The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tenbrock, K.
Right arrow Articles by Tsokos, G. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tenbrock, K.
Right arrow Articles by Tsokos, G. C.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH
The Journal of Immunology, 2005, 175: 5975-5980.
Copyright © 2005 by The American Association of Immunologists

The Cyclic AMP Response Element Modulator Regulates Transcription of the TCR {zeta}-Chain1

Klaus Tenbrock2,*,{dagger},{ddagger}, Vasileios C. Kyttaris§,¶, Martina Ahlmann*,{dagger}, Jan Mauno Ehrchen{dagger}, Mate Tolnay||, Harutyun Melkonyan{dagger}, Christian Mawrin§, Johannes Roth*,{dagger}, Clemens Sorg{dagger}, Yuang-Taung Juang§ and George C. Tsokos§

* Department of Pediatrics, Division of Rheumatology, University Hospital, {dagger} Institute of Experimental Dermatology, and {ddagger} Interdisciplinary Center for Clinical Research, Research Group 5, University of Muenster, Muenster, Germany; § Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910; Section of Rheumatology, Washington Hospital Center, Washington, D.C. 20010; and || Division of Monoclonal Antibodies, Food and Drug Administration, Rockville, MD 20857


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemic lupus erythematosus T cells display decreased amounts of TCR {zeta} mRNA that results in part from limited binding of the transcriptional enhancer Elf-1 to the TCR {zeta} promoter. We have identified a new cis-binding site for the cAMP response element (CRE) modulator (CREM) on the TCR {zeta} promoter, centered on the –390 nucleotide. Transfection of T cells with an antisense CREM {alpha} plasmid reduced the binding of CREM to the TCR {zeta} promoter, as shown by chromatin and reporter chromatin immunoprecipitation assays, and enhanced the production of TCR {zeta} mRNA and protein. Mutagenesis of the –390 CRE site prevented the binding of CREM to the TCR {zeta} promoter. The mechanism of CREM-mediated repression appears to be chromatin dependent, because antisense CREM promotes the acetylation of histones on the TCR {zeta} promoter. Finally, we established an enhanced binding of CREM to the TCR {zeta}-chain promoter in systemic lupus erythematosus cells compared with control T cells. Our studies demonstrate that CREM {alpha} binds to the TCR {zeta} promoter and repress its activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemic lupus erythematosus (SLE)3 is an autoimmune disease of unknown etiology that is characterized by multiple T cell signaling abnormalities (1). T cells from patients with SLE have been shown to have decreased levels of TCR {zeta} protein and mRNA (2). The decreased levels of TCR {zeta} mRNA have been shown to be the result of decreased TCR {zeta} promoter activity (3). In normal T cells, the transcriptional activity of the TCR {zeta} promoter is regulated through the binding of Elf-1, a member of the Ets family of transcription factors, to two distinct binding motifs defined by the TCR {zeta} promoter (4). Mutagenesis of these motifs greatly diminishes the promoter activity of this TATA-less promoter (4, 5). No other obvious cis sites have been noted on the proximal TCR {zeta} promoter.

While studying the causes for the transcriptional repression of the TCR {zeta} promoter in SLE T cells, we identified two groups of patients. In the first group the 98-kDa form of Elf-1 that binds to the TCR {zeta} promoter and accounts for its activity was absent despite the fact that the 80-kDa form was present at levels comparable to those in normal T cells. In the second group, the 98-kDa form was present, but it was not able to bind to the TCR {zeta}-chain promoter. In these patients the 98-kDa form resolved into two bands in isoelectric focusing gels, whereas the DNA binding 98-kDa form resolved into three bands (3). In the same experiments we noticed that although TCR {zeta} promoter activity in SLE T cells was decreased, mutagenesis of the 2 Elf-1 cis sites of the TCR {zeta} promoter further decreased its activity (3). This stepwise decrease in promoter activity suggested the presence of additional transcription factors involved in the transcriptional repression of the TCR {zeta} promoter. The presumed additional transcriptional repression could reflect the result of transcriptional enhancers that were either absent or defective in SLE T cells or the presence of transcriptional repressors. In addition, inspection of the proximal TCR {zeta}-chain promoter revealed seven cAMP response element (CRE) half-sites (TGAC or GTCA) to which members of the CRE-binding protein/CRE modulator (CREM) family of transcription factors could bind.

Because SLE T cells have been shown to express increased amounts of CREM {alpha} that binds to the –180 site of the IL-2 promoter and represses its activity (6, 7), we considered that CREM {alpha} may bind to an as yet unidentified site on the TCR {zeta} promoter and repress its activity. In this study we demonstrate that indeed CREM {alpha} binds to a palindromic CRE half-site (TGAC or GTCA) centered at the –390 nt of the TCR {zeta} promoter and represses its activity. In addition, we show increased binding of CREM {alpha} to the TCR {zeta} promoter in SLE T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Lymphocyte isolation

Heparinized peripheral venous blood was obtained from healthy volunteers, patients with rheumatoid arthritis, and five patients with the diagnosis of SLE according to American College of Rheumatology criteria (8). 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 by a Lymphoprep gradient (Nycomed). The purified T cells were >98% positive for CD3, as tested by flow cytometry.

Antibodies

Abs against CREM and TCR {zeta}-chain were purchased from Santa Cruz Biotechnology.

Preparation of mRNA and cDNA, PCR, and real-time PCR

One million T cells were used for extracting RNA (RNA Easy Mini kit; Qiagen). RNA was quantified, and 500 ng of total RNA was used for cDNA synthesis by RT (RT-PCR kit; Promega). A total of 50 ng of cDNA was used for each PCR. PCR primers were synthesized by Sigma Genosys.

PCR beads were used for amplification (BD Biosciences). Real-time PCR was conducted with a Cepheid Smart Thermocycler by adding SYBR Green to the reaction mixture. Primers used for PCR were as follows: {zeta}-chain, 5' 3'; reverse, 5' 3'; CREM, 5'-GAA ACA GTT GAA TCC CAG CAT GAT GGA AGT-3'; and reverse, 5'-TGC CCC GTG CTA GTC TGA TAT ATG-3'. PCR products of semiquantitative RT-PCR were separated on a 1.5% agarose gel, and the OD was quantified using QuantityOne software (Bio-Rad) after background subtraction from each band.

Chromatin immunoprecipitation (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 with primers flanking the TCR {zeta} promoter, including the –390 site (forward, 5'-CAACGCCACACAGCAAGTAGG-3'; reverse, 5'-GGCTGGCAAAATGACAGAACC-3'). DNA from ~1 million cells was used per PCR. PCR products were run on a 1.5% agarose gel and quantified with QuantityOne software.

Transfection of T cells or Jurkat cells

Approximately 5–10 x 106 freshly isolated T cells or PBMCs were transfected by electroporation with an AMAXA electroporator. Plasmids encoding CREM {alpha} antisense (a gift from Dr. P. Sassone-Corsi, Institut de Genetique et de Biologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Sante et de la Recherche Medicale, Universite Louis Pasteur, Strasbourg, France) or corresponding empty vector plasmid were used for transfection. One microgram of each plasmid was used per transfection. Alternatively, Jurkat cells were used and transfected with a Bio-Rad electroporator (250 V, 975 µF). After 18 h, T cells were harvested for different purposes, such as FACS analysis, Western blot, ChIP assay, or RT-PCR.

Reporter ChIP

This technique enables study of the in vivo binding of transcription factors to a specific binding site on a promoter of a reporter construct. The TCR {zeta} promoter-luciferase construct used in this study has been described previously (4). Mutagenesis of the –390 site into luciferase was performed with a site-directed mutagenesis kit (Stratagene) and primers including the mutagenesis site (5'-GGACTCTAATCCAGGGTTCTGGTATTTTGCCAGCCACCAT-3') (mutated nucleotides are underlined). The mutated construct was transfected into 5 million Jurkat cells, and the nonmutated construct was transfected into another 5 million cells for control purposes. Eighteen hours after transfection, ChIP of the cells was performed with an anti-CREM Ab (Santa Cruz Biotechnology). PCR was performed with a primer specific for the TCR {zeta}-chain promoter and another one specific for the luciferase plasmid to ensure that only reporter-specific DNA, and not genomic DNA, is amplified.

Preparation of nuclear extracts and EMSA

Five to 10 million T cells were used for preparation of nuclear extracts as previously described (9). The dsDNA probe of the –390 site on the TCR {zeta}-chain promoter was 5'-CAGGGTTCTGTCATTTTGCCAGCCA-3' in shift assays as previously described (9). Recombinant GST-tagged CREM (plasmid was a gift from M. Montminy, Peptide Biology Laboratories, The Salk Institute for Biological Studies, La Jolla, CA) and recombinant human ribonucleoprotein-D (hnRNP-D) were purified as previously described (10).

Flow cytometric analysis

Cells were transfected with antisense CREM or an empty vector (psGV) and a plasmid containing GFP. Twenty-four hours after transfection, 1 million cells/assay were permeabilized with a BD Cytofix/Cytoperm Plus kit (BD Biosciences) according to the manufacturer’s instructions and stained with PE-labeled anti-TCR {zeta}-chain Ab or an isotype-specific control (Santa Cruz Biotechnology). A minimum of 10,000 events were collected for evaluation on a FACSCalibur (BD Biosciences), and data were analyzed using CellQuestPro software. The mean fluorescence intensity was calculated, and the samples were compared by paired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CREM binds to the TCR {zeta} promoter in vivo

Because CREM {alpha} is up-regulated in SLE T cells (6, 7) and suppresses IL-2 production in these cells, we were interested to find out whether CREM binds to the TCR {zeta} promoter that also displays decreased activity in SLE (3, 11). To investigate whether CREM binds to the TCR {zeta} promoter in live cells, unstimulated normal T cells were fixed with formalin to cross-link DNA to proteins, then sonicated, and the DNA-protein complexes were immunoprecipitated. The DNA was extracted and amplified with sets of primers specific to the {zeta} promoter. As shown in Fig. 1a (representative of four experiments), we detected enhanced binding of CREM to the TCR {zeta} promoter, although minimal binding of c-Fos was detected.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 1. CREM binds to the TCR {zeta} promoter. a, Enhanced binding of CREM to the TCR {zeta} promoter. T cells were fixed with formalin, washed, lysed, and sonicated. The DNA-protein complexes were immunoprecipitated with the indicated Ab (anti-CREM or anti-c-Fos) and extracted by protein A/G agarose-Sepharose beads. The DNA was purified and amplified with primers flanking the TCR {zeta} promoter, including the –390 CRE site. DNA from 1 x 106 cells was used for each PCR. PCR products were run on a 1.5% agarose gel and quantified with QuantityOne software. The picture shows enhanced binding of CREM to the TCR {zeta}-chain promoter compared with the binding of c-Fos to the same promoter. b, Antisense CREM decreases the binding of CREM protein to the TCR {zeta} promoter. Normal T cells were transfected with antisense CREM {alpha} (AS) or empty vector plasmids (EV). Cells were fixed with formalin and sonicated. The anti-CREM Ab precipitates were subjected to PCR using TCR {zeta} promoter-specific primers and were visualized on 1.5% agarose gel. Left panel, Representative experiment; right panel, mean ± SEM densitometric readings from four experiments.

 
We have previously reported that the activity of a luciferase reporter construct driven by the IL-2 promoter is reduced by a CREM {alpha} plasmid, whereas it is enhanced by an antisense CREM plasmid in T cells (12). We thus transfected normal T cells with the antisense CREM {alpha} or control (PSG5) plasmids. In Fig. 1b (left panel, representative experiment; right panel, cumulative data from four experiments), we show that transfection of T cells with the antisense CREM plasmid prevents the binding of CREM to the TCR {zeta} promoter.

CREM binds to the –390 site of the TCR {zeta} promoter

We searched the proximal TCR {zeta} promoter for CRE binding motifs to which CREM could bind. As shown in Fig. 2, seven such sites were identified, representing one part of the canonical CRE palindrome. To identify which of the seven sites served as a binding sites(s) of CREM, we performed EMSA using seven oligonucleotides defining the palindromic half-sides within –1000 bp of the TCR {zeta} promoter and recombinant CREM {alpha} protein. CREM {alpha} was found to bind only to the CRE site centered on the –390 nt site (Fig. 3a), but not to sites centered on the –219, –352, –439, –483, and –631 sites (Fig. 3b). The binding was specific, because it was not inhibited by a 50-fold increase in nonspecific oligonucleotides (Elf-1; Fig. 3a). In addition, the –390 site did not bind hnRNP protein, which, in contrast, bound to the a previously identified hnRNP binding site (13) (Fig. 3c).



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 2. The TCR {zeta}-chain promoter defines seven putative CRE sites and two Elf-1 sites.

 


View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 3. Recombinant CREM protein binds to the –390 site on the TCR {zeta} promoter. a, A radiolabeled oligonucleotide identical with the –390 site of the TCR {zeta} promoter was incubated with recombinant CREM protein in the absence or the presence of the indicated cold oligonucleotides and separated in gels. b, Radiolabeled oligonucleotides identical with the other putative CRE sites were incubated with recombinant CREM protein in the absence or the presence of the indicated cold oligonucleotides and separated in gels. c, Recombinant hnRNP-D protein was incubated with the –390 CRE site oligonucleotide and with a consensus hnRP-D site-defined oligonucleotide.

 
Using a modified reporter construct-based ChIP method, we explored whether the –390 site was also important for in vivo binding. The –390 site of a TCR {zeta} promoter-luciferase construct was mutagenized and transfected into Jurkat T cells. As a control, we transfected the wild-type construct into the same number of cells. Subsequently, we performed ChIP assays using an anti-CREM {alpha}-specific Ab. The PCR was performed using primers specific to the reporter construct to ensure that only plasmid DNA is amplified. As shown in Fig. 4 (upper panel, representative experiment; lower panel, cumulative data from three experiments), mutagenesis of the –390 site prevented the binding of CREM to the reporter construct in vivo. Mutagenesis of the –219-centered TGAC half-site did not affect the binding of CREM {alpha} to the TCR {zeta} promoter (Fig. 4).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 4. Mutagenesis of the –390 site prevents the binding of CREM to the TCR {zeta} promoter. Potential CRE sites were mutated at –390 and –219 of the TCR {zeta} promoter. Normal T cells were transfected with either one of the mutated (M) or a wild-type (WT) TCR {zeta} promoter-driven reporter construct. Cells were fixed with formalin and sonicated. The anti-CREM Ab precipitates were subjected to PCR using TCR {zeta} promoter and reporter gene-specific primers and were visualized on 1.5% agarose gel. Upper panel, Representative experiment; lower panel, mean densitometric values ± SEM from three experiments using the –390 site-defined oligonucleotide.

 
Increased CREM binding to the TCR {zeta} promoter in live SLE T cells

Because CREM is known to be up-regulated in SLE T cells (7), we asked whether CREM binding to the TCR {zeta} promoter was also increased in SLE T cells. We purified T cells from the peripheral blood of patients with SLE, performed ChIP assays using an anti-CREM Ab, and compared the binding of CREM to the TCR {zeta} promoter in SLE T cells (n = 5) with that in normal T cells (n = 7) and that in patients with rheumatoid arthritis (n = 3). The PCR was conducted in real-time mode. A representative experiment is shown in Fig. 5a, and cumulative data are shown in Fig. 5b. The data show that more CREM binds to the TCR {zeta} promoter in SLE T cells compared with normal T cells and T cells form patients with rheumatoid arthritis (mean real-time PCR cycle threshold, 34 (SLE) vs 36.7 (normal) vs 38.1 (rheumatoid arthritis)).



View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 5. Increased CREM binding to the TCR {zeta} promoter in SLE T cells. T cells from SLE patients and normal controls were fixed with formalin, washed, lysed, and sonicated. The DNA-protein complexes were immunoprecipitated with anti-CREM Ab and extracted by protein A/G agarose-Sepharose beads. The DNA was purified and amplified with primers flanking the TCR {zeta} promoter, including the –390 CRE site, by real-time PCR. DNA from 1 x 106 cells was used for each PCR. a, Representative experiment that was run on a 1.5% agarose gel; b, the threshold cycle values from five SLE patients, seven control individuals, and three patients with rheumatoid arthritis. Horizontal lines depict mean values.

 
CREM binding to the TCR {zeta} promoter is up-regulated after T cell stimulation and enhances chromatin remodeling

We have previously shown that CREM protein is up-regulated after T cell stimulation and binds to the IL-2 promoter in vivo to terminate IL-2 production by a chromatin-dependent mechanism (14). We therefore conducted experiments to determine whether CREM uses a similar mechanism to regulate the activity of the TCR {zeta} promoter. We first stimulated T cells with CD3 and CD28 Abs and determined TCR {zeta}-chain mRNA and protein expression as well as CREM mRNA expression. As shown in Fig. 6a (representative; n = 3), {zeta}-chain protein expression decreased dramatically after 24 h. Furthermore, although CREM mRNA expression was strongly enhanced (~4.4-fold, representing approximately two cycles difference), {zeta}-chain mRNA synthesis decreased by ~50% (Fig. 6b; n = 8). In addition, we observed an enhanced binding of CREM protein to the TCR {zeta} promoter by ChIP assay (Fig. 6c, representative experiment).



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 6. T cell stimulation decreases the expression and transcription of {zeta}-chain and enhances CREM production and its binding to the TCR {zeta} promoter. T cells were treated with CD3 and CD28 Abs for 24 h. a, Representative Western blot is shown using an anti-TCR {zeta}-chain Ab. b, RNA was extracted from stimulated cells and reverse transcribed. cDNA was subjected to real-time PCR using TCR {zeta}-chain and CREM-specific primers. The data from eight experiments were normalized against the ribosomal protein RPL 13A cDNA (bars represent the SEM). CREM mRNA shows a 4.4-fold increased expression, representing about two cycle differences, whereas the TCR {zeta} mRNA decreased by 50%. c, Cells were fixed with formalin, washed, lysed, and sonicated. The DNA-protein complexes were immunoprecipitated with anti-CREM and extracted by protein A/G agarose-Sepharose beads. The DNA was purified and amplified with primers flanking the TCR {zeta} promoter including the –390 CRE site. DNA from 1 x 106 cells was used for each PCR. PCR products were run on a 1.5% agarose gel and quantified with QuantityOne software.

 
Next we transfected Jurkat T cells or naive T cells with either an antisense CREM or an empty vector and determined whether it affected the binding of acetylated histone 4 to the TCR {zeta} promoter 24 h after transfection. As shown in Fig. 7 (representative of four experiments), antisense CREM enhanced the acetylation of histone 4 next to the TCR {zeta} promoter, although in the empty vector, which does not prevent the binding of CREM, enhanced deacetylation of histone 4 occurred. These data suggest a chromatin-dependent mechanism by which CREM influences TCR {zeta} promoter activity.



View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 7. Antisense CREM promotes the acteylation of histones next to the TCR {zeta} promoter. T cells were transfected with either antisense CREM (AS) or an empty vector (EV) control plasmid. Cells were fixed with formalin after 24 h, washed, lysed, and sonicated. The DNA-protein complexes were immunoprecipitated with the indicated Ab (anti-histone 4 (H4), anti-acetylated histone 4 (AH4), and anti-acetylated histone 3 (AH3)) and extracted by protein A/G agarose-Sepharose beads. The DNA was purified and amplified with primers flanking the TCR {zeta} promoter, including the –390 CRE site. DNA from 1 x 106 cells was used for each PCR. PCR products were run on a 1.5% agarose gel and quantified with QuantityOne software.

 
Antisense CREM enhances TCR {zeta}-chain expression

After showing that CREM binds to the TCR {zeta} promoter and influences chromatin remodeling, we asked whether CREM is able to regulate the expression of the endogenous TCR {zeta} gene. To this end we transfected T cells with antisense CREM or empty vector constructs and determined the levels of cell surface expression of TCR {zeta} protein in gated GFP-positive cells and the levels of TCR {zeta}-chain mRNA by PCR. We performed flow cytometric analysis on transfected cells and gated on the GFP-positive cells. As shown in Fig. 8a (representative) and Fig. 8b (cumulative data; n = 12; p = 0.0015), transfection of T cells with an antisense CREM construct resulted in a 26% up-regulation of TCR {zeta}-chain expression on the cell surface membrane, which was associated with parallel increase in the levels of TCR {zeta}-chain mRNA (n = 5; Fig. 8c).



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 8. Antisense CREM enhances TCR {zeta} mRNA and protein expression. Normal T cells were transfected with antisense CREM {alpha} (AS) or empty vector plasmids (EV) and a GFP expression plasmid. Twenty-four hours after transfection, cells were permeabilized and stained with PE-labeled anti-{zeta}-chain Ab or an isotypic control, and FACS analysis was performed. a, Representative experiment with a moderate increase (25%) in TCR {zeta}-chain after transfection of antisense CREM construct. b, Cumulative data (n = 12; p = 0.0015) are shown. c, RNA was extracted and reverse transcribed. cDNA was subjected to real-time PCR using TCR {zeta}-specific primers and was normalized against the ribosomal protein RPL 13A cDNA. Mean cycle differences are shown (error bar represents the SEM; n = 5).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this communication we present evidence that besides Elf-1, CREM {alpha} is involved in regulation of the activity of the TCR {zeta} promoter. Rellahan et al. (4) have established that the proximal TCR {zeta} promoter defines two Elf-1 sites that are important regulators of TCR {zeta} gene expression. CREM is a transcriptional repressor known to be of importance in SLE T cell pathophysiology and to repress the transcription of both IL-2 (6, 7) and c-fos genes (15). TCR {zeta} mRNA and protein expression are diminished in patients with SLE (2), and we have shown that decreased DNA binding of Elf-1 to contribute to the decreased activity of the TCR {zeta} promoter (3). In this study we show that CREM {alpha} binds to the –390 site of the TCR {zeta} promoter using shift, ChIP, and reporter construct-based ChIP assays. We have also shown that this binding represses transcription of the TCR {zeta}-chain gene and expression of the {zeta}-chain.

It is unclear at this point whether and how CREM{alpha} interacts with Elf-1 before or after binding to the TCR {zeta}-chain promoter. Elf-1 undergoes phosphorylation and O-GlcNAc glycosylation in two phases: during the first phase, an 80-kDa form is produced that does not bind to DNA, whereas a 98-kDa form is produced after T cell activation (3). Elf-1 defines at least 13 phosphorylation and O-GlcNAc glycosylation sites, and the order and significance of each modification are unknown. More importantly, posttranscriptional modification defects in SLE T cells lead to either defective production of the DNA-binding 98-kDa form or a wrongly phosphorylated 98-kDa form, which, again, does not bind to DNA. Although it is possible that in SLE T cells the mere absence of Elf-1 binding to the TCR {zeta} promoter permits the unbalanced repressive action of CREM, it is unclear though how Elf-1 and CREM interact in normal T cells.

This study demonstrates CREM to be involved in the transcription repression of a third gene in immune cells. Previously, we had demonstrated that CREM binds to the IL-2 promoter (6) and represses its activity. Indeed, antisense CREM can effectively augment the transcriptional activity of the IL-2 promoter (7). Similarly, CREM binds to the c-fos promoter and represses its activity, and a decrease in the levels of CREM by siRNA CREM enhances the expression of c-fos mRNA and protein and the formation of AP1 heterodimers (15).

We have shown previously that CREM is induced in normal human T cells after stimulation, and we have proposed that it is involved in the termination of IL-2 production (14). The data presented in this study demonstrate that CREM can exert the same effect on TCR {zeta} gene transcription, a mechanism that could help terminate the T cell response. Under physiologic conditions, the T cell response after stimulation of TCR leads to the up-regulation of cytokines, such as IL-2. At the same time, mechanisms that will eventually silence the expression of these proinflammatory genes are activated, thus making sure that the T cell activation will be terminated in a timely fashion. Along the same lines, the expression of TCR {zeta} mRNA and protein is down-regulated shortly after activation of the T cell, a mechanism that limits further TCR-mediated stimulating signals.

How does binding of CREM to the –390 site of the TCR {zeta}-chain promoter enable the repression of its activity? CREM {alpha} exerts its transcriptional repression activity by the mere fact that it misses the Q1 and Q2 domains that are responsible for its association with the transcription coactivators, CREB binding protein and p300 (16). We have shown previously that binding of CREM to the IL-2 promoter enhances the deacetylation of histones associated with the promoter, an effect that limits the accessibility of the promoter to endonucleases (14). In this communication we show that similar to the IL-2 promoter, binding of CREM to the –390 site of the TCR {zeta} promoter facilitates the deacetylation of histones next to the TCR {zeta} promoter, rendering the promoter inaccessible to basal transcription machinery and thus terminating transcription.

In conclusion, we have demonstrated the involvement of a second transcription factor in regulation of the activity of the TCR {zeta} promoter. The enhancing effect of Elf-1 is apparently balanced, or even negated, after binding of the repressor, CREM. Because CREM is induced after T cell activation, its binding to the TCR {zeta} promoter may contribute to termination of the T cell response.


   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 Grants RO1AI42269 and RO149954 (to G.C.T.) and Deutsche Forschungsgemeinschaft Grant TE 339/1-1. The opinions expressed in this study are those of the authors and do not represent those of the Department of Defense. Back

2 Address correspondence and reprint requests to Dr. Klaus Tenbrock, University Hospital, Department of Pediatrics, Division of Rheumatology, Institute of Experimental Dermatology, Interdisciplinary Center for Clinical Research, Research Group 5, University of Muenster, Roentgenstrasse 21, 48149 Muenster, Germany. E-mail address: ktenbroc{at}uni-muenster.de Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; ChIP, chromatin immunoprecipitation analysis; CRE, cAMP response element; CREM, CRE modulator; hnRNP-D, heterogeneous nuclear ribonucleoprotein-D. Back

Received for publication May 11, 2005. Accepted for publication August 18, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Liossis, S. N., P. P. Sfikakis, G. C. Tsokos. 1998. Immune cell signaling aberrations in human lupus. Immunol. Res. 18:27.-39. [Medline]
  2. Liossis, S. N., X. Z. Ding, G. J. Dennis, G. C. Tsokos. 1998. Altered pattern of TCR/CD3-mediated protein-tyrosyl phosphorylation in T cells from patients with systemic lupus erythematosus: deficient expression of the T cell receptor {zeta} chain. J. Clin. Invest. 101:1448.-1457. [Medline]
  3. Juang, Y. T., K. Tenbrock, M. P. Nambiar, M. F. Gourley, G. C. Tsokos. 2002. Defective production of functional 98-kDa form of Elf-1 is responsible for the decreased expression of TCR {zeta}-chain in patients with systemic lupus erythematosus. J. Immunol. 169:6048.-6055. [Abstract/Free Full Text]
  4. Rellahan, B. L., J. P. Jensen, T. K. Howcroft, D. S. Singer, E. Bonvini, A. M. Weissman. 1998. Elf-1 regulates basal expression from the T cell antigen receptor {zeta}-chain gene promoter. J. Immunol. 160:2794.-2801. [Abstract/Free Full Text]
  5. Juang, Y. T., E. E. Solomou, B. Rellahan, G. C. Tsokos. 2002. Phosphorylation and O-linked glycosylation of Elf-1 leads to its translocation to the nucleus and binding to the promoter of the TCR {zeta}-chain. J. Immunol. 168:2865.-2871. [Abstract/Free Full Text]
  6. Solomou, E. E., Y. T. Juang, M. F. Gourley, G. M. Kammer, G. C. Tsokos. 2001. Molecular basis of deficient IL-2 production in T cells from patients with systemic lupus erythematosus. J. Immunol. 166:4216.-4222. [Abstract/Free Full Text]
  7. Tenbrock, K., Y. T. Juang, M. F. Gourley, M. P. Nambiar, G. C. Tsokos. 2002. Antisense cyclic adenosine 5'-monophosphate response element modulator up-regulates IL-2 in T cells from patients with systemic lupus erythematosus. J. Immunol. 169:4147.-4152. [Abstract/Free Full Text]
  8. Hochberg, M. C.. 1997. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 40:1725.[Medline]
  9. Solomou, E. E., Y. T. Juang, G. C. Tsokos. 2001. Protein kinase C-{theta} participates in the activation of cyclic AMP-responsive element-binding protein and its subsequent binding to the –180 site of the IL-2 promoter in normal human T lymphocytes. J. Immunol. 166:5665.-5674. [Abstract/Free Full Text]
  10. Chrivia, J. C., R. P. Kwok, N. Lamb, M. Hagiwara, M. R. Montminy, R. H. Goodman. 1993. Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855.-859. [Medline]
  11. Nambiar, M. P., E. J. Enyedy, V. G. Warke, S. Krishnan, G. Dennis, H. K. Wong, G. M. Kammer, G. C. Tsokos. 2001. T cell signaling abnormalities in systemic lupus erythematosus are associated with increased mutations/polymorphisms and splice variants of T cell receptor {zeta} chain messenger RNA. Arthritis Rheum. 44:1336.-1350. [Medline]
  12. Nambiar, M. P., S. Krishnan, G. C. Tsokos. 2004. T-cell signaling abnormalities in human systemic lupus erythematosus. Methods Mol. Med. 102:31.-48. [Medline]
  13. Tolnay, M., Y. T. Juang, G. C. Tsokos. 2002. Protein kinase A enhances, whereas glycogen synthase kinase-3{beta} inhibits, the activity of the exon 2-encoded transactivator domain of heterogeneous nuclear ribonucleoprotein D in a hierarchical fashion. Biochem. J. 363:127.-136. [Medline]
  14. Tenbrock, K., Y. T. Juang, M. Tolnay, G. C. Tsokos. 2003. The cyclic adenosine 5'-monophosphate response element modulator suppresses IL-2 production in stimulated T cells by a chromatin-dependent mechanism. J. Immunol. 170:2971.-2976. [Abstract/Free Full Text]
  15. Kyttaris, V. C., Y. T. Juang, K. Tenbrock, A. Weinstein, G. C. Tsokos. 2004. Cyclic adenosine 5'-monophosphate response element modulator is responsible for the decreased expression of c-fos and activator protein-1 binding in T cells from patients with systemic lupus erythematosus. J. Immunol. 173:3557.-3563. [Abstract/Free Full Text]
  16. Asahara, H., B. Santoso, E. Guzman, K. Du, P. A. Cole, I. Davidson, M. Montminy. 2001. Chromatin-dependent cooperativity between constitutive and inducible activation domains in CREB. Mol. Cell. Biol. 21:7892.-7900. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
I. A. Parish, S. Rao, G. K. Smyth, T. Juelich, G. S. Denyer, G. M. Davey, A. Strasser, and W. R. Heath
The molecular signature of CD8+ T cells undergoing deletional tolerance
Blood, May 7, 2009; 113(19): 4575 - 4585.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
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]

Home page
 Ann Rheum DisHome page
J. C Crispin, V. Kyttaris, Y.-T. Juang, and G. C Tsokos
Systemic lupus erythematosus: new molecular targets
Ann Rheum Dis, November 1, 2007; 66(suppl_3): iii65 - iii69.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y.-T. Juang, L. Sumibcay, M. Tolnay, Y. Wang, V. C. Kyttaris, and G. C. Tsokos
Elf-1 Binds to GGAA Elements on the FcR{gamma} Promoter and Represses Its Expression
J. Immunol., October 1, 2007; 179(7): 4884 - 4889.
[Abstract] [Full Text] [PDF]

Home page
 Rheumatology (Oxford)Home page
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]

Home page
 J. Immunol.Home page
K. Tenbrock, Y.-T. Juang, N. Leukert, J. Roth, and G. C. Tsokos
The Transcriptional Repressor cAMP Response Element Modulator {alpha} Interacts with Histone Deacetylase 1 to Repress Promoter Activity
J. Immunol., November 1, 2006; 177(9): 6159 - 6164.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tenbrock, K.
Right arrow Articles by Tsokos, G. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tenbrock, K.
Right arrow Articles by Tsokos, G. C.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS