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J. Biol. Chem., Vol. 281, Issue 37, 26802-26812, September 15, 2006

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Negative Regulation of the RelA/p65 Transactivation Function by the Product of the DEK Proto-oncogene^*

Morgan Sammons¹, Shan Shan Wan¹, Nancy L. Vogel, Edwin J. Mientjes^¶, Gerard Grosveld^¶, and Brian P. Ashburner²

From the Department of Biological Sciences and Undergraduate Honors Program, University of Toledo, Toledo, Ohio 43606 and ^¶Department of Genetics and Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

Received for publication, January 30, 2006 , and in revised form, July 7, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
NF-B-mediated transcriptional activation is controlled at several levels including interaction with coregulatory proteins. To identify new proteins capable of modulating NF-B-mediated activation, a cytoplasmic two-hybrid screen was performed using the p65 C-terminal transactivation domain as bait and identified the product of the DEK proto-oncogene. DEK is a ubiquitous nuclear protein that has been implicated in several types of cancer and autoimmune diseases. DEK appears to function in several nuclear processes including transcriptional repression and modulation of chromatin structure. Our data indicate that DEK functions as a transcriptional corepressor to repress NF-B activity. DEK expression blocked p65-mediated activation of an NF-B-dependent reporter gene and also inhibited TNF-induced activation of the reporter gene. Chromatin Immunoprecipitation (ChIP) assays showed that DEK associates with the promoters of the NF-B-regulated cIAP2 and IL-8 genes in untreated cells and dissociates from these promoters upon NF-B binding in response to TNF treatment. Moreover, the expression levels of an NF-B-dependent reporter gene as well as the NF-B-regulated Mcp-1 and IB genes is increased in DEK^–/– cells compared with wild-type cells. ChIP assays on these promoters show enhanced and prolonged binding of p65 and increased recruitment of the P/CAF coactivator. Overall, these data provide further evidence that DEK functions to negatively regulate transcription.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
NF-B plays an important role in many cellular processes by regulating the expression of genes involved in immune and inflammatory responses, cell adhesion, cell cycle regulation, angiogenesis, and apoptosis (1, 2). In mammals the NF-B family includes five members: RelA (referred to as p65 from here on), c-Rel, and RelB as well as p105 and p100, which are processed into the p50 and p52 subunits, respectively (2, 3). Each subunit includes a Rel homology domain (RHD) containing the DNA binding and protein dimerization domains as well as the nuclear localization sequence. In addition, p65, c-Rel, and RelB contain an acidic transactivation domain (TAD)³ at the C terminus. The most well studied and predominant form of NF-B consists of a heterodimer composed of the p50 and p65 subunits (4). In unstimulated cells most NF-B resides in the cytoplasm bound by a member of the IB family of inhibitory proteins (2, 3). Upon stimulation, IB is phosphorylated by the IB kinase (IKK) complex, which targets IB for polyubiquination and degradation by the 26 S proteasome, allowing NF-B to translocate to the nucleus to activate transcription of its target genes (2, 3).

Upon entry into the nucleus, NF-B binds to the promoters of its target genes to induce high levels of transcription of these genes. Activation of transcription requires interaction with coactivator proteins, such as those that possess histone acetyltransferase (HAT) activity (3). HAT enzymes function to facilitate alteration of chromatin structure that allows recruitment of the basal transcription factors and RNA polymerase II to the promoter to initiate transcription. NF-B interacts with several different HAT enzymes including the CREB-binding protein (CBP) and its homolog, p300 (5–7), the p300/CBP-associated factor (P/CAF) (7–9), and members of the SRC/p160 family (7–9). These HAT enzymes function to acetylate the N-terminal tails of the core histones as well as to acetylate the p50 and p65 subunits of NF-B (8, 10, 11) to stimulate high levels of NF-B-dependent gene expression.

NF-B-dependent gene expression is also modulated through interaction with histone deacetylase (HDAC) enzymes including the Class I HDACs: HDAC1, HDAC2, HDAC3 (10, 12, 13), and the Class III HDAC, SIRT1 (14). These HDAC proteins function to negatively regulate NF-B transcriptional activity by deacetylating the N-terminal tails of the core histones as well as by deacetylating the p50 and p65 subunits of NF-B. In support of this, treatment of cells with specific HDAC inhibitors results in potentiation of TNF-induced NF-B activity (12, 13, 15, 16). This indicates that negative regulation of NF-B activity through interaction with transcriptional corepressor proteins plays an important role in the overall control of NF-B activity.

DEK is a 375-amino acid nuclear protein that is ubiquitously expressed in most mammalian cells (17, 18) and was initially identified as a fusion protein with the nucleoporin CAN in a minority of acute myeloid leukemia (AML) patients (19). Expression of the dek gene is increased in multiple tumor cell types including bladder cancer, hepatocellular carcinoma, glioblastoma, melanoma, and various forms of leukemia (17, 20). DEK has also been implicated in multiple autoimmune diseases such as lupus and juvenile rheumatoid arthritis (21). Homology searches indicate that DEK is unrelated to any known family of proteins and contains no known enzymatic domains (18). Very little is known about the actual function of DEK, however several studies have implicated DEK in transcriptional regulation, regulation of HIV-2 replication (22), mRNA splicing (23), and chromatin remodeling (24). Much of the recent research on DEK has focused on its role in mediating changes in DNA structure. However, DEK can exist in a complex with the HDAC2 and Daxx transcriptional corepressor proteins (25), indicating a role for DEK in regulating transcription. In addition, DEK also can interact with the AP-2 transcription factor to enhance its ability to activate transcription (26).

Previously DEK was shown to interact with the pets (peri-ets) site within the HIV-2 promoter and that expression from this promoter is enhanced by disassociation of DEK (22, 27), suggesting DEK acts as a transcriptional repressor (27). DEK was recently shown to be acetylated by the P/CAF transcriptional coactivator protein within its N terminus resulting in a decreased affinity for DNA and accumulation in interchromatin granule clusters (IGCs) (28). Based on these results, it was proposed that coactivator-mediated acetylation of DEK displaces DEK from promoters to allow transcriptional activation (28).

We identified DEK as a p65-interacting protein through a cytoplasmic yeast two-hybrid screen utilizing the C-terminal TAD (aa 313–551) of the p65 subunit as the bait protein. Based on this, we sought to determine the potential role of DEK in regulating NF-B-mediated gene expression. Our data confirm that DEK interacts with the p65 subunit of NF-B and can repress NF-B-dependent gene expression in a concentration-dependent manner in transient transfection reporter gene assays. ChIP analysis demonstrates that DEK associates with the NF-B-regulated IL-8 and cIAP2 promoters in uninduced cells and dissociates in response to TNF stimulation. In addition, quantitative real time PCR (QRT-PCR) shows that basal expression of the NF-B-regulated Mcp-1 and IB genes is increased in fibroblasts isolated from dek^–/– mice relative to cells from wild-type mice. Taken together, these data indicate that DEK can function as a transcriptional corepressor protein to negatively regulate NF-B-dependent gene expression.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cells, Plasmids, and Other Reagents—HeLa and HEK293T cells were obtained from ATCC and maintained in DMEM-H (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Atlanta Biologicals) and penicillin/streptomycin. Immortalized DEK^+/+ and DEK^–/– MEFs were isolated from E14.5 embryos and were maintained in DMEM-H (Invitrogen) with 10% bovine calf serum (HyClone Laboratories, Logan, UT) and penicillin/streptomycin. CMV-p65, 3x B-luc, GAL4-p65, and 5x GAL4-luc have been described previously (12). The DEK expression plasmid (FLAG-DEK) was provided by D. Markovitz (University of Michigan). The human androgen receptor (AR) and GAL4-AR(N terminus) expression plasmids (29) and the PSA-luciferase reporter plasmid (30) were obtained from L. Shemshedini. CMV-p53 (31, 32) is originally from B. Vogelstein (Johns Hopkins University). p53ConA.luc is described in Ref. 33 and was obtained from W. Taylor (University of Toledo). The Stat5-dependent luciferase reporter plasmid containing three repeats of an IFN-activated site element derived from the IRF-1 gene is described in Ref. 34, and the Stat5b expression plasmid is described in Ref. 35. Antibodies used in these studies include: p65 (Rockland Immunochemicals, Inc, Gilbertsville, PA), RNA Pol II large subunit and IB (Santa Cruz Biotechnology, Santa Cruz, CA), DEK (BD Biosciences Pharmingen, San Diego, CA), -actin and P/CAF (Abcam Inc., Cambridge, MA), nonspecific mouse and rabbit IgG and anti-FLAG M2 antibody (Sigma-Aldrich). Recombinant human tumor necrosis factor (TNF) (BIOSOURCE, Camarillo, CA) was used at a final concentration of 10 ng/ml.

Transient Transfection/Reporter Gene Assays—Transient transfection/reporter gene assays were performed in 24-well plates with Fugene 6 (Roche Applied Science) according to the manufacturer's recommendations. Cells were harvested 48 h after transfection, lysed with M-PER cell lysis buffer (Pierce) and assayed for luciferase activity using a LMax luminometer (Molecular Devices, Sunnyvale, CA). All experiments were performed a minimum of three times in triplicate. Data were normalized to total protein assayed.

Coimmunoprecipitation and Western Blot Analysis—For coimmunoprecipitations, the cells were lysed in cell lysis buffer (50 mM Tris, pH 7.5, 85 mM KCl, 0.5% Nonidet P-40 with protease inhibitors) for 10 min on ice and the intact nuclei/membrane fraction was pelleted by centrifugation at 3,300 x g for 5 min. The supernatant was discarded and the nuclei lysed in nuclear lysis/IP buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1.0% Triton X-100; 0.5% Nonidet P-40; 10 mM EDTA with protease inhibitors) for 10 min on ice. The soluble nuclear extract was recovered by centrifugation at 16,000 x g for 15 min and protein concentration determined using the Bio-Rad protein assay reagent. Immunoprecipitations were performed using equal amounts of nuclear extracts with the indicated antibodies and protein A/G-agarose (Santa Cruz Biotechnolgy). The immunoprecipitated complexes were washed four times with 500 µl of nuclear lysis/IP buffer and resuspended in 15 µl of 2x SDS-PAGE sample buffer. The samples were boiled for 3 min and the proteins separated on a SDS-10% polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore). Membranes were blocked in 1x TBST with 5% milk and probed with the indicated antibodies. Proteins were visualized using either anti-rabbit IgG-horseradish peroxidase or anti-mouse IgG-horseradish peroxidase (Promega) and PicoWest chemiluminescent reagent (Pierce) followed by exposure to film.

Chromatin Immunoprecipitation (ChIP) Assays—ChIP analysis was performed essentially as described (36). Cellular proteins and DNA were cross-linked by adding formaldehyde to the growth medium to a final concentration of 0.1%. Cells were harvested in cold phosphate-buffered saline and lysed in cell lysis buffer (50 mM Tris, pH 8.0, 85 mM KCl, 0.5% Nonidet P-40 with protease inhibitors) on ice for 10 min. Nuclei were recovered by centrifugation at 3,300 x g for 5 min at 4 °C. Nuclei were lysed in nuclear lysis buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 1% SDS with protease inhibitors). Lysates were sonicated using a Sonic Dismembranator, Model 500 (Fisher Scientific) and precleared with protein A-agarose or protein A/G-agarose (Santa Cruz Biotechnology) and rabbit or mouse IgG (Sigma). Lysates were then rocked overnight at 4 °C with the immunoprecipitation antibody. Protein A-agarose or protein A/G-agarose were added and incubated at 4 °C for 1 h with rocking. Complexes were washed one time in low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris, pH 8.0, 150 mM NaCl), one time in high salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20 mM Tris, pH 8.0, 500 mM NaCl), and one time in LiCl buffer (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1mM EDTA, 10 mM Tris, pH 8.0) followed by two washes in TE buffer (20 mM Tris and 2 mM EDTA). Washed complexes were eluted with freshly prepared elution buffer (1% SDS and 100 mM NaHCO₃) and the Na⁺ concentration was adjusted to 200 mM by adding NaCl followed by incubation at 65 °C overnight to reverse cross-links. DNA was purified utilizing a PCR purification kit (Qiagen, Valencia, CA) and one-tenth of the eluate was used to amplify the indicated promoter regions. The primers used for PCR were: IL-8: forward, 5'-GGGCCATCAGTTGCAAATC-3' and reverse, 5'-TTCCTTCCGGTGGTTTCTTC-3'; cIAP2: forward, 5'-GTGTGTGTGGTTATTACCGC-3' and reverse, 5'-AGCAAGGACAAGCCCAGTCT-3'; GAPDH: forward, 5'-AGCGCAGGCCTCAAGACCTT-3' and reverse, 5'-AAGAAGATGCGGCTGACTGT-3'; Mouse IB: forward, 5'-CGCTAAGAGGAACAGCCTAG-3' and reverse, 5'-CAGCTGGCTGAAACATGGC-3'; mouse MCP-1: forward, 5'-CACCCCATTACATCTCTTCCCC-3' and reverse, 5'-TGTTTCCCTCTCACTTCACTCTGTC-3'. Amplified DNA was visualized by agarose gel electrophoresis using SYBR Gold (Invitrogen/Molecular Probes). Images were captured using a Kodak EAS290 Digital Camera and images were inverted using the software program provided by the manufacturer (Kodak).

View larger version (30K): [in this window] [in a new window]   FIGURE 1. DEK interacts with p65 in vivo. A, coimmunoprecipitations were performed on whole cell extracts from HEK293T cells transfected with the indicated plasmids. The upper panel shows coimmunoprecipitation of p65 with DEK. Lower panels show input extracts probed for p65 (middle panel) and DEK (lower panel). B, coimmunoprecipitation between endogenous DEK and endogenous p65. Nuclear extracts from HeLa cells that were either untreated or treated with TNF (10 ng/ml) for the indicated times were immunoprecipitated with an antibody specific for DEK, and immunoprecipitated proteins were subjected to Western blot analysis probing for p65.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
DEK Interacts with the p65 Subunit of NF-B—A cytoplasmic yeast two-hybrid screen initially identified DEK as a potential protein that can interact with the C-terminal TAD of p65. To confirm the interaction between DEK and p65 HEK293T cells were transfected with the indicated plasmids and immunoprecipitations were performed (from equal amounts of nuclear extracts) with FLAG antibody. As shown in Fig. 1A, lane 1, p65 coimmunoprecipitated with FLAG-DEK from nuclear extracts of cells transfected with both DEK and p65. In addition, a weak but reproducible interaction was observed between the FLAG-DEK and endogenous p65 (Fig. 1A, lane 3). This weak interaction is likely caused by the low basal level of nuclear p65 in uninduced cells and may indicate that DEK inhibits the ability of this nuclear p65 to activate transcription. No nonspecific interactions were observed from control transfected cells (Fig. 1A, lanes 2 and 4), indicating the specificity of the DEK-p65 interaction. The lower panels (Fig. 1A) show the input extracts used for the immunoprecipitation, probed for both p65 (middle panel) and DEK (lower panel). Overall these results suggest that an interaction between p65 and DEK occurs in the nuclei of intact cells.

Endogenous DEK and p65 Interact in a TNF-inducible Manner—To further confirm this interaction, coimmunoprecipitations were performed to demonstrate an interaction between endogenous DEK and endogenous p65. Nuclear extracts from cells that were treated with TNF for 20 or 120 min (or no TNF) were immunoprecipitated with DEK antibody and the immunoprecipitates analyzed by immunoblotting. In untreated cells, a low level of nuclear p65 coprecipitated with DEK (Fig. 1B, lane 1). In contrast, there was a strong interaction between p65 and DEK detected after a 20-min TNF treatment (Fig. 1B, lane 2). At the 120-min TNF treatment, a reduced amount of p65 coprecipitated with DEK despite a similar level of nuclear p65 to the 20 min treatment, (Fig. 1B, lane 3). This decrease is possibly caused by the association of p65 with transcriptional coactivators or with IB, both of which may displace DEK. Taken together, the data in Fig. 1 show that DEK and p65 interact and confirm our two-hybrid results.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. DEK inhibits p65-mediated transcriptional activation. A, HeLa and HEK293T cells were transiently transfected with the B-luc reporter plasmid and the indicated plasmids. For CMV-F-DEK, lanes 3, 4, and 5 were transfected with 10, 25, and 50 ng of plasmid, respectively. Lanes 7 and 8 were transfected with 25 and 50 ng of the plasmid, respectively. HeLa cells were also transiently transfected with PSA-luc (B), p53-luc (C), and IRF-luc (D) and the indicated plasmids. In all cases, lanes 3, 4, and 5 were transfected with 10, 25, and 50 ng of plasmid, respectively. Cells were harvested 48 h after transfection and extracts assayed for luciferase activity. Luciferase activities were normalized to total protein assayed and expressed as the average RLU/µg of extract ± S.D. of a representative experiment performed in triplicate.

Repressive Effect of DEK Is Not Cell Type-specific—Similar experiments were also performed in HEK293T cells to show that the ability of DEK to repress p65-mediated transcription is not cell type-specific. HEK293T cells (Fig. 2A, right panel) were transfected as indicated and assayed for reporter gene activity 48-h post-transfection. Similar to the effects observed in HeLa cells, coexpression of DEK with p65 in HEK293T cells caused a concentration-dependent decrease in p65-mediated activation of reporter gene expression (Fig. 2A, right panel, compare lanes 3–5 with lane 2). These results further demonstrate that DEK is able to repress p65-mediated transcriptional activation and that the effect is not cell type-specific.

DEK Represses the Ability of Other Transcription Factors to Activate Transcription—To determine if the ability of DEK to repress transcription is specific to p65 or if DEK is also able to repress the activity of other transcription factors, transient transfection reporter gene assays were performed using reporter genes driven by the androgen receptor (PSA-luc), p53 (p53-luc), and Stat5B (IRF-luc),. These results show that DEK also is able to repress AR-driven PSA-luc reporter gene expression and p53-driven expression of the p53-dependent reporter gene in a concentration-dependent manner (Fig. 2, B and C). In addition, DEK was also able to modestly repress Stat5B-mediated expression of the IRF-luc reporter gene but only at the highest concentration of transfected plasmid (Fig. 2D, lane 5). These data indicate that similar to other transcriptional corepressors, the repressive effect mediated by DEK is more general and not specific to repression of p65-mediated transcription.

DEK Represses TNF-induced NF-B-dependent Transcriptional Activity—We next wanted to determine whether DEK could repress the ability of endogenous TNF-induced NF-B to activate expression of the B-luc reporter gene. HeLa cells were transfected with the B-luc reporter gene plasmid and either the vector control plasmid (CMV-FLAG) or increasing amounts of the DEK expression plasmid. About 40 h after the transfection, the cells were either left untreated or were treated for 6 h with TNF (10 ng/ml), the cells harvested and extracts assayed for reporter gene activity. In the absence of DEK expression, TNF treatment resulted in a 35-fold increase in reporter gene expression (Fig. 3). Transfection of 10 ng of the DEK plasmid had only a negligible affect on TNF-induced reporter gene activity (Fig. 3). However, increasing the amount of transfected DEK expression plasmid to 25 ng and 50 ng resulted in a 50% (for 25 ng) and 90% (for 50 ng) reduction in TNF-induced reporter gene activity (Fig. 3). In contrast, when a mutant B-luc reporter plasmid was used, as expected there was no TNF-induced increase in reporter gene activity, and DEK expression had no effect on reporter gene expression in the presence or absence of TNF stimulation (data not shown). Because the NF-B binding sites in this promoter are mutated to prevent binding of NF-B, this indicates that the DEK-mediated repression is dependent on binding of NF-B to the promoter.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. DEK represses TNF-induced NF-B activity in a concentration-dependent manner. HeLa cells were transfected with the B-luc reporter plasmid and either the control parental vector (CMV-F) or the indicated amounts of the CMV-F-DEK plasmid. Approximately 40 h after transfection, cells were either left untreated or treated with TNF (10 ng/ml) for 6 h. Extracts were prepared and relative luciferase activities were determined by normalizing to total protein assayed and expressed as the average RLU/µg of extract ± S.D. of a representative experiment performed in triplicate.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. DEK inhibits NF-B activity through the p65 transactivation domain. A, HeLa cells were transiently transfected with the indicated plasmids and the 5x GAL4-luc reporter plasmid. Extracts were assayed for luciferase activity 48 h after the transfection. In addition, HeLa cells were transfected with a GAL4-p53 fusion (B) or a GAL4-AR(N terminus) fusion protein (C) and the indicated plasmids and extracts assayed for luciferase activity. Luciferase values were normalized to total amount of protein assayed and expressed as average RLU/µg of extract ± S.D. All experiments were performed in triplicate and performed three times with similar results.

View larger version (43K): [in this window] [in a new window]   FIGURE 5. DEK associates with the NF-B-regulated cIAP2 and IL-8 promoters. A, ChIP assays were performed on nuclear extracts from HeLa cells according to the protocol described under "Materials and Methods." Immunoprecipitated DNA was subjected PCR using primers specific to the indicated promoters and amplified DNA visualized by agarose gel electrophoresis. B, real-time PCR analysis of the cIAP2 and IL-8 genes. HeLa cells were either untreated or treated with TNF for the indicated times and total RNA was harvested, reverse-transcribed, and subjected to real-time PCR analysis in triplicate. Expression cIAP2 and IL-8 was normalized to -actin and data expressed as fold activation relative to the untreated sample. All experiments were performed a minimum of three times with similar results.

To correlate the dissociation of DEK from the cIAP2 and IL-8 promoters with the TNF-induced expression of these genes, expression was analyzed by real-time PCR analysis. For cIAP2, low levels of expression was observed in untreated cells and cells treated with TNF for 15 and 30 min (Fig. 5B, left panel). However, as DEK began to dissociate from the promoter at 15 and 30 min (Fig. 5A), expression of cIAP2 gradually increased (Fig. 5B). At 60 min, where DEK binding is at its lowest level, cIAP2 expression is maximal (Fig. 5, A and B). After a 2-h TNF treatment, DEK binding to the promoter is slightly higher (Fig. 5A) and expression is lower (Fig. 5B) compared with that at 60 min. A similar pattern is seen with IL-8 where maximal TNF-induced expression is detected at 30 and 60 min (Fig. 5B, right panel) coinciding with the lowest level of DEK association with the promoter (Fig. 5A). Taken together, these data show that DEK associates with the promoters of two NF-B-regulated genes (but not the constitutively expressed GAPDH gene) and that dissociation of DEK from these promoters in response to TNF treatment correlates with increased expression of these genes.

The Absence of DEK Results in Increased Basal Expression of NF-B-regulated Genes—To further analyze the role of DEK in repression of NF-B-dependent gene expression we utilized immortalized fibroblasts from dek^–/– knock-out mice and wild-type control littermates. To analyze the effect of loss of DEK on NF-B-dependent gene expression, wild type and DEK^–/– cells were transiently transfected with theB-luc reporter plasmid. About 36 h after the transfection, cells were either left untreated or treated with TNF (10 ng/ml) for 6 h, harvested and assayed for luciferase activity. In the absence of TNF treatment, basal reporter gene expression was 5.6-fold higher in the DEK^–/– cells compared with the wild-type cells (Fig. 6A). TNF treatment of the wild-type cells resulted in a 4.4-fold induction of reporter gene activity, whereas TNF treatment of the DEK^–/– cells resulted in a 24-fold increase in reporter gene activity (Fig. 6A). Thus, in the absence of DEK, TNF-induced NF-B activity increases about 5-fold compared with wild-type cells, consistent with our observation that DEK represses the ability of NF-B to activate transcription.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Increased expression of NF-B-dependent genes in DEK–/– cells. A, MEFs from DEK–/– mice or wild-type littermates were transiently transfected with the B-luc reporter plasmid. Approximately 40 h after the transfection the cells were treated with TNF (10 ng/ml) for 6 h and extracts were harvested and assayed for luciferase activity. Experiments were performed three times in triplicate. Data were normalized to total protein assayed and is expressed as average fold activation compared with the wild-type untreated ± S.D. Numbers above each bar indicate the average fold activation. B, real-time PCR analysis of the Mcp-1 and IB genes. Wild-type and DEK–/– cells were either untreated or treated with TNF (10 ng/ml) for the indicated times and total RNA was harvested, reverse-transcribed, and subjected to real-time PCR analysis in triplicate. Expression Mcp-1 and IB was normalized to -actin and data expressed as fold activation relative to the untreated sample. Experiments were performed a minimum of three times with similar results. A representative experiment is shown. C, Western blot analysis using whole cell extracts (25 µg/lane) from wild-type and DEK–/– cells showing increased basal and TNF-induced expression of the IB gene in the DEK–/– cells compared with the wild-type cells.

To further demonstrate the role of DEK in regulating expression of an NF-B-regulated gene, IB protein expression was monitored in wild type and DEK^–/– cells in response to TNF treatment by immunoblotting. In the wild-type cells a typical pattern of IB degradation and resynthesis was observed (Fig. 6C, lanes 1–5). In the DEK^–/– cells a basal level of IB was detected that was similar to wild-type cells and once again the typical pattern of degradation and resynthesis was observed (Fig. 6C, lanes 6–10). However, the amount of IB that was resynthesized in the DEK^–/– cells was consistently greater at all of the time points compared with the wild-type cells (Fig. 6C, lanes 7–10 versus 2–5). These data correlate well with the increased level of IB expression observed by real-time PCR in the DEK^–/– cells (Fig. 6B, lower panel). Taken together, these data further support a role for DEK in repressing NF-B-dependent gene expression. It should also be noted that in both the immunoblotting and real-time PCR, expression of the NF-B-regulated genes was compared with expression of the constitutively expressed -actin. No detectable difference in -actin expression (protein or mRNA) between the DEK^–/– cells and the wild-type cells was observed, indicating that there may be some promoter specificity for DEK-mediated repression.

Effect of the Absence of DEK on Binding of p65 to the IB and Mcp-1 Promoters—To assess the mechanism by which DEK represses the ability of NF-B to activate transcription, we performed ChIP assays on the Mcp-1 and IB promoters in the wild-type and DEK^–/– cells to determine if the absence of DEK affects binding of p65 to these promoters. The cells were untreated or treated with TNF (10 ng/ml) for the indicated times and after cross-linking of protein-DNA complexes, immunoprecipitations were performed from nuclear extracts using an antibody specific for p65. Primers specific for the Mcp-1 and IB promoters were used to amplify immunoprecipitated DNA. In wild-type cells, a typical pattern of recruitment of p65 to both the Mcp-1 (Fig. 7, lanes 1–5) and IB (Fig. 7, lanes 11–15) promoters was observed in response to TNF treatment with peak binding occurring at 30 min (Fig. 7, lanes 3 and 13) and a gradual decrease in binding at the 60 and 120 min (Fig. 7, lanes 4 and 5 and 14 and 15). Interestingly, in the absence of DEK, the TNF-induced recruitment of p65 to both the Mcp-1 and the IB promoters was enhanced and prolonged (Fig. 7, lanes 6–10 versus lanes 1–5 and lanes 16–20 versus lanes 11–15).

View larger version (22K): [in this window] [in a new window]   FIGURE 7. ChIP analysis of Mcp-1 and IB promoters in DEK–/– cells. ChIP assays were performed on nuclear extracts from the wild-type and DEK–/– MEFs according to the protocol described under "Materials and Methods." Protein-DNA complexes were immunoprecipitated with the indicated antibodies and immunoprecipitated DNA was subjected to PCR using primers specific to the mouse Mcp-1 or IB promoters. PCR products were visualized by agarose gel electrophoresis. Each IP was performed a minimum of three times with similar results.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Regulation of transcription by NF-B requires a number of different proteins including transcriptional coactivator and corepressor proteins. In order to gain a better understanding of how NF-B transcriptional activity is regulated we initiated a cytoplasmic yeast two-hybrid screen to identify proteins that interact with the C-terminal TAD of the p65 subunit. Previously, three proteins that interact with the p65 TAD were identified in transcription-based two-hybrid screens, however the bait proteins in these screens were deletion mutants that removed a significant portion of the TAD (37–39). This deletion was necessary because the p65 TAD can potently stimulate transcription in yeast. Interestingly, within the TAD are three LXXLL (LXD) motifs (aa 436–441; 450–454; and 523–528). LXD motifs are frequently found in transcription factors as well as in transcriptional coactivator proteins and function to mediate protein-protein interactions (40, 41). For example, LXD motifs within the p160/SRC family of coactivators mediate the interaction between these proteins and the AF2 domain of ligand-bound nuclear receptors (42, 43). The three LXD motifs found within the p65 TAD appear to be important since mutation of any one of them results in a p65 protein that cannot activate transcription of a reporter gene in transient transfection reporter gene assays.⁴ Based on this, we felt it was necessary to utilize a bait protein that includes the entire TAD in order to identify new proteins that interact with and regulate the transcriptional activity of NF-B. By using a cytoplasmic two-hybrid screen, we were able to use the intact TAD as our bait and were successful in identifying several proteins that can specifically interact with p65. In support of this strategy, another group used a similar C-terminal fragment from p65 as bait to identify the RING finger protein AO7 as a transcriptional coactivator of NF-B-mediated transcription (44).

One of the proteins we identified and the focus of the present study is the protein encoded by the DEK proto-oncogene. DEK is a nuclear protein that has been associated with a number of human diseases including autoimmune diseases and cancer (17, 18). Identification of the precise cellular function of DEK has been elusive since DEK does not belong to any characterized protein family and its similarity to other proteins is limited to the 34-amino acid scaffold attachment (SAF) box (18). DEK has been proposed to be involved in a number of different nuclear processes including chromatin organization, mRNA processing, and transcription (18). Previously DEK was shown to bind to the peri-ets (pets) site in the HIV-2 promoter where it functions to repress transcription (22, 27). DEK also interacts with a protein complex that includes hDaxx and HDAC2 (25), further suggesting a role for DEK in transcriptional repression. Because we identified DEK in a two-hybrid screen as a protein that interacts with the C-terminal TAD of the p65 subunit of NF-B, we wanted to determine if DEK could function to regulate the transcriptional activity of NF-B. Our data strongly indicate that DEK functions as a transcriptional corepressor protein to repress the ability of NF-B to activate transcription. We have confirmed the interaction between DEK and p65 by coimmunoprecipitations from transiently transfected cells as well as between endogenous DEK and p65. Transient overexpression of DEK results in a concentration-dependent repression of p65-mediated activation of a NF-B-dependent reporter gene as well as TNF-induced activation of the NF-B-dependent reporter gene. In addition, DEK expression also resulted in repression of the ability of the androgen receptor, p53, and Stat5B to activate transcription indicating DEK functions as a general repressor of transcription. Consistent with the identification of DEK as a protein that interacts with the p65 C-terminal TAD, DEK was able to repress transcription of a heterologous fusion between the GAL4 DNA binding domain and the C-terminal TAD of p65. DEK was also able to repress the ability of the GAL4-AR (N-terminal domain) and GAL4-p53 fusion proteins to activate transcription.

Because the reporter gene assays relied on overexpression of DEK, which could result in nonspecific repression, we further confirmed that DEK functions as a transcriptional corepressor protein that can inhibit expression of NF-B-regulated genes by using MEFs isolated from DEK knock-out mice or from wild-type littermates. Using quantitative real-time PCR we showed that the basal expression of two NF-B-regulated genes (Mcp-1 and IB) was elevated in the DEK^–/– cells compared with the wild-type cells. In addition, TNF-induced expression was also elevated in the DEK^–/– cells compared with the wild-type cells. Therefore, our data strongly indicate that DEK functions as a transcriptional corepressor protein to repress both basal and TNF-induced NF-B-dependent transcription through a direct interaction with the C-terminal TAD of the p65 subunit. Our observations in the DEK^–/– cells strongly support our hypothesis that DEK has a specific function in transcriptional repression. Furthermore, we and others have shown that overexpression of other corepressor proteins, including HDAC1, HDAC2, HDAC3, and SMRT can repress NF-B activity in transient transfection reporter gene assays (10, 12, 13, 45) and that HDAC1, HDAC3, and SMRT all directly interact with the p65 subunit of NF-B (10, 12, 13, 45), in addition to directly interacting with many other transcription factors. The specific interaction of transcriptional coregulatory proteins with a large number of transcription factors is not uncommon. For example, the CBP/p300 interactome includes more than 300 physical or functional interactions with mammalian and viral proteins (46).

Using ChIP assays, we showed that DEK associates with the promoters of the IL-8 and cIAP2 genes in unstimulated HeLa cells. Upon TNF stimulation DEK gradually dissociates from these promoters as binding of the p65 subunit of NF-Btothe promoters increases. Real-time PCR analysis showed that maximal expression of these genes was detected when the lowest level of DEK association with the promoters was detected. Thus it appears that at the early TNF time points a somewhat lower level of DEK remains associated with the promoters and functions to maintain a repressed or low level of expression of these genes. At the later time points most of the DEK dissociates from the promoters, likely correlating with maximal recruitment of coactivator proteins and resulting in maximal levels of gene expression. The fact that a low level of DEK remains associated with the promoters after TNF treatment indicates that DEK may function to not only repress basal (uninduced) expression of these genes, but may also contribute to regulation of induced expression of these genes. This is further supported by the enhanced basal and TNF-induced expression of both Mcp-1 and IB in the DEK^–/– MEFs. In fact, several corepressors, including HDAC2, HDAC3, SMRT, and N-CoR remain associated with the IB promoter in response to TNF induction (47). Moreover, coactivators (P/CAF and GCN5) and corepressors (HDAC1) can physically interact (48). Therefore, it is likely that many different corepressors are associated with promoters even under activation conditions, implying that these proteins function during activation, possibly to help maintain a proper level of transcription and to counteract the activity of coactivators.

The precise mechanism by which DEK represses transcription is not yet known. Our data indicate that DEK functions through a direct interaction with the p65 subunit of NF-B. However it is also possible that DEK functions to repress transcription through other mechanisms including by binding directly to DNA. Our ChIP data show a high level of DEK association with both the cIAP2 and IL-8 promoters in unstimulated cells at a time when p65 association with these promoters is minimal, which may indicate a direct association of DEK with DNA. Another potential mechanism for DEK-mediated repression is through interactions with transcriptional coactivator proteins. A recent manuscript showed that DEK is able to repress the histone acetyltransferase activity of the P/CAF and p300 coactivators through a direct interaction with these proteins resulting in decreased levels of histone H3 and H4 acetylation (49). Interestingly, both P/CAF and p300/CBP are required for NF-B-dependent transcriptional activation (5, 7, 50). Based on our coimmunoprecipitation experiments, DEK is associated with the low level of basal nuclear p65 in untreated cells. In this capacity DEK may function to repress the ability of this basally nuclear NF-B to activate transcription. This may provide an important function to ensure that genes, such as those encoding proinflammatory cytokines are not expressed in the absence of an activating signal. In support of this, an increase in basal expression of both the Mcp-1 and IB genes are observed in untreated DEK^–/– cells compared with the wild-type cells. Upon stimulation of cells with TNF, there is an increase in the nuclear concentration of p65 and a subsequent increase in the amount of DEK associated with p65. Results from ChIP assays show that DEK dissociates from the cIAP2 and IL-8 promoters in response to TNF treatment and that this dissociation occurs as NF-B binding to these promoters increases. Although DEK dissociates from these promoters, there is still some DEK that remains associated after TNF stimulation indicating that DEK not only functions to repress basal expression of these genes but also contributes to repression of activated NF-B. This is further supported by the higher level of expression of both the Mcp-1 and IB genes in the DEK^–/– cells compared with the wild-type cells in response to TNF treatment.

It has been well established that DEK is capable of binding directly to DNA (17, 18); however, it appears to bind DNA in a non-sequence specific, but structure-specific manner (51). This characteristic is similar to the DNA binding preference of the HMG family of non-histone proteins (18) that play a role in a wide variety of nuclear processes including regulation of transcription (52). The HMGI(Y) family plays an important role in regulation of the interferon- (IFN-) gene, which is regulated by the binding of NF-B, members of the IRF family, and ATF-2/c-Jun to its promoter (53). Binding of these transcription factors recruits several coactivator proteins including CBP/p300 and P/CAF to the promoter resulting in the formation of an enhanceosome complex that includes the HMGI(Y) protein (53). In this context HMGI(Y) appears to function to mediate both enhanceosome assembly and disassembly (53). For example, acetylation of HMGI(Y) by P/CAF potentiates transcription of the IFN- gene by stabilizing the enhanceosome whereas acetylation of a different lysine residue on HMGI(Y) by CBP destabilizes the enhanceosome leading to termination of IFN- transcription (54). Much like the HMGI(Y) proteins, DEK may interact directly with DNA as well as with proteins bound to specific promoter elements. However, unlike the HMGI(Y) proteins, association of DEK with these promoters results in repression of transcription, possibly in association with corepressors such as HDAC2 (25) and/or through the inhibition of p300/CBP and P/CAF HAT activity (49). Interestingly, it was recently shown that P/CAF acetylates DEK at lysine residues within the first 70 amino acids of DEK resulting in decreased affinity of DEK for DNA and its accumulation in IGCs (28). Since P/CAF is required for NF-B-dependent activation of transcription (7), it is possible that the recruitment of P/CAF to promoters by activated NF-B results in the subsequent acetylation of DEK and its dissociation from the promoter. In support of this, we observed enhanced recruitment of p65 and P/CAF to the Mcp-1 and IB promoters in DEK^–/– cells compared with the wild-type cells. Thus it appears that DEK-mediated repression is regulated, at least in part by coactivator-mediated acetylation of DEK and that DEK in turn can regulate the HAT activity associated with the p300/CBP and P/CAF coactivators.

Our data support previously published reports that DEK functions at least in part as a transcriptional corepressor protein, possibly through a direct interaction with DNA as well as in association with sequence-specific transcription factors such as NF-B and with other transcriptional corepressor proteins such as HDAC2. Recent data demonstrating coactivator-mediated acetylation of DEK, resulting in its dissociation from DNA suggest a mechanism for overcoming DEK-mediated repression of transcription, which may in turn allow assembly of transcription factor-coactivator complexes to mediate activation of transcription. Future experiments will be directed at gaining a better understanding of the mechanism by which DEK functions as a transcriptional corepressor protein and how this contributes to regulation of transcription by NF-B.

	FOOTNOTES

 
^* This work was supported in part by grants from the Ohio Division of the American Cancer Society and from Grant 1 R15 GM071405-01 from the NIGMS, National Institutes of Health (to B. P. A.). 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 Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Biological Sciences, MS 601, 2801 W. Bancroft St., Toledo, OH 43606. Tel.: 419-530-1542; Fax: 419-530-7737; E-mail: brian.ashburner{at}utoledo.edu.

³ The abbreviations used are: TAD, transactivation domain; ChIP, chromatin immunoprecipitation assay; IL, interleukin; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; HAT, histone acetyltransferase; HDAC, histone deacetylase; aa, amino acid(s); IFN, interferon; Stat, signal transducer and activator of transcription; AR, androgen receptor; TNF, tumor necrosis factor.

⁴ D. Guttridge, personal communication.

	ACKNOWLEDGMENTS

 
We thank Drs. David Markovitz for providing the CMV-FLAG-DEK expression construct, Lirim Shemshedini for the human AR, GAL4-AR and PSA-luciferase reporter plasmids, Fan Dong for the Stat5B and IRF-luciferase plasmids, and William Taylor for the p53-luciferase reporter plasmid. We would also like to thank Drs. Denis Guttridge, Richard Komuniecki, and Fan Dong for critical reading of the manuscript and for many helpful discussions.

	REFERENCES

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