolabs) in the presence of [{alpha}-32P]dCTP (NEN PerkinElmer).

ChIP assay

Human peripheral blood T cells were prepared as described (14). ChIP was performed according to the manufacturer’s instructions (Upstate Biotechnology). Briefly, cellular proteins that contact DNA were cross-linked to DNA with 1% formaldehyde. T cell lysate containing chromatin was prepared and precleared with a salmon sperm DNA-related protein G-agarose slurry. The aliquots of the lysate were incubated with Abs to GABP{alpha}, GABPβ1, pro-IL-16, and HDAC3 at 4°C for 16 h, respectively. The immunocomplexes were harvested using protein G-agarose. After reversing the cross-linking, the eluted immunocomplexes were treated with proteinase K and DNA was purified by phenol extraction. Amplification of extracted DNA was done using primers specific for the GABP-target site on the promoter region of Skp2 in PCR (5'-SKP2, 5'-AAAATCCGTCTACAGTCCAG-3'; 3'-SKP2, 5'-CCTTCGAGATACCCACAACC-3'). The 127-bp PCR product was analyzed by electrophoresis on a 1.5% agarose gel. The PCR product from the input reaction was used to normalize the PCR band intensity found from the corresponding immunoprecipitated samples.

Cell culture and treatment with trichostatin A (TSA)

Jurkat cells expressing GFP-pro-IL-16 or GFP alone under the control of doxycycline-inducible promoter have been previously described (10). They were maintained in RPMI 1640 medium with 10% FBS plus 2 µg/ml doxycycline, 100 µg/ml neomycin, and 200 µg/ml hygromycin B, and cultured at 37°C in a humidified atmosphere of 5% CO2. Forty-eight hours after removing doxycycline, resulting in the expression of GFP-pro-IL-16 or GFP alone, 50 ng/ml TSA (Sigma-Aldrich) was added to the culture medium. Cells were collected at 0-, 6-, and 12-h time points. One-third of the collected cells were used for cell cycle analysis by FACS, one-third for Western blot analysis, and one-third to isolate total RNA for semiquantitative RT-PCR.

Cell cycle analysis

Cell cycle analysis was performed as previously described (10). Briefly, 1 million Jurkat cells or murine thymic T cells were washed with PBS buffer and fixed in 35% ethanol at 4°C overnight. After fixation, cells were washed twice with PBS before being resuspended in propidium iodide/RNase A solution (50 µg/ml propidium iodide and 100 µg/ml RNase A) at room temperature in the dark for 1 h. Propidium iodide-stained cells were analyzed by flow cytometry (FACScan; BD Biosciences).

RT-PCR

Jurkat cells expressing GFP-pro-IL-16 or GFP alone were treated with 50 ng/ml TSA and collected at 0-, 6-, and 12-h time points. Total RNA was extracted using an RNeasy mini kit (Qiagen). A semiquantitative RT-PCR was conducted in triplicate using the Access RT-PCR System (Promega) with the primers to detect full-length Skp2 cDNA (forward, 5'-ATGCACAGGAAGCACCTCCAG-3'; reverse. 5'-TCATAGACAACTGGGCTTCCG-3') and p27KIP1 cDNA (forward, 5'-ATGTCAAACGTGCGAGTGTCT-3'; and reverse, 5'-TTACGTTTGACGTCTTCTGAG-3').

Cell fractionation

Jurkat cells were induced to express GFP-Pro-IL-16 or nuclear GFP alone, harvested by centrifugation, and washed two times with cold PBS before incubation with buffer I (see below) on ice for 15 min. Twenty microliters of 10% Nonidet P-40 was added into the 400 µl cell suspension, hand mixed for 10 s, and then centrifuged at 2000 RPM for 5 min at 4°C to pellet the nuclei. The supernatants were further fractionated by centrifugation at 14,000 RPM for 20 min at 4°C. The supernatant fraction of this centrifugation was collected and classified as the cytoplasmic fraction. The nuclear pellet was washed twice with ice cold PBS before adding 50 µl of buffer II (see below) to lyse the nuclei. After 15 min on ice, nuclear lysates were centrifuged at 14,000 RPM for 10 min at 4°C, and this supernatant fraction was classified as nuclear. The integrity of a nuclear fraction was determined by the absence of tubulin using Western analysis. The protein concentration in each fraction was quantified (Bio-Rad). Buffer I contained 20 mM Tris-HCl, 0.5 mM DTT, 10 mM β-glycerol phosphate, 300 mM sucrose, 0.2 mM EGTA, 5 mM MgCl2, and 10 mM KCl. Buffer II contained 10 mM Tris-HCl, 0.5 mM DTT, 10 mM β-glycerol phosphate, 0.2 mM EGTA, 5 mM MgCl, 350 mM KCl, and 25% glycerol. Both buffers contain the protease inhibitors aprotinin, chymostatin, antipain, and pepstatin at 10 µg/ml and PMSF at 1 mM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Identification of HDAC3, GABPβ1, and HSC70 as pro-IL-16 binding proteins

To identify proteins bound to pro-IL-16 we used GST pull-down assays. To identify selective binding to each PDZ domain we generated three truncated constructs in which each PDZ domain’s coding region of pro-IL-16 was ligated to a GST expression vector (as shown in the diagram of Fig. 1A), expressed in E. coli, and purified using glutathione S-agarose chromatography (Fig. 1B). After incubating GST-PDZ fusion proteins with lysates of human peripheral blood T cells, the cellular proteins that bind and coelute with GST-PDZ fusion proteins were subjected to SDS-PAGE followed by PVDF membrane transfer and visualized by Ponceau S staining. As shown in Fig. 1C, there were detectable proteins bound to PDZ domain 1 corresponding to the 70- and 40-kDa bands (lane 1) and to PDZ domain 2 corresponding to the 47- and 32 kDa bands (lane 2). No proteins were identified as interacting with PDZ domain 3 (Fig. 1C, lane 3). This last result is consistent with nuclear magnetic resonance spectroscopy, which predicted that the occluded GLGF motif in PDZ domain 3 would not bind other proteins (13).


Figure 1
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  		FIGURE 1. Identification of pro-IL-16 binding proteins in normal human T cells. A, Schematic representation of GST-pro-IL-16 PDZ 1, 2, and 3 fusion constructs. B, Coomassie blue staining of purified GST fusion proteins (as depicted in A) separated by SDS-PAGE. C, GST fusion proteins and their binding proteins eluted from a glutathione-Sepharose column were separated by SDS-PAGE, transferred onto a PVDF membrane, and stained with Ponceau S. The asterisk (*) depicts the input GST fusion proteins. The 40-kDa band bound to GST-PDZ1 was identified as GABPβ1, the 70kDa band was identified as HSC70, and the 47kDa band bound to GST-PDZ2 was identified as HDAC3. These are representative figures from four separate experiments.

 
Following transfer to PVDF membranes, the 70-kDa (p70) and 40-kDa (p40) polypeptides bound to PDZ 1 and the 47-kDa (p47) polypeptide bound to PDZ 2 were successfully sequenced and unambiguously identified as the heat shock cognate protein 70 designated HSC70 (p70), the β1 subunit of GA binding protein designated GABPβ1 (p40), and histone deacetylase 3 designated HDAC3 (p47), respectively. No definitive sequence could be derived from the 32-kDa band bound to PDZ 2. PDZ domains recognize specific C-terminal sequence motifs that are characteristically four residues in length and are categorized on the basis of the class of the C-terminal residues of their binding proteins. After reviewing the C-terminal sequences, we found that the last four amino acids of the C-termini of HDAC3 and GABPβ1 fall into class II and class III PDZ domain-binding consensus residues, respectively (Table I). Neither GABP{alpha}, nor any other histone deacetylase family member contains C-terminal PDZ binding motifs (17, 18). Interestingly, HSC70 also does not contain a C-terminal PDZ binding motif. Because the protein product of the GST-PDZ 1 construct also contains an additional sequence of pro-IL-16 that lies outside the binding cleft of PDZ domain 1, it is possible that HSC70 binds via an alternate mechanism to pro-IL-16. Independent of the binding site, the lack of association with either PDZ2 or PDZ3 indicates a specific interaction with the fragment of pro-IL-16 that contains PDZ1 and could involve a nonclassical PDZ binding interaction.


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  		Table I. Summary of identified binding proteins of pro-IL-16

 
Because HSC70 has been shown to facilitate nuclear transport and protein stability but not directly affect T cell proliferation, we focused the remaining studies on the interactions between pro-IL-16 with GABPβ1 and HDAC3.

Association of pro-IL-16 with GABPβ1 and HDAC3 in cotransfected COS cells

To confirm the binding of pro-IL-16-PDZ1 to GABPβ1 and pro-IL-16-PDZ2 to HDAC3, we coexpressed FLAG-tagged pro-IL-16 PDZ-domain derivatives, as depicted in Fig. 2A, with GABPβ1 and HDAC3 in COS cells followed by immunoprecipitation and Western blotting. As shown in Fig. 2B, both GABPβ1 (top panel) and HDAC3 (second panel from top) immunoprecipitated with full-length pro-IL-16 (lane 1) and the truncated pro-IL-16 derivatives that contain both the PDZ1 domain and the PDZ2 domain (lanes 2 and 3). GABPβ1 only associates with truncated pro-IL-16 that contains PDZ1 (lane 4), and HDAC3 only binds to the pro-IL-16 derivative that contains PDZ2 (lane 5). Taken together with GST pull-down data, pro-IL-16 appears to interact with GABPβ1 and HDAC3 in T cells and cotransfected COS cells and confirms that GABPβ1 binds to the PDZ1 domain of pro-IL-16, whereas HDAC3 binds to the PDZ2 domain of pro-IL-16.


Figure 2
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  		FIGURE 2. Association of GABPβ1 and HDAC3 with pro-IL-16 in cotransfected COS cells. A, Schematic shows the structures of the human FLAG-tagged pro-IL-16 constructs. B, Pro-IL-16 associates with GABPβ1 and HDAC3 in a COS cell lysate. To confirm and map the GABPβ1 and HDAC3 interacting domains, transiently expressed FLAG-tagged pro-IL-16 protein derivatives along with coexpressed GABPβ1 and HDAC3 proteins were analyzed for association by immunoprecipitation (IP) with an anti-FLAG Ab followed by immunoblotting with either anti-GABPβ1 (top panel) or anti-HDAC3 (second panel from top). Input GABPβ1 and HDAC3 protein levels are shown in the bottom two panels, and the coexpressed pro-IL-16 protein derivatives are shown in the middle panel. C, A far-Western analysis reveals direct interaction between the GABPβ1 and pro-IL-16. Binding of the GST-GABPβ1 to the anti-FLAG immunoprecipitated full-length FLAG-pro-IL-16 (a, anti-pro-IL-16; b, anti-FLAG) was visualized by first probing the membrane with purified GST-GABPβ1 protein (shown in d) and then incubating with anti-GST Ab (c, far-Western blot).

 
To confirm that GABPβ1 binds directly to pro-IL-16, we expressed and purified a GST-GABPβ1 fusion protein using an E. coli expression system (Fig. 2Cd) and analyzed its ability to recognize the FLAG-tagged full-length pro-IL-16 in a far-Western analysis. In this experiment, anti-FLAG Ab-immunoprecipitated FLAG-pro-IL-16 was resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with purified GST-GABPβ1 protein. Binding of GST-GABPβ1 to FLAG-pro-IL-16 was visualized by chemiluminescence following incubation of the blot with anti-GST Ab (Fig. 2Cc). Far-Western analysis revealed that purified GABPβ1 binds directly to FLAG-pro-IL-16 that is separated by SDS-PAGE and confirmed by both anti-pro-IL-16 and anti-FLAG Abs (Fig. 2C, a and b).

Association of pro-IL-16 with GABPβ1 and HDAC3 in normal human T cells

To investigate these interactions further, we next examined whether GABPβ1 and HDAC3 can bind intact endogenous pro-IL-16 in normal human T cells. Because functional GABP transcriptional units exist as heterotetrameric complexes containing two {alpha} subunits bearing DNA binding domains and two β1 subunits bearing transactivation domains (19, 20), we tested lysates of normal resting T cells for the presence of each of these components and for the presence of HDAC3. Our result showed that each protein is present in T cells (Fig. 3, left lane) and that they are present in the anti-pro-IL-16 immunoprecipitates as well (Fig. 3; right lane). These data confirmed that a complex comprised of pro-IL-16, HDAC3, and GABP transcription factor exists in resting T cells. Note that while both GABP{alpha} and β1 were detected in the immunoprecipitates, GABP{alpha} was detected at an expression level that was lower than that of GABPβ1. This finding is consistent with the concept that there is a direct interaction of pro-IL-16 with GABPβ1 and an indirect interaction with GABP{alpha} through the GAPBβ1 subunit.


Figure 3
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  		FIGURE 3. Association of HDAC3, GABP{alpha}, and GABPβ1 with Pro-IL-16 in primary human T cells. Left panel, Immunoblots (IB) of lysate from normal resting human T cells for detecting the presence of HDAC3, GABP{alpha}, and GABPβ1. Following T cell lyses, Western blots were performed using the appropriate Abs. These are representative Western blots from four separate experiments. Right panel, Immunoprecipitation (IP) followed by immunoblotting (IB) for studying pro-IL-16 binding proteins. The immunoprecipitates were resolved by SDS-PAGE, transferred onto nitrocellulose membrane, and incubated with the indicated Abs.

 
The pro-IL-16/HDAC3 complex is targeted to the Skp2 promoter by GABP

Previously we have shown that nuclear expression of pro-IL-16 results in the repression of Skp2 expression (10). Thus far we have demonstrated that pro-IL-16 forms a complex with HDAC3 and GABPβ1. We next sought to confirm that pro-IL-16 is part of GABP-DNA complex of the Skp2 promoter by using EMSA followed by an Ab supershift assay. The GABP complex has been reported to bind to the core promoter region of the Skp2 gene in HeLa cells (21), but this has not been demonstrated in T cells. The functional GABP binding motif in the core promoter of Skp2 resides between nucleotides –175 and –152 (21). As shown in Fig. 4A, the nuclear extract prepared from resting normal human T cells generated four gel shift complexes (I-IV) with the [{alpha}-32P]dCTP-labeled oligonucleotides spanning the region from nucleotide positions –175 to –152 of the Skp2 promoter. The addition of a cold specific competitor effectively inhibited the formation of four gel shift complexes (Fig. 4A, lane 3), but these DNA-protein complexes were not affected by a nonspecific oligonucleotide (Fig. 4A, lane 4). To determine the composition of four gel shift complexes, we performed supershift EMSA with Abs specific for GABP{alpha} or GABPβ1 to determine whether the gel shift complexes contained GABP. As shown in Fig. 4B, the intensity of complexes I, III, and IV are decreased and supershifted by both anti-GABP{alpha} (Fig. 4B, lane 2) and GABPβ1 (Fig. 4B, lane 3) Abs whereas II was not affected by either Ab, suggesting that the I, III, and IV complexes contain both the {alpha} and β1 subunits. To investigate whether pro-IL-16 is a component of the DNA-GABP complex, supershift EMSA with anti-pro-IL-16 Ab was also performed. It is apparent that anti-pro-IL-16, but not anti-actin, caused the appearance of a slow-migrating band (Fig. 4B, right panel, lane 4), indicating that pro-IL-16 is part of the DNA-GABP complex.


Figure 4
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  		FIGURE 4. Pro-IL-16, HDAC3, and GABP form protein complexes capable of binding to the core promoter of Skp2 gene. A, EMSA. The nuclear extracts prepared from resting T cells were incubated with 32P end-labeled probe at room temperature for 20 min (lane 2). The resultant DNA-protein complexes were incubated with a 200-fold molar excess of the unlabeled cold probe (lane 3) or unrelated cold oligonucleotides (lane 4) for an additional 10 min. Lane 1 is the control of 32P-labeled probe. All reactions were separated on a 5% nondenaturing gel. Dried gel was exposed to film. B, EMSA supershift analysis were performed as in (A) in the absence (lane 1) or presence of anti-GABP{alpha} (lane 2), anti-GABPβ1 (lane 3), anti-pro-IL-16 (lane 4), or anti-actin (lane 5) Abs. The positions of the supershifted bands are indicated (SS). A longer exposed film was also presented (right panel). C, ChIP assay was performed using nuclear extracts of resting human T cells and polyclonal Abs specific to GABP{alpha} (lane 3), GABPβ1 (lane 4), pro-IL-16 (lane 5), HDAC3 (lane 6), and normal rabbit IgG (lane 2). Input for PCR contains 0.5% of total amount of chromatin used for respective immunoprecipitation (lane 1). The DNA was analyzed by PCR with primers specific for the GABP-binding region in the human Skp2 core promoter.

 
We next performed ChIP (Fig. 4C) to further investigate whether pro-IL-16 and HDAC3 are the components of the core promoter of Skp2. Primers flanking the GABP binding region were used to amplify DNA bound in complexes precipitated by anti-GABP{alpha}, anti-GABPβ1, anti-pro-IL-16, and anti-HDAC3 Abs (Fig. 4C, lane 3, 4, 5, and 6). Specific DNA fragments were observed from complexes immunoprecipitated by anti-GABP{alpha} and anti-GABPβ1 as predicted by EMSA and also from the complexes immunoprecipitated by anti-pro-IL-16 and anti-HDAC3. Because it is GABP{alpha} that directly binds DNA, it is strongly suggest that pro-IL-16, GABPβ1, HDAC3, and GABP{alpha} form a multiprotein complex associated with the Skp2 core promoter.

Inhibition of pro-IL-16-mediated growth repression by the HDAC inhibitor TSA

We next sought to define the functional significance of a HDAC in the repression of Skp2 transcription using the HDAC inhibitor TSA (22, 23). For these experiments we used tetracycline-responsive Jurkat cells stably transfected to express either wild-type GFP-pro-IL-16, GFP-pro-IL-16 with the nuclear localization signal (NLS) deleted ({Delta}NLS), or GFP fused to a NLS sequence (10). As shown in Fig. 5A, HDAC3 is present in the cytoplasm and nucleus of Jurkat cells (upper left panel). Importantly, HDAC3 bound to pro-IL-16 as demonstrated by immunoprecipitation and Western blotting (lower left panel). Notably, {Delta}NLS pro-IL-16 defective in nuclear localization did not affect the nuclear localization of HDAC3 (Fig. 5A, right panels), indicating that pro-IL-16 does not function as a nuclear chaperone for HDAC3. Because transcription of Skp2 is highly active in Jurkat cells that lack nuclear pro-IL-16 (10), it is clear that nuclear HDAC3 alone is incapable of regulating Skp2 transcription in the absence of pro-IL-16. We next examined whether the catalytic activity of HDAC3, bound to pro-IL-16, is required for the effects of pro-IL-16 on the transcription of Skp2 and on cell growth. This was determined by first measuring Skp2 mRNA in nuclear pro-IL-16-positive or -negative Jurkat cells treated with the HDAC inhibitor TSA. As shown in Fig. 5B, top panel, Skp2 mRNA was barely detectable in cells expressing nuclear pro-IL-16. However, by 6 h of treatment with TSA, Skp2 mRNA was readily detected at the levels approximating those seen in the control cells that lack nuclear pro-IL-16 (Fig. 5B, bottom panel). These data indicate that the activity of HDAC can affect Skp2 transcription, but the presence of nuclear pro-IL-16 is required. As expected, p27KIP1 mRNA levels were high in both circumstances and not affected by TSA treatment. In addition, protein levels of Skp2 increased in the pro-IL-16-expressing cells following TSA treatment (Fig. 5C, left panel); as expected, rising levels of Skp2 protein were associated with decreasing levels of p27KIP1.


Figure 5
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  		FIGURE 5. HDAC catalytic activity is required for the nuclear effects of pro-IL-16. A, Nuclear localization of HDAC3 occurs in the absence of pro-IL-16. Nuclear (N) and cytoplasmic extracts (C) from Jurkat cells, stably transfected to express pro-IL-16 in both nucleus and cytoplasm (wild type) or cytoplasm alone ({Delta}NLS) were separated by SDS-PAGE and blotted with the Abs as indicated. The top panels are the results of immunoblotting with Abs to pro-IL-16 and HDAC3. The bottom panels are the results of immunoprecipitation with anti-IL-16 Ab followed by immunoblotting with either anti-pro-IL-16 or anti-HDAC3 Abs. B, RT-PCR analysis of p27KIP and Skp2 expression from Jurkat cells treated with TSA. Stably transfected Jurkat cells expressing either GFP alone or GFP-pro-IL-16 were treated with the HDAC inhibitor TSA (50 ng/ml). Cells were harvested at 0 or 6 h. Total RNA was extracted and subjected to one-step RT-PCR analysis using primers specific for p27KIP1, Skp2, or β-actin, respectively. C, Western blot analysis of p27KIP and Skp2 expression from Jurkat cells treated with TSA. The total proteins of Jurkat cells as described in B were extracted and subjected to Western blot analysis with Abs as indicated. D, Cell cycle analysis of Jurkat cells treated with TSA. The TSA treated Jurkat cells as described in B were fixed and stained with propidium iodide. The cell cycle distributions were analyzed by FACS analysis, showing that TSA treatment led to a reduced G1 cell cycle arrest caused by pro-IL-16. This set of experiment was repeated three times with similar results. These are representative figures from those experiments.

 
Next, we investigated whether the changes in Skp2 and p27KIP1 protein levels in the presence of TSA were associated with changes in the cell cycle distribution (Fig. 5D). Because TSA is known to cause apoptotic cell death, we elected to use low dose TSA (50 ng/ml) and short-time (6 h) treatment. Under these conditions <10% cell death was observed in keeping with several reports (24, 25). As shown in Fig. 5D, the expression of pro-IL-16 in Jurkat cells caused a significant accumulation of G0/G1 cells (62%) in comparison with the control cells (45%). Upon TSA treatment, which induced Skp2 accumulation and up-regulation of p27KIP1 (Fig. 5C), Jurkat cells expressing pro-IL-16 that had not yet undergone apoptosis exhibited a cell cycle distribution pattern similar to that of control cells, suggesting that the loss of the growth suppression function of pro-IL-16 is due to the inhibition of HDAC activity. Taken together, cell growth arrest of Jurkat cells induced by the nuclear expression of pro-IL-16 can be reversed by the HDAC inhibitor TSA, suggesting that the enzymatic activity of HDAC is essential for both pro-IL-16-mediated transcription repression of Skp2 and cell growth control.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Identifying HDAC3 and GABPβ1 as unique proteins bound to pro-IL-16 provides a potential mechanism for the repression of Skp2 transcription in resting T cells, namely that HDAC3 is recruited to the Skp2 core promoter by interacting with pro-IL-16, which targets it to GABP transcription factors (Fig. 6). Our studies indicate that HDAC3 is present in the nucleus of T cells independently of pro-IL-16 expression (Fig. 5A, right upper panel); however, HDAC3-mediated regulation of Skp2 transcription is dependent on the nuclear expression of pro-IL-16 (Fig. 5B). These findings, along with the reversal of G1 arrest induced by nuclear expression of pro-IL-16 by the HDAC inhibitor TSA (Fig. 5D), define pro-IL-16 as a nuclear scaffold for recruiting HDAC3 to the Skp2 promoter as schematically represented in Fig. 6. Although these studies demonstrate that this complex can target the core promoter of the Skp2 gene in T cells, it is likely that transcription of other genes with suitable GABP transcriptional complexes might also be repressed. In fact, by comparing mRNA expression arrays of Jurkat cells that express nuclear vs cytoplasmic pro-IL-16, we identified that nuclear pro-IL-16 is also associated with the repression of PCNA transcription (W. W. Cruikshank and D. M. Center, unpublished observations). The PCNA core promoter contains a canonical GABP binding motif that, interestingly, has not been functionally characterized (26, 27, 28). Along these lines there is an inverse relationship between pro-IL-16 expression and PCNA in neural cells and, thus, it is possible that similar functions for pro-IL-16 exist in non-T cells (29).


Figure 6
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  		FIGURE 6. A working model for pro-IL-16-mediated repression of Skp2 gene expression. In resting T cells, pro-IL-16 recruits HDAC3 to the Skp2 promoter through binding to GABP and allows HDAC3 to block Skp2 transcription. Following T cell activation, pro-IL-16 expression is diminished and results in dissociation of the transcription repressor complex and transcription of Skp2, leading to degradation of p27KIP1 and cell cycle progression.

 
In addition to HDAC3 and GABPβ1, we identified HSC70 as another unique protein bound to the PDZ domain 1 of pro-IL-16. HSC70 lacks a PDZ binding consensus sequence, suggesting that binding occurs through a different region of PDZ domain 1 or alternatively, through an adjacent sequence contained within the construct used. The role of HSC70 bound to pro-IL-16 has not been determined; however, HSC70 has been shown to function as a molecular chaperone for proteins such as NF-{kappa}B (30), temperature-sensitive p53 (31), and nucleoplasmin (32) and as a stabilizing factor for HDAC3 (33). The role of HSC70, when bound to pro-IL-16, is under investigation. Of note, our experiments were not designed to detect high molecular mass proteins like myosin phosphatase-targeting subunits, which have previously been shown to associate with pro-IL-16 in the cytoplasm when coexpressed in COS cells (9). Presumably, this cytoplasmic interaction has a function independent of nuclear events such as transcriptional regulation.

Our overall concept is that maintaining adequate levels of nuclear pro-IL-16 to function as a scaffold for HDAC3 and GABPβ1 may be an important component for maintaining T cell quiescence. If the GABP/pro-IL-16/HDAC3 complex plays a role in normal T cell activation and cell cycle progression, some component of this complex should be highly susceptible to regulation following T cell activation. Clearly HDAC3, GABP{alpha}, and GABPβ1 are all present in both actively dividing and quiescent cells, and none of these components appear to be regulated or altered during active cell proliferation. In contrast, pro-IL-16 is markedly down-regulated following T cell activation by the acceleration of protein degradation, nuclear export (34), and the silencing of transcription (35, 36). In the mRNA array studies, (pro)-IL-16 mRNA has been shown to be one of the most highly down-regulated genes following T cell activation (35, 36). Taken together with existing literature, our current data suggest that pro-IL-16, functioning as an HDAC3 scaffold, plays a key regulatory function in permitting T cell transition from the G1 phase to the S phase. Consistent with this hypothesis, constitutive overexpression of nuclear pro-IL-16 is sufficient to prevent T cell proliferation in response to TCR activation (34), and re-expression of nuclear pro-IL-16 in pro-IL-16-negative Jurkat cells is also sufficient to induce cell cycle arrest (10). Because pro-IL-16 is required for HDAC3 regulation of Skp2 transcription, constitutively expressed at high levels in the nuclei of quiescent T cells, and lost in association with T cell activation and proliferation, we believe that these studies provide novel insights into the regulation of T cell quiescence and proliferation.


   Acknowledgment
 
We thank Dr. W. J. Leonard for providing the Abs to GABP{alpha} and GABPβ1.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants R01 HL32802 and R01 AI35680 (to D.M.C.), and R01 CA100925 (to Y.Z.). Back

2 Address correspondence and reprint requests to Dr. Yujun Zhang, Pulmonary Center, R-304, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. E-mail address: yujunz{at}bu.edu Back

3 Abbreviations used in this paper: GABP, GA-binding protein; ChIP, chromatin immunoprecipitation; HDAC3, histone deacetylase; HSC70, heat shock cognate protein 70; NLS, nuclear localization signal; PVDF, polyvinylidene difluoride; TSA, trichostatin A. Back

Received for publication September 26, 2006. Accepted for publication October 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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