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Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain, activation of ZAP-70, and an increase of bioactive intermediates such as intracellular Ca2+, inositol 3-phosphate, and diacylglycerol (1). The cascade results in the activation of kinases such as protein kinase C or phosphatases like calcineurin, which promote the binding of several transcription factors to the IL-2 promoter (1). These transcription factors include NF-
B, NFAT, AP1, octamer binding protein, and CREB, and there is evidence that all binding sites on the IL-2 promoter need to be occupied to ensure maximal transcription and production of IL-2 (1, 2). These factors represent transcription enhancers and bind minutes after T cell engagement within 300 bp upstream of the start codon (1). IL-2 production reaches a maximum 8–12 h after stimulation, declines steadily thereafter, and terminates after 24–48 h (3, 4). Costimulation with CD28 can expand IL-2 production mainly by enhancing transcription of the IL-2 gene and increasing IL-2 mRNA stability (1, 5). The kinetics of IL-2 production parallel that of chromatin remodeling in murine cell lines. Thus, full activation of the IL-2 promoter requires open chromatin (2).
In contrast to the activation, termination and negative regulation of IL-2 production is not well defined and has been perceived as a passive process dependent on the dissociation and dephosphorylation of activating transcription factors (3). Our group has recently established the importance of the cAMP response element modulator (CREM)3 as a negative regulator of IL-2 production in systemic lupus erythematosus (SLE) T cells (6). T cells from SLE patients express increased amounts of CREM 
protein. CREM acts as a transcriptional repressor, and we have proposed that this is central for decreased IL-2 production in SLE T cells (6, 7, 8). CREM can form homoduplexes or heteroduplexes with CREB and bind to the -180 site of the IL-2 promoter. The -180 site of the IL-2 promoter is important in IL-2 production because mutations of this site almost completely abolish IL-2 transcription (6, 9). T cells of mice that express a dominant negative form of CREB show a marked decrease in the production of IL-2 (10).
CREM shares a high level of sequence homology with CREB. They both belong to the family of cAMP responsible factors. While CREB is a transcriptional activator, CREM is a transcriptional repressor. CREM is present in many tissues and has been shown to be constitutively active in spermatocytes and the brain (11, 12, 13). The expression of various isoforms of CREM is regulated by four different promoters and alternative splicing (14). The isoforms function either as transcriptional repressor or activator, depending on the presence or absence of the transactivating domains (Q1 and/or Q2) (13, 15, 16). The second promoter is responsible for the regulation of an inducible CREM, inducible cAMP early repressor (ICER), which is a transcriptional repressor. ICER is expressed in the brain (17) and has been suggested to play a role in T cells (18, 19). However, CREM is bigger than ICER protein (36 vs 16 kDa), possesses a kinase inducible domain, and is regulated by promoter 1. The regulation of CREM expression has been investigated extensively in spermatocytes (13, 14), but little is known about its expression and regulation in T cells. CREM and CREB are both able to bind the histone acetyltransferases (HAT), protein 300 (p300) and CREB binding protein (CBP), and through this association they likely influence chromatin remodeling (20, 21). The kinase inducible domains of CREM and CREB contain the p300 binding properties, but the transactivating domain Q2, which is lacking in CREM , is necessary to activate p300 (22). Furthermore, Q2 is necessary to activate the TATA box binding factor IID (TFIID) complex of the RNA polymerase II (23).
In this study we show that CREM is transcriptionally induced in normal T cells after TCR/CD3 stimulation. CREM binds to the CRE site of the IL-2 promoter, recruits p300 and CBP, and interferes with their HAT activities. CREM binding promotes chromatin deacetylation, limits promoter accessibility, and decreases its transcriptional activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Heparinized peripheral venous blood was obtained from healthy volunteers (Walter Reed Army Institute of Research Human Use Protocol number 776). Non-T cells were selected by a tetrameric Ab mixture against CD14, CD16, CD19, CD56, and glyA (StemCell Technologies, Vancouver, Canada) and bound to erythrocytes. These complexes were separated from the T cells by a lymphoprep gradient (Nycomed, Oslo, Norway). The purified T cells were >98% positive for CD3 as tested by flow cytometry.
Antibodies
Abs against CREM, CREB, Jun, p300, and CBP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Abs against pCREB, histone 3 (H3), histone 4 (H4), acetylated H3, and acetylated H4 were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-CD3 Ab was purchased from Ortho-McNeil Pharmaceuticals (Raritan, NJ), anti-CD28 was purchased from BD Biosciences (Franklin Lakes, NJ), and goat anti-mouse cross-link Ab was purchased from Sigma Genosys (The Woodlands, TX).
Preparation of mRNA and cDNA, PCR, and real-time PCR
One million T cells were used for extracting RNA (RNA Easy Mini kit; Qiagen, Hilden, Germany). RNA was quantified, and 500 ng of total RNA was used for cDNA synthesis by reverse transcription (Reverse Transcription PCR kit; Promega, Madison, WI). 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 (Pharmacia Biotech, Piscataway, NJ). Real-time PCR was conducted with a Cepheid Smart Thermocycler by adding Sybr Green to the reaction mixture. Primers used for PCR were IL-2: 5'-CAC TAC TCA CAT TAA CCT CAA CTC CTG-3', reverse 5'-CTG GGA AGC ACT TAA TTA TCA AGT TAG TG-3'; CREM: 5'-GAA ACA GTT GAA TCC CAG CAT GAT GGA AGT-3', reverse 5'-TGC CCC GTG CTA GTC TGA TAT ATG-3'. PCR products were separated on a 1.5% agarose gel, and the OD was quantified by using QuantityOne software (Bio-Rad, Hercules, CA) after background subtraction from each band.
For sequencing of CREM isoforms, RT-PCR products were excised from the gel, extracted (Gel extraction kit; Qiagen), and cloned into a TOPO cloning system (Topo TA; Invitrogen, Carlsbad, CA). Plasmids were isolated from recombinant clones and sequenced on an ABI Prism sequencer.
Chromatin immunoprecipitation analysis (ChIP)
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 a desired 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 IL-2 promoter, including the -180 site (forward 5'-CTA AGT GTG GGC TAA TGT AAC-3', reverse 5'-TGT AAA ACT GTG GGG GT-3'); -1014 to -1233 (forward 5'-AAA TAG GAG CCA TCA CTT CAC AA-3', reverse 5'-GCC TTA TTT ATT TCT TAC CTG GAC-3'); -562 to -758 (forward 5'-TAA AAA CGA GAA ACA TGG ACT GGT-3', reverse 5'-TTG AGG TAC TGT TTA ACG CTA TTT-3'); +301 to +510 (forward 5'-TTA CAA GAA TCC CAA ACT-3', reverse 5'-TAG AGG CTT CAT TAT CAA A-3'). DNA from 
1,000,000 cells was used per PCR. PCR products were run on a 1.5% agarose gel and quantified with QuantityOne software.
DNA-accessibility assay (chromatin assembly by RT-PCR (CHART))
A modified protocol described by Rao et al. (24) was used. T cells were either transfected with antisense CREM or an empty vector plasmid. Twenty-four hours after transfection, T cells were stimulated with CD3 and CD28 for 1–18 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, 0.1 mM EGTA) with addition of NP40. Nuclear pellets were harvested and washed in restriction enzyme buffer. Digestion was conducted at suggested temperatures with EcoRI, DdeI or Tsp509I for 1 h. DNA was extracted and amplified by real time method with primers flanking the IL-2 promoter that contained the digestion sites.
Transfection, stimulation, and IL-2 ELISA
Freshly isolated normal T cells were rested for 1 h in RPMI and 10% FBS without stimulation. Plasmids encoding CREM antisense (a kind gift from Dr. P. Sassone-Corsi, Laboratoire de Génétique Moleculaire des Eucaryotes, Institute National de la Santé et de la Recherche Médical, Strasbourg, France) and corresponding empty vector plasmid were used for transfection. Five micrograms of each plasmid were used per transfection. Approximately 5–10 x 106 T cells were transfected by electroporation with an AMAXA electroporator (AMAXA, Cologne, Germany). After different time points, T cells and supernatant were harvested for ELISA (R&D Systems, Minneapolis, MN). For stimulation, CD3 Ab (10 µg/ml), CD28 Ab (2.5 µg/ml), and a goat anti-mouse cross-linking Ab (25 µg/ml) were used, and T cells were stimulated after transfection. The supernatant was harvested at different time points up to 125 h after stimulation. As control, the mitogen PMA and the calcium ionophore A23178 (ionomycin) were used for stimulation.
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Results |
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To demonstrate that CREM is up-regulated in stimulated T cells, we cross-linked CD3 and CD28 on human T cells and collected mRNA, protein, and cell supernatant at different time points. We found that stimulated T cells express increased amounts of CREM mRNA and protein. CREM mRNA can be detected within 1 h and rapidly rises within the first 4 h. The peak of CREM mRNA production occurs at 48–72 h after a slow but steady increase. IL-2 production rises rapidly during the first 4–8 h and steadily declines afterward. Both return to baseline levels within 96–124 h. Increases in CREM protein can be detected within 2 h, and they reach maximal levels at 72 h (Fig. 1). The IL-2 production is prolonged compared with previous reports of T cells being stimulated with PHA and PMA (25), and this is probably the consequence of costimulation with CD28, which elongates RNA stability (1, 5). We cloned and sequenced the PCR products to determine the CREM isoforms that are expressed after stimulation in T cells. The most dominant form of CREM mRNA in stimulated T cells was the transcriptional repressor CREM (data not shown).
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Because CREM was found up-regulated in stimulated T cells, we wanted to determine whether it is able to bind to the IL-2 promoter in live T cells. T cells were treated with CD3 and CD28, then fixed with formalin to cross-link DNA to proteins, sonicated, and the DNA-protein complexes were immunoprecipitated. The DNA was extracted and amplified with primers flanking the -180 CRE site of the IL-2 promoter. As shown in Fig. 2, we detected increased binding of CREM to the IL-2 promoter in stimulated T cells within 2 h of stimulation, which is shortly after it is transcriptionally induced. Increased binding was observed 6 and 24 h after stimulation too (Fig. 2). CREB bound to the IL-2 promoter in unstimulated T cell, but the binding was much reduced after stimulation. In contrast, pCREB binding was noted immediately after stimulation and was maintained during the first 2–6 h but decreased subsequently (Fig. 2). Thus, CREM remains bound for at least 24 h, while pCREB binding decreases after 6 h and is not detectable after 24 h.
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treatment inhibits the binding of CREM to the IL-2 promoter in activated T cells and enhances the production of IL-2
To investigate whether CREM is indeed involved in the suppression of IL-2 transcription, we transfected an antisense CREM plasmid into normal T cells and stimulated them with CD3 and CD28 Abs. An empty vector plasmid (PGS5) served as control. We have previously reported that the activity of a luciferase reporter construct driven by the IL-2 promoter is reduced by a CREM plasmid while enhanced by an antisense plasmid in T cells (8). In Fig. 3 we show that transfection of T cells with the antisense CREM plasmid prevented the binding of CREM to the IL-2 promoter and resulted in enhanced production of IL-2. Similar results were obtained when the cells were stimulated either with PMA or ionomycin. Stimulated T cells transfected with antisense CREM produced two times more IL-2 after 24 h as measured by ELISA (Fig. 4). The effect of antisense CREM on the levels of IL-2 protein and mRNA was detectable within 2 h (Figs. 3 and 4), which represents the time point when CREM binding to the IL-2 promoter in vivo is detectable. In addition, IL-2 levels in the antisense CREM-transfected T cells persisted at higher levels for 48 h (Fig. 4), while at 96 h they leveled out because of an accumulation of IL-2 in the medium (4). Thus, diminishing CREM levels in stimulated T cells leads to an enhanced and prolonged production of IL-2.
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To address the possibility that CREM inhibits IL-2 production by enhancing chromatin condensation, we conducted ChIP and restriction enzyme accessibility experiments (CHART) (24). As shown in Fig. 5A, antisense CREM significantly enhanced accessibility of the IL-2 promoter to EcoRI, TSP509I, and DdeI as compared with an empty vector 24 h following stimulation with anti-CD3 and CD28 Abs. The difference in the accessibility was not detectable at 30 min, that is before CREM is up-regulated, but it became detectable after 2 h. This corresponds to the differences observed in the changes of IL-2 mRNA and protein levels. Thus, antisense CREM prevents chromatin remodeling and increases the restriction enzyme accessibility to the IL-2 promoter. The accessibility was restricted only to the core promoter, because primers flanking 301 to 510 bp downstream and 562 to 758 and 1014 to -1233 bp upstream of the transcription initiation site of the IL-2 promoter were inaccessible to digestion, although they contained several DdeI and TSP509I sites (Fig. 5B). Furthermore, transfection of cells with the antisense CREM vector failed to change the XhoI digestion pattern. Because XhoI does not have a recognizable restriction site on the core IL-2 promoter, this finding excludes nonspecific effects of the antisense CREM treatment (Fig. 5C).
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Because the restriction enzyme accessibility was increased in T cells treated with antisense CREM, we looked for acetylation patterns of histones in these cells. As predicted, we found enhanced binding of acetylated H3 and H4 and reduced binding of the nonacetylated H3 to the IL-2 promoter in T cells transfected with antisense CREM (Fig. 6A) 24 h after stimulation. Additionally, after transfection with antisense CREM we saw a decreased binding of CBP and p300 to the IL-2 promoter (Fig. 6A). The difference in the acetylation was not observed during the first hour, that is before CREM is effectively produced (Fig. 6B). Furthermore, this binding pattern was restricted to the core promoter of IL-2, because PCR with primers flanking -1014 and -562 of the IL-2 promoter showed no difference in the acetylation pattern after transfection with antisense CREM after 24 h of T cell stimulation (Fig. 6C). Thus, CREM recruits CBP and p300 and increases the binding of nonacetylated histone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we demonstrate for the first time a negative regulation of IL-2 promoter activity after T cell activation. We show that CREM is transcriptionally induced in normal T cells after stimulation with CD3 and CD28; a similar fact has been previously shown for ICER (18). The transcriptional induction of CREM in T cells is novel and contrasts with other cell types like those in the brain and the testis where CREM is constitutively active (13, 16). Immediately after its production, CREM starts to bind to the IL-2 promoter and represses transcriptional activity. The suppression of CREM production by an antisense plasmid enhanced and lengthened IL-2 production. The binding of CREM to the IL-2 promoter is followed by an increased association of nonacetylated histones and the inaccessibility of the proximal promoter to endonucleases. The binding of CREM retains the transactivational cofactors p300 and CBP located on the IL-2 promoter, but because CREM lacks the Q2 domain it is unable to stimulate their HAT activity (22), and as a consequence, chromatin is remodeled. Furthermore, CREM does not activate TFIID, which is a critical step in DNA transcription (23).
Both CREM and IL-2 mRNA are induced in a parallel fashion with a sharp rise during the first 2–4 h. This could suggest that similar transcription factors regulate the initiation of the transcription process in both promoters. But while the IL-2 mRNA levels decline after 8 h, CREM levels increase slowly during the next 48–72 h. The factors that contribute to its rise are yet unknown. The slow decline of the IL-2 mRNA levels between 24 and 100 h after T cell stimulation can be explained by increased RNA stability, which has been reported in T cells that have been costimulated with an anti-CD28 Ab (1, 25).
At this moment we cannot exclude any posttranslational modifications of CREM that might contribute to its repressor activity. However, Foulkes et al. (30) have shown that phosphorylation of CREM does not increase its binding activity to the CRE site of the c-Fos promoter, a fact that we were able to reproduce using an oligonucleotide defining the -180 site of the IL-2 promoter in Biacore experiments. These facts indicate that phosphorylation of CREM does not increase its DNA binding activity of CREM to the IL-2 promoter (our unpublished data).
The similarity in the transcriptional up-regulation of +IL-2 and CREM during the first hours, the increased binding of CREM together with the decline of IL-2 production, and the prevention of this decline after transfection of an antisense CREM suggests the following two-step model for the negative transcriptional regulation of IL-2 by CREM (Fig. 7). During the first step of T cell stimulation, kinases and phosphatases become activated, which lead to the phosphorylation of CREB. Phosphorylated CREB recruits p300 and CBP and binds to the -180 (9) and possibly other sites on the IL-2 promoter (31). The binding of other factors like AP1 and NF-B together with pCREB and their ability to recruit CBP and p300 lead to the building of an enhanceosome that associates with the RNA polymerase II complex (32, 33, 34) and activates the transcription of IL-2 mRNA and the production of protein. The binding of pCREB declines after 2–6 h promoted by increased dephosphorylation by phosphatases PP1 and PPA2 (35). We suggest that as a second step in T cell activation, CREM mRNA is induced and the CREM protein binds to the IL-2 promoter within 2 h after stimulation, gradually replaces pCREB, and shuts down IL-2 production. CREM and pCREB are both able to recruit p300 and CBP, but by lacking the transactivator domain Q2, CREM does not activate the histone acetylating function of p300 (22) and does not activate TFIID. As a consequence, transcription ceases (23).
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to activate the HAT function of p300, which represents a passive mechanism, an active repression by CREM or the CREM/p300 complex might be considered. p300 contains a repression domain called cell cycle regulatory domain 1 (36), which is conserved in other proteins like the erythroblast transformation-specific domain and transcription factor ELK-1 as well, (37) and is able to actively repress transcription (36) in a manner that is dependent on the abundance of activating or repressing transcription factors. Furthermore, other mechanisms of transcriptional repression by CREM are possible and could include glycosylation or methylation of p300, CBP, or histones as well as a recruitment of inhibitors of transcription like histone deacetylating proteins (38, 39, 40).
In conclusion, we have demonstrated a significant correlation between IL-2 and CREM protein expression after T cell stimulation. The direct binding of CREM to the CRE site of the IL-2 promoter endows CREM with a central role in the repression of IL-2 gene expression. After induction, it gradually replaces pCREB at the CRE site and recruits p300 and CBP, but is not able to activate their histone acetylase capabilities. The consequence is chromatin remodeling and reduced transcription (Fig. 7). The prevention of the production and binding of CREM to the IL-2 promoter by an antisense CREM plasmid and the ensuing increase of IL-2 production clearly demonstrates the importance of CREM in the negative regulation of IL-2 transcription in stimulated T cells.
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Acknowledgments |
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antisense plasmid. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. George C. Tsokos, Department of Cellular Injury, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Building 503, Room 1A32, Silver Spring, MD 20910. E-mail address: gtsokos{at}usuhs.mil
3 Abbreviations used in this paper: CREM, cAMP response element modulator; SLE, systemic lupus erythematosus; ICER, inducible cAMP early repressor; p300, protein 300; HAT, histone acetyltransferases; CBP, CREB binding protein; H3, histone 3; H4, histone 4; ChIP, chromatin immunoprecipitation analysis; pCREB, phosphorylated CREB; TFIID, TATA box binding factor IID; CHART, chromatin assembly by RT-PCR.
Received for publication October 16, 2002. Accepted for publication January 6, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
-cells. Diabetes 49:1681.[Abstract]
isoforms expressed by two newly identified cAMP-responsive promoters active in the testis. Endocrinology 141:3923. enhanceosome is required for synergistic activation of transcription. Mol. Cell 1:277.[Medline]
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