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Interacts with Histone Deacetylase 1 to Repress Promoter Activity1
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* Department of Pediatrics, Division of Rheumatology, University Hospital, University of Muenster, Muenster, Germany;
Institute of Experimental Dermatology, University of Muenster, Muenster, Germany;
Interdisciplinary Center for Clinical Research, Research Group 5, University of Muenster, Muenster, Germany; and
Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910
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Abstract |
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is an ubiquitously expressed transcription factor and a counterpart of the activator CREB. In T cells, CREM is responsible for the termination of the IL-2 expression by a chromatin-dependent mechanism. We demonstrate in this study that CREM associates with histone deacetylase (HDAC)1 through its H domain, which is located between the kinase inducible and DNA binding domains. The CREM-mediated recruitment of HDAC1 to the CRE sites of the IL-2 and c-Fos promoter causes histone deacetylation and inaccessibility to restriction enzymes and limited transcriptional activity. Importantly, the CRE sites of these promoters are crucial for the activity and binding of HDAC1. Therefore, CREM exerts its repressor activity by a mechanism that involves recruitment of HDAC1, increased deacetylation of histones, and repression of promoter activity. |
Introduction |
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binds to promoters that contain CRE sites (1) and regulates their activity. CREM has been shown to be important in the regulation of the immune response because its expression is induced following TCR engagement (2) and it is increased in T cells from patients with systemic lupus erythematosus (SLE) (3). In activated normal T cells, increased CREM expression leads to enhanced binding to the CRE site centered at the –180 site of the IL-2 promoter and replacement of bound phosphorylated CREB. This exchange in the occupancy of the –180 site of the IL-2 promoter appears to contribute to the termination of the production of IL-2 mRNA and protein (2). In patients with SLE, CREM binds to the –180 site of the IL-2 promoter (4) and the –57 CRE site of the c-Fos promoter (5), resulting in repression of their activity. Therefore, it appears that CREM is important in the regulation of the immune response in health and disease.
CREM, unlike CREB, lacks the transactivator domain Q2, and therefore it is unable to recruit TATA box binding factor IID of the RNA polymerase II complex that is important for the initiation of transcription (6, 7). In addition, CREM fails to recruit the histone acetyltransferase activity of p300 (7). CREM binding to the IL-2 promoter causes changes to the acetylation pattern of histones that limits the accessibility of restriction enzymes in an area-specific manner (2). These observations suggest a second, chromatin-dependent mechanism whereby CREM negatively regulates gene transcription. Because histone acetylation is a critical step that enables transcription factors to regulate promoter activity (8, 9), we hypothesized that binding of CREM to CRE-defining promoters recruits histone deacetylases (HDAC), which promote the presence of deacetylated histones and the repression of transcriptional activity. HDACs are critical enzymes for the deacetylation of histones and are involved in transcriptional repression resulting from nucleosome remodeling (10). In this study, we present evidence that binding of CREM to immune response gene promoters results in active recruitment of HDAC1, enhanced deacetylation of histones, and transcriptional repression.
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Materials and Methods |
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The GST-tagged HDAC1 plasmid was a gift from Dr. C. Seiser (Max F. Perutz Laboratories, Vienna, Austria) and has been described elsewhere (10). We recloned it into a His-tagged construct (pQE30; Qiagen). The eukaryotic CREM plasmid was a gift from Dr. P. Sassone-Corsi (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France). From this construct, we performed PCR amplification and generated several full-length and truncated constructs and cloned them into GST-tagged (p-Gex; Pharmacia) and His-tagged (pQE30 and 32; Qiagen) bacterial expression plasmids. The plasmids were grown in Escherichia coli and after isopropyl 
-D-thiogalactoside-induction, purification of the recombinant protein was performed. The IL-2 promoter plasmid was a gift from A. Rao (Harvard Medical School, Boston, MA) (11), and the c-Fos plasmid was a gift from Dr. G. Bowden (Department of Radiation Oncology, Arizona Cancer Center, University of Arizona, Tucson, AZ) (12).
Immunoprecipitation
Cells were lysed in Kyriakis lysis buffer (20 mM HEPES (pH 7.4), 50 mM -glycerophosphate, 2 mM EGTA, 10 mM NaF, 1% Triton X-100, 10% glycerol) supplemented with fresh proteinase inhibitors for 30 min on ice. Cells were spun down at 14,000 rpm for 10 min at 4°C. Supernatant was incubated with protein G-Sepharose beads (Pharmacia) and appropriate Ab overnight. Pellets were washed three times with lysis buffer and subjected to Western blot analysis or HDAC assays.
HDAC assays
A commercial HDAC assay (Upstate Biotechnology) was used and modified by combining it with a nonradioactive ELISA-based histone acetyltransferase (HAT) assay (Upstate Biotechnology). The immunoprecipitated material was subjected to the streptavidin-coated ELISA-plates together with biotinylated acetyl-histone, and HDAC buffer was used instead of HAT buffer in the assay.
GST pull-down assay
Recombinant GST-HDAC1 was incubated with recombinant His-tagged CREM and vice versa in 1 ml of GST-binding buffer (20 mM Tris (pH 8.0), 0.5 M NaCl, 1 mM EDTA, and 0.5% Nonidet P-40 (NP-40)) and glutathione-Sepharose (Pharmacia) overnight at 4°C. The precipitate was washed three times with binding buffer (20 mM Tris (pH 8.0), 0.5 M NaCl, 1 mM EDTA, and 0.5% NP-40) and subjected to Western blot analysis.
His pull-down assay
Recombinant GST-HDAC1 was incubated with recombinant His-tagged CREM and vice versa in 1 ml of binding buffer (20 mM Tris (pH7.8), 0.5 M NaCl, 5 mM imidazole, and 0.5% NP-40) and nickel-affinity resin (Talon) overnight at 4°C. The precipitate was washed three times with binding buffer and subjected to Western blot analysis.
DNA-affinity chromatography
Recombinant HDAC1 (10 µg) was incubated with 10 µg of recombinant CREM constructs in 1 ml of EMSA buffer (20 mM HEPES (pH 7.5), 1 mM MgCl2, 75 mM KCl, 1 mM DTT, and 0.2% NP-40) together with 1 µM biotinylated dsDNA containing a consensus CRE (TGACGTCA) and streptavidin-coated magnetic beads (Dynal Biotech) for 1 h. The precipitate was collected on a magnet (Dynal Biotech) and washed three times with EMSA buffer. Precipitates were subjected to Western blot analysis.
Lymphocyte isolation
Heparinized peripheral venous blood was obtained from healthy volunteers. Non-T cells were selected by a tetrameric Ab mixture against CD14, CD16, CD19, CD56, and glyA (StemCell Technologies) and bound to erythrocytes. These complexes were separated from the T cells on lymphoprep gradient (Nycomed). The purified T cells were >98% positive for CD3 as tested by flow cytometry.
Transfection of T cells, Raji B cells, and Jurkat cells
Approximately 5–10 x 106 freshly isolated T cells or PBMCs were transfected by electroporation with an AMAXA electroporator. Plasmids encoding CREM sense or antisense (a gift from Dr. P. Sassone-Corsi), or the corresponding empty vector plasmid were used for transfection. One µg of each plasmid was used per transfection. Alternatively, 3–5 x 106 Jurkat cells or Raji cells were used and transfected with a Bio-Rad electroporator (250V, 975uF). The transfection efficiency was 
30% in Jurkat T cells and 50% in normal T cells.
After 18 h, T cells were harvested for different purposes like Western blot, luciferase assays, and chromatin immunoprecipitation (ChIP) assays. For stimulation, PMA and ionomycin (both obtained from Sigma-Genosys) were used. Sodium butyrate was obtained from Upstate Biotechnology, and superoylanilide hydroxamic acid (SAHA) was a gift from Aton Pharmaceuticals.
Antibodies
Abs against CREM were purchased from Santa Cruz Biotechnology. Abs against HDAC1, H3, and acetylated H3 were purchased from Upstate Biotechnology.
DNA accessibility assay (chart)
Jurkat T cells were either transfected with antisense CREM or an empty vector plasmid. Twenty-four hours after transfection, T cells were stimulated with PMA and ionomycin for 8 h. T cells were harvested, resuspended in ice-cold PBS, and lysed in buffer A (10 mM HEPES-KOH (pH 7.9), 10 mM KCl, 0.1 mM EDTA, and 0.1 mM EGTA) with addition of NP-40. Nuclear pellets were harvested and washed in restriction enzyme buffer. Digestion was conducted at suggested temperatures with DdeI or XhoI for 1 h. DNA was extracted and amplified by real-time method with primers flanking the c-Fos promoter that contained the digestion sites.
ChIP analysis
Five million T cells were used per investigated Ab. The cells were treated with formalin (1%) for 10 min, washed, lysed, and sonicated. The DNA protein complexes were immunoprecipitated with an Ab and extracted by protein A/G-Sepharose beads (Santa Cruz Biotechnology). After several washing steps, the protein was digested with proteinase K, the cross-link between DNA and protein was reversed at 65°C overnight, and the DNA was extracted (QiaAmp DNA Extraction kit; Qiagen). The DNA was amplified in semiquantitive PCR with primers flanking the c-Fos promoter including the CRE –57 site: forward, 5'-GAGCCCGTGACGTTTACACTCA-3' and reverse, 5'-TGAGTGTAAACGTCACGGGCTC-3'. DNA from approximately one million cells was used per PCR. PCR products were run on a 1.5% agarose gel and quantified with QuantityOne software (Bio-Rad). For real-time PCR, an ABI Prism thermocycler (Applied Biosystems) was used. PCR primers flanking the c-Fos CRE site forward 5'-GAAGCGCCCAGGCCC-3' and reverse 5'-CGCAGCCACTGCTTTTATAACAA-3' were used in a SYBR Green reaction mixture.
Reporter ChIP
This technique enables the study of in vivo binding of transcription factors to a specific binding site on a promoter of a reporter construct (13). The IL-2 promoter-luciferase construct has been described elsewhere (11). Mutagenesis of the –180 site into luciferase was performed with a site-directed mutagenesis kit (Stratagene) and primers including the mutagenesis site (5'-ATCCATaaAGTCAGTCTTTGGGGGTTTAAA-3'). The mutated construct was transfected into 5 x 106 Jurkat cells, and the nonmutated construct was transfected into another 5 x 106 cells for control purpose. Eighteen hours after transfection, ChIP of the cells was performed with anti-CREM, anti-HDAC1, and p65 Abs (Santa Cruz Biotechnology). The DNA was amplified with primers flanking the proximal IL-2 promoter (reverse) and the luciferase gene (forward): 5'-tcacctcgatatgtgcatctgta-3' (forward), 5'-ccaaagagtcatcagaagaggaa-3' (reverse).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We first asked whether changes in histone acetylation pattern, which we observed in human T cells following modulation of CREM levels (2), were unique to the IL-2 promoter or whether they occurred in other CRE-containing promoters. We investigated the c-Fos promoter because it defines a CRE site that binds CREM (14, 15). Like CREM, c-Fos is induced in T cells after stimulation and forms AP1 complexes by forming heterodimers with Jun. AP1 enhances IL-2 transcription by cooperating with NFAT1 (16). We have previously shown that the presence of antisense CREM results in significant decrease of CREM protein in normal and SLE T cells (2, 4). In agreement with these findings, the decrease of CREM by transfection of Jurkat T cells with an antisense CREM plasmid enhanced c-Fos production (Fig. 1A). Similarly to the IL-2 promoter data (2), antisense CREM increased accessibility of DdeI restriction enzyme to the c-Fos promoter-defined DdeI site (Fig. 1B) but not to XhoI restriction enzyme, which has no cutting site within the c-Fos promoter. Accordingly, antisense CREM enhanced the acetylation of histone 3, whereas the binding of CREM (empty vector) promoted histone deacetylation, as determined in ChIP assays (Fig. 1C).
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Increased deacetylation of histones following binding of CREM to DNA implies the recruitment of HDACs, which are known to repress transcription by deacetylating histones and closing chromatin structure. To test this possibility, we determined the HDAC activity in nuclear extracts from Jurkat T cells immunoprecipitated with anti-CREM, anti-c-Jun (negative control), or anti-HDAC1 (positive control) Abs. The CREM Ab-generated immunoprecipitates contained significantly higher HDAC activity compared with the c-Jun Ab-generated immunoprecipitates. The HDAC activity was less in the anti-CREM Ab immunoprecipitates than in the anti-HDAC immunoprecipitates (Fig. 2A). Western blot analysis of the immunoprecipitated material identified the interacting HDAC as HDAC1 (Fig. 2B). Membranes were also blotted with an anti-HDAC9 Ab (data not shown) but failed to reveal a positive band.
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Next, we sought to determine the CREM domain that was responsible for binding HDAC1. To this end, we constructed truncated forms of the GST- tagged CREM expression constructs and incubated them with His-HDAC1. Following GST pull-down, the separated immunoprecipitates were blotted with an anti-HDAC1 Ab. In Fig. 3A, we show experiments which demonstrate that HDAC1 interacts with the domain designated H and which lies between the kinase inducible domains (KID) and DNA binding domains (DBD) (Fig. 3A). These data were further confirmed by GST pull-down assays using recombinant His-tagged CREM-truncated constructs and GST-HDAC1. Only the H domain carrying constructs of CREM interacted with HDAC1, whereas the N-terminal domain and KID-domains were not necessary for this interaction (Fig. 3B). The binding of His-CDBD CREM, a C-terminal construct containing both DBDs and the H domain to HDAC1, was reproducibly faint due to little protein expression in the His system (Fig. 3B). We therefore performed DNA-affinity chromatography with a better expressed GST-tagged CDBD CREM and additional truncated forms, one containing the H domain and DBD2 and a full-length CREM together with a GST-HDAC1. In Fig. 3C, we show that the DBD1 is dispensable for the interaction. Therefore, the binding domain lies within exon H and—unlike CREB—the transactivator domains are not necessary for this binding (17). Interestingly, this domain is glutamine-rich, but it shares <50% sequence homology with CREB (18), suggesting that CREB might lack a part of this structure, which is capable of binding HDAC1 and therefore needs the transactivator domains, which are able to bind HDAC1, but which are not a part of CREM.
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Next, we studied the functional interaction between CREM and HDAC in vivo to determine how CREM influences the binding of HDAC1 to the IL-2 and c-Fos promoters. ChIP assays using normal T cells transfected with a CREM plasmid demonstrated increased binding of CREM to the c-Fos promoter that is associated with increased binding of HDAC1 (Fig. 4A). Similar data were obtained with the IL-2 promoter (Fig. 4A). Reversely, transfection of T cells with an antisense CREM plasmid prevented the binding of HDAC1 to the c-Fos and the IL-2 promoters after T cell stimulation with anti-CD3 and anti-CD28 Abs (Fig. 4, B and C).
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B p65 remained unaffected, suggesting that the interaction between CREM and HDAC1 is specific (Fig. 5A). This experiment further indicates a crucial role for the recruited HDAC1 to the IL-2 promoter by CREM after T cell stimulation. Next, we performed the same experiment using a CRE –57-mutated c-Fos promoter construct, but we were unable to obtain results comparable to those obtained using the –180 site-mutated IL-2 promoter construct. One possible explanation for this result is the fact that the c-Fos promoter contains a Rb binding site, and Rb itself is able to recruit HDAC1 (19, 20). If the CRE site is mutated, the Rb binding site is apparently sufficient to mediate the binding of HDAC1 to the c-Fos promoter luciferase construct. However, when we treated Raji B cells or Jurkat T cells that had been transfected with the c-Fos-luciferase construct with the HDAC-inhibitors SAHA and Na-butyrate, the promoter activity was enhanced, which corroborates previous results (21). But this enhanced activity was at least partially dependent upon a functional CRE site, because a mutagenesis of the CRE site prevented a part of the up-regulation of luciferase activity by SAHA and Na-Butyrate (Fig. 5, B and C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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) and repressor (, , 
) isoforms of transcriptional regulation. The two groups of CREM differ in the number of exons that are excluded by alternative splicing (22). The repressor isoforms are shorter and lack exons (generated by alternative splicing, alternative initiation codon, and alternative polyadenylation) that contribute to the transactivating, N terminus of the protein. The transcriptional repressor CREM binds to CRE sites, which define palindromic sites (TGACGTCA) in susceptible promoters. A characteristic feature of CREM is its pronounced cell- and development-specific expression. We have shown that CREM is transcriptionally up-regulated in T cells after stimulation (2) and terminates the IL-2 production. The up-regulation of CREM can also be observed in other hemopoietic cells like monocytes and B cells (our unpublished observation) or macrophages (23) after appropriate stimulation like PMA and ionomycin or LPS. Therefore, CREM represents an important player in the regulation of the immune response. Moreover, CREM is increased in T cells from SLE patients and has been shown to be involved in the transcriptional repression of the IL-2 (4), c-Fos (5), and TCR-
(13) promoter activity. Interestingly, HDAC inhibitors have been shown to improve disease pathology in the lupus-prone MRL-lpr mice (24). In this study, we show that the CRE site is important for the recruitment of HDAC activity to the c-Fos and IL-2 promoters. Thus, we speculate that CREM exerts part of its biological function through HDAC1. The ability of CREM to recruit HDAC1 to CRE sites appears to be generalized because it occurs, besides the IL-2 and c-Fos promoters, in the CD86 promoter, which also contains a CRE site (data not shown). It is known that treatment with trichostatin A, which is another potent HDAC inhibitor, decreases IL-2 production in Jurkat as well as naive T cells (21, 25). We were able to reproduce these findings with SAHA (data not shown); however, the reason for the down-regulation is not clear. It is known that trichostatin A affects the NF-KB signaling pathway in CD4 T cells and therefore primarily inhibits IL-2 activation (25).
In conclusion, we have shown a novel mechanism whereby CREM influences the transcription of important cellular genes like IL-2 and c-fos. It has been known that CREM fails to activate the HDAC activity of p300 and that it does not interact with TATA box binding factor IID. Both mechanisms though are passive and occur because CREM lacks the Q1 and Q2 transactivation domains. In this study, we show for the first time an active mechanism whereby CREM interacts with and recruits HDAC1 to gene promoters. The recruitment of HDAC1 leads to occupation of the promoter areas of at least two genes by nonacetylated histones, which is known to lead to closed chromatin structure and limited transcriptional activity.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 The work was supported by the Interdisciplinary Center for Clinical Research (Interdisziplinäres Zentrum für Klinische Forschung), Research Group 5, and Public Health Service Grant RO1 AI49954 (to G.C.T.). 
2 Address correspondence and reprint requests to Dr. Klaus Tenbrock, University of Muenster, Experimental Dermatology, Röntgenstrasse 21, 48149 Muenster, Germany. E-mail address: ktenbroc{at}uni-muenster.de
3 Abbreviations used in this paper: CRE, cAMP response element; CREM, CRE modulator; SLE, systemic lupus erythematosus; HDAC, histone deacetylase; ChIP, chromatin immunoprecipitation; KID, kinase inducible domain; DBD, DNA binding domain; R-ChIP, reporter-based ChIP; HAT, histone acetyltransferase; SAHA, superoylanilide hydroxamic acid; NP-40, Nonidet P-40.
Received for publication April 21, 2006. Accepted for publication August 11, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter. J. Exp. Med. 175: 853-862. -chain. J. Immunol. 175: 5975-5980. This article has been cited by other articles:
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M. Ahlmann, G. Varga, K. Sturm, R. Lippe, K. Benedyk, D. Viemann, T. Scholzen, J. Ehrchen, F. U. Muller, M. Seidl, et al. The Cyclic AMP Response Element Modulator {alpha} Suppresses CD86 Expression and APC Function J. Immunol., April 1, 2009; 182(7): 4167 - 4174. [Abstract] [Full Text] [PDF]
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 T. Suzuki and K. Nohara Regulatory Factors Involved in Species-specific Modulation of Arylhydrocarbon Receptor (AhR)-dependent Gene Expression in Humans and Mice J. Biochem., October 1, 2007; 142(4): 443 - 452. [Abstract] [Full Text] [PDF]
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 K. Tenbrock, Y.-T. Juang, V. C. Kyttaris, and G. C. Tsokos Altered signal transduction in SLE T cells Rheumatology, October 1, 2007; 46(10): 1525 - 1530. [Abstract] [Full Text] [PDF]
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