The Journal of Immunology

Methylation Status of CpG Islands Flanking a cAMP Motif on the Protein Phosphatase 2Ac␣ Determines CREB Binding and Activity1

Katsue Sunahori, Yuang-Taung Juang,2 and George C. Tsokos2

Protein phosphatase 2A (PP2A) is a major serine/threonine protein phosphatase in eukaryotic cells and is involved in many essential aspects of cell function. The catalytic subunit of the enzyme (PP2Ac), a part of the core enzyme, has two isoforms, ␣ (PP2Ac␣) and ␤ (PP2Ac␤), of which PP2Ac␣ is the major form expressed in vivo. Deregulation of PP2A expression has been linked to several diseases, but the mechanisms that control the expression of this enzyme are still unclear. We conducted experiments to decipher molecular mechanisms involved in the regulation of the PP2Ac␣ promoter in human primary T cells. After preparing serially truncated PP2Ac␣ promoter luciferase constructs, we found that the region stretching around 240 bases upstream from the translation initiation site was of functional significance and included a cAMP response element motif flanked by three GC boxes. Shift assays revealed that CREB/phosphorylated CREB and stable protein 1 could bind to the region. Furthermore, we demonstrated that methylation of deoxycytosine in the CpG islands limited binding of phos- phorylated CREB and the activity of the PP2Ac␣ promoter. In contrast, the binding of stable protein 1 to a GC box within the core promoter region was not affected by DNA methylation. Primary T cells treated with 5-azacitidine, a DNA meth- yltransferase inhibitor, showed increased expression of PP2Ac␣ mRNA. We propose that conditions associated with hy- pomethylation of CpG islands, such as drug-induced lupus, permit increased PP2Ac expression. The Journal of Immunol- ogy, 2009, 182: 1500–1508.

rotein phosphatase 2A (PP2A)3 is a major serine/threonine mosome 5q23–q31 for ␣ and in 8p12–p11.2 for ␤. The promoters phosphatase with complex composition and is involved in of both genes are GC rich and lack the TATA and CCAAT se- P many essential aspects of cell function (1–3). The het- quences typical of many housekeeping genes. However, the 5Ј erodimeric PP2A core enzyme consists of a well-conserved 36- upstream regions as well as the regions encoding the 5Ј and 3Ј kDa catalytic subunit (C subunit; PP2Ac) and a 65-kDa scaffold untranslated sequences of each mRNA are quite different. The ac- subunit (A subunit) (4). To gain full activity toward specific sub- tivity of the PP2Ac␣ gene promoter is 7(Ϯ10)-fold stronger than strates, the PP2A core enzyme associates with a variable regula- that of the PP2Ac␤ gene promoter, which may explain why mRNA tory subunit (B subunit) to form a heterotrimeric holoenzyme level of PP2Ac␣ is ϳ10 times higher than that of PP2Ac␤ (6). The (1–3). expression of PP2Ac is tightly controlled through autoregulation to Molecular cloning has revealed the existence of two mammalian ensure the presence of relatively constant levels of PP2A (7), but PP2Ac isoforms, ␣ (PP2Ac␣) and ␤ (PP2Ac␤), which share 97% many essential aspects of PP2Ac regulation remain poorly identity in their primary sequence (5). Both isoforms are ubiqui- understood. tously expressed and these genes are composed of seven exons and Abnormal expression of PP2A has been linked to many diseases six introns encoded by different genes, localized in human chro- such as cancer (8, 9), Alzheimer disease (10), and Opitz BBB/G syndrome (11). We have reported that the message, protein, and Division of Rheumatology in Department of Medicine, Beth Israel Deaconess Med- enzymatic activity of PP2Ac are increased in the peripheral T cells ical Center, Harvard Medical School, Boston, MA 02115 from the patients with systemic lupus erythematosus (SLE) com- Received for publication October 24, 2008. Accepted for publication November 20, pared with normal T cells (12). Phosphorylated CREB (pCREB), 2008. which is an important factor in the regulation of the The costs of publication of this article were defrayed in part by the payment of page expression of IL-2 (13), is known to be one of the substrates of the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. PP2Ac (14), and decreased production of IL-2 by SLE T cells is considered to be of pathogenic importance (15–20). Treatment of 1 This work was supported by Public Health Service National Institutes of Health Grant RO1 AI068787. SLE T cells with PP2Ac-small interfering RNA decreases the pro- 2 Address correspondence and reprint requests to Dr. George C. Tsokos or Dr. Yuang- tein levels and activity of PP2Ac in a specific manner and in- Taung Juang, Department of Medicine, Division of Rheumatology, Beth Israel Dea- creases the levels of pCREB and its binding to the IL-2 and c-fos coness Medical Center, Harvard Medical School, 330 Brookline Avenue, E/CLS 937, Boston, MA 02115. E-mail addresses: [email protected] or yjuang@bidmc. promoters, as well as increases AP-1 activity, causing normaliza- harvard.edu tion of IL-2 production. 3 Abbreviations used in this paper: PP2A, protein phosphatase 2A; PP2Ac, PP2A SLE is an autoimmune disease that affects multiple organs in- catalytic subunit; PP2Ac␣, PP2Ac ␣ isoform; PP2Ac␤, PP2Ac ␤ isoform; SLE, sys- cluding the joints, skin, kidneys, and brain. Several signaling ab- temic lupus erythematosus; pCREB, phosphorylated CREB; DNMT, DNA methyl- transferase; dC, deoxycytosine; CRE, cAMP response element; 5-azaC, 5-azacitidine; normalities of the immune system have been described in SLE and Sp1, stable protein 1; M. SssI, CpG methyltransferase; SAM, S-adenosyl methionine; are thought to be central in the pathophysiology of this disease. At CREM, cAMP response element modulator; ChIP, chromatin immunoprecipitation the cellular level, T lymphocytes orchestrate the altered immune assays; dmC, deoxymethylcytosine; Mt, mutant. responses in SLE (21, 22). One of the well-known abnormalities of Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 T cell signal transduction in SLE is the defective signaling via www.jimmunol.org The Journal of Immunology 1501

Table I. Sequence of oligonucleotides used in EMSA and mutant constructsa

2 Ϫ280 GC box GC box CRE GC box 2 Ϫ218 Sequence of core promoter 5Ј-TTTTCCCCTCCGCTCCCCGCCGCCTCCTGACGCCGGGCGTGAC GTCACCAC GCCC GGCGGCCGCC-3Ј EMSA probe Wild-type probe 1 (unmethylated, Ϫ250 to Ϫ226) CGCCGGGCGTGAC GTCACCAC GCCC Mutant probe 1 CGCCGGGCGTGCG TACACCAC GCCC Methylated probe 1 CGCCGGGCGTGACMeGTCACCAC GCCC Wild-type probe 2 (unmethylated, Ϫ240 to Ϫ216) GAC GTCACCAC GCCC GGCGGCCGCC Mutant probe 2 GAC GTCAATGT GCCC GGCGGCCGCC Mutant probe 3 GAC GTCACCAC ATGT GGCGGCCGCC Mutant probe 4 GAC GTCACCAC GCCC ATCTGCCGCC Methylated probe 2 GAC GTCACCACMeGCCCMeGGCGGCCGCC Mutant construct sequence ⌬-468-MtCREB ϳTTTTCCCCTCCGCTCCCCGCCGCCTCCTGACGCCGGGCGTGCG TACACCAC GCCC GGCGGCCGCCϳ ⌬-468-MtSp1 ϳTTTTCCCCTCCGCTCCCCGCCGCCTCCTGACGCCGGGCGTGAC GTCAATGT GCCC GGCGGCCGCCϳ

a Three GC boxes and a CRE motif in PP2Ac␣ core promoter region (nt Ϫ280 to Ϫ218) are noted by underlining. Boldface characters indicate mutated bases. 5-methyl- cytosine is indicated as CMe.

ERK-1 and ERK-2 (23), which contribute to the DNA hypomethy- CCGGGCGTGCGTACACCACGCCCGG-3Ј,R,5Ј-CCGGGCGTGGTG lation by the decreased expression of DNA methyltransferase TACGCACGCCCGGCGTCAGGAGG-3Ј; ⌬-468-MtSp1, F, 5Ј-GGGCGTG Ј Ј (DNMT-1) (21–26). Because methylation of deoxycytosine (dC) ACGTCAATGTGCCCGGCGGCC-3 ,R,5-GGCCGCCGGGCACATTG ACGTCACGCCC-3Ј (mutated bases are underlined). Plasmids were in regulatory sequences can suppress transcription of the associ- sequenced to detect any errors introduced by PCR. ated genes, abnormal hypomethylation may result in the overex- Twenty micrograms of ⌬-468 construct or empty vector was methylated pression of certain genes such as lymphocyte function-associated using 16 U of CpG methyltransferase (M. SssI) and S-adenosyl methionine Ag 1 (LFA-1; CD11a) and CD70 in SLE T cells (27, 28). (SAM; New England Biolabs) at 37°C for 16 h, with subsequent inacti- vation of enzyme at 65°C for 20 min. Mock-methylation reactions were Because PP2Ac mRNA was found to be increased in T cells also performed in the absence of M. SssI and SAM. The methylated or from patients with SLE and its stability was not compromised (12), mock-methylated constructs were purified using QIAquick PCR purifica- we have initiated studies to define the transcriptional control of the tion kit (Qiagen), and the methylation status of each construct was deter- PP2Ac gene. We report herein that the PP2Ac␣ promoter defines mined by methylation-senstive restriction enzyme Aat⌱⌱ (New England ⌬ ⌱ a cAMP response element (CRE) site flanked by CpG motifs and Biolabs) for -468 construct or Sal (New England Biolabs) for empty vector. that methylation controls the binding of pCREB and the activity of the promoter. Cell transfection Materials and Methods The transfection of plasmids into human primary T cells was done using an Cells and reagents Amaxa nucleoporator according to the manufacturer’s instructions. In Primary T cells were purified from peripheral venous blood obtained from some experiments, a plasmid encoding CREB (Clontech), Sp1 (Invivo- healthy volunteers. The blood was incubated for 30 min with a rosette T Gen), or corresponding empty vector (pCMV or pORF9) were cotrans- fected into primary T cells with PP2Ac␣-PGL3 basic construct. Then, 0.25 cell purification kit (StemCell Technologies) that contained a tetrameric Ab ␮ mixture against CD14, CD16, CD19, CD56, and glyA that attaches non-T g of pRL-TK (Promega) was cotransfected as an internal control for transfection efficiency in all cases. The total amount of plasmid used in cells to erythrocytes. Ficoll containing Lymphoprep gradient (Greiner Bio- ␮ One) was subsequently used to separate these complexes from T cells. each sample was up to 5 g. ϫ 6 Using flow cytometry, we established that the purified cells were Ͼ98% Following transfection, 3 10 T cells were cultured in 12-well plates positive for CD3. and harvested after 24 h incubation. Cytoplasmic extracts were prepared T cells (up to 10 ϫ 106/sample) were plated onto appropriate size plates using a luciferase assay kit (Promega), and luminescence was measured in RPMI 1640 medium (Mediatech) supplemented with 10% heat-inacti- immediately for 30 s using AutoLumat LB 953 (EG&G Berthold, Bad Wildbad, Germany). The luciferase activity was normalized using Renilla vated FBS (Quality Biological) and 2 mM L-glutamine and maintained in readings. a humidified incubator (37°C, 5% CO2). For some experiments, T cells were preincubated with 200 U/ml human recombinant IL-2 (R&D Sys- tems) overnight and treated with 0–10 ␮M 5-azacytidine (5-azaC; Sigma- Aldrich) for 24 or 48 h. Studies were approved by the human use com- EMSA mittee of our institution. The purification of nuclear proteins and the conduction of EMSA have Plasmids been described previously with minor modification (12). Briefly, the nu- clear extracts of normal T cells were obtained by treatment with lysis buffer The PP2Ac␣ gene promoter region was amplified using GoTaq DNA poly- (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 10 merase (Promega) and human genomic DNA template (Roche) with primers mM NaF, 1 mM Na3VO4, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluo- containing NheI, XhoI, or HindIII restriction sites at the 5Ј ends. The PCR ride, 2 mM aprotinin, and 1 mM leupeptin) after the collection of cyto- primers used in these amplification reactions were: forward (F)1 (Ϫ1104), plasmic extract. Oligonucleotides and methylated oligonucleotides were 5Ј-GCGCTAGCGACAGGTGCATCCATCTTCTCT-3Ј;F2(Ϫ781), 5Ј-GGG purchased from Biotechnologies. The sequences of the oligonu- CTAGCGGTGGGGGTGGTTAATCCAA-3Ј;F3(Ϫ468), 5Ј-CCGCTAG cleotides used are shown in Table I. All listed oligonucleotides included CATGCTCCAGCTCCATCCTTC-3Ј;F4(Ϫ368), 5Ј-GCCTCGAGTGC antiparallel partner oligonucleotides. In case of the methylated oligonucle- GCTTTGACCCCCAGTTT-3Ј;F5(Ϫ280), 5Ј-GCCTCGAGTTTTCCCCTC otides, the antisense oligonucleotides pair was also methylated at the cor- CGCTCCCC-3Ј;F6(Ϫ218), 5Ј-CGGCTAGCGCCATTACAGAGAGCCGA responding cytosine as the sense strand. Each pair of these complementary GCT-3Ј; reverse (R) (Ϫ83), 5Ј-CCAAGCTTGCCGGTTCCTCGTGTACTT oligonucleotides was annealed and used as probe or competitor. The nu- CT-3Ј. The PCR-generated DNA fragments were then gel purified and ligated clear extracts (1 ␮g) were incubated with 1 ␮g of poly(dI-dC) in the bind- ␮ with the pGL3-basic luciferase constructs (Promega). The QuickChange ing buffer (20 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl2,10 M ␮ Lightning site-directed mutagenesis kit (Stratagene) was used to mutate the ZnCl2, 4% glycerol, and 100 g of BSA per ml) for 15 min at room CREB or stable protein 1 (Sp1) binding site in the construct including the temperature (29). For some samples, unlabeled double-stranded oligonu- 386-bp length of the PP2Ac␣ promoter region. Each primer that we used for cleotides were added as competitors at 10-fold molar excess. For supershift mutagenesis is as below. ⌬-468-mutant (Mt) CREB, F, 5Ј-CCTCCTGACG assays, the nuclear proteins were incubated with 1 ␮g of anti-CREB Ab, 1502 METHYLATION DETERMINES PP2Ac␣ PROMOTER ACTIVITY

anti-pCREB Ab (Upstate Biotechnology), anti-cAMP response element high GC content and revealed the existence of several potential modulator (CREM) Ab (Aviva Systems Biology), or anti-Sp1 Ab (Santa sites and a complete CRE motif. The ma- Cruz Biotechnology) at this time. After incubation, the radiolabeled DNA jor transcription start site is located at Ϫ211 position, which was probe was added and incubated for 20 min at room temperature. The re- action mixture was then subjected to separation in a 6% nondenaturing gel identified first using the primer extension method (6) and was con- (Invitrogen). The dried gel was then autoradiographed. firmed by information from GenBank database (accession no. AK097599) (30). Reverse transcription and real-time PCR To determine whether this fragment (nt Ϫ1104 to Ϫ83) defined RNA extracted from T cells (3 ϫ 106) (RNeasy Mini kit; Qiagen) was an active promoter, we cloned it into pGL3-basic luciferase con- treated with DNase I (Qiagen). Total RNA (300 ng) was transcribed in struct and transiently transfected it into human normal T cells us- cDNA in a conventional thermocycler using avian myeloblastosis virus ing the empty vector and a Renilla construct as controls. We also reverse transcriptase and oligo(dT) primer (RT-PCR kit; Promega). Real- time RT-PCR was performed in triplicate for every sample with a Light- tested the promoter activity of several constructs that had different Cycler 480 system by adding SYBR Green (Roche) to the reaction mixture. 3Ј ends. Although the longer construct (nt Ϫ1104 to Ϫ2) had sim- Primers used were: PP2Ac␣,F,5Ј-TCCGAGTCCCAGGTCAAGAG-3Ј, ilar levels of promoter activity, the shorter one (nt Ϫ1104 to Ϫ180) Ј Ј Ј R, 5 -GCTACAAGCAGTGTAACTGTTTCA-3 ; and GAPDH, F, 5 - displayed reduced activity compared with the ⌬-1104 construct CAACTACATGGTTTACATGTTCC-3Ј,R,5Ј-GGACTGTGGTCATG Ј (nt-1104 to Ϫ83) (data not shown). Subsequently, we generated AGTCCT-3 . The averaged cycle threshold (CT) values of each reaction derived from the target gene, determined with LightCycler 480 system several truncated constructs by progressive deletion of nucleotides ⌬ software, were normalized to GAPDH levels. CT was calculated using the from the 5Ј end and placed these fragments in the luciferase re- ⌬ ϭ Ϫ following equation: CT CT of target CT of GAPDH. porter construct to define the minimal sequences required for the Methylation-specific PCR transcriptional initiation of PP2Ac␣ expression. Compared with the basal activity of the empty vector, the pro- ␮ Genomic DNA (1 g) from primary T cells treated with 5-azaC for 48 h moter activity of all constructs was higher ( p Ͻ 0.05), and among was purified and treated with the methylation-sensitive enzyme AatII (New ⌬ Ϫ Ϫ England Biolabs). After DNA repurification, 50 ng of DNA was used as a them construct -468 (nt 468 to 83) always displayed the template for PCR. To distinguish between methylated and nonmethylated highest activity (Fig. 1C). Moreover, the region between Ϫ280 and status within the CRE motif in the PP2Ac␣ promoter, two sets of primers Ϫ218 was determined to be sufficient to support most of the pro- Ϫ were used. F3 ( 468) primer was used as forward primer of both control moter activity because the construct without this region showed and methylation-sensitive products as described above. Reverse primer for methylation-sensitive product was the same as R (Ϫ83). Reverse primer for significantly reduced promoter activity. Within this region there is control product, R (Ϫ286), was the following oligonucletotide: 5Ј- a complete CRE motif surrounded by three potential Sp1 binding CGAAGCTTATGCCACCCGCCCCAG-3Ј. The AatII recognition site sequences immediately preceding the major transcription start site. was contained in the fragments generated by F3 and R (Ϫ83), but not in When compared with the human sequence of the region preceding Ϫ those by F3 and R ( 286). PCR products were electrophoresed on 1% the transcription start site to that of the rat, we found that this agarose gels, visualized by ethidium bromide staining, and semiquantified with QuantityOne software. region is evolutionarily well preserved and therefore of importance (Fig. 1A). Chromatin immunoprecipitation assays (ChIP) Binding of CREB and Sp1 proteins to the core promoter region ChIP analysis was done using a ChIP assay kit (Upstate Biotechnology) ␣ according to the manufacturer’s instructions. T cells (3 ϫ 106) with or of PP2Ac without 5-azaC treatment were used per immunoprecipitating Ab. The To identify whether these cis-acting elements physically interacted cells were fixed with 1% formaldehyde for 10 min, lysed, and sonicated. The DNA-protein complexes were immunoprecipitated with anti- with trans factors, we incubated nuclear protein from normal T pCREB Ab or its control Ab (rabbit normal IgG; Santa Cruz Biotech- cells with two labeled double-stranded oligonucleotides containing nology), and, after a series of washing steps, the cross-linking was either the CRE motif or the Sp1 site at the center. The sequence of reversed and the protein was digested with proteinase K (Qiagen). The the oligonucleotides that we used in EMSA is shown in Table I. DNA was extracted by a QIAquick PCR purification kit (Qiagen). Im- Shift assays shown in Fig. 2 demonstrate the formation of one band munoprecipitated and purified DNA was analyzed by PCR using the primers F3 (Ϫ468) and R (Ϫ83) described as above, which are specific (lane 1; indicated by asterisk). Although there are two candidates for the human PP2Ac␣ promoter and were used to amplify a promoter for Sp1 binding upstream of the CRE motif, no nuclear protein fragment containing the CRE motif. The corresponding nonimmuno- binding to the labeled probe was recorded (data not shown). precipitated DNA (input DNA) was also analyzed by same method as We conducted competitive EMSA using unlabeled wild-type used for the control. PCR products were semiquantified using the same method as above in the methylation-specific PCR. oligonucleotides or several oligonucleotides mutated around the consensus binding site to determine the exact binding requirements Statistical analysis for nuclear protein. Both (W1 and W2) unlabeled wild-type probes Data are presented as mean values Ϯ SD. A paired two-tailed Student’s t outcompeted the binding between the labeled wild-type probe and test and the Pearson product moment correlation coefficient were used for nuclear protein, and this confirmed the specificity of the bands statistical analysis. Statistical significance was defined as p Ͻ 0.05. (lanes 2 and 3 in Fig. 2A and lane 2 in Fig 2B). The observation that unlabeled mutant oligonucleotides (Mt1 in CRE motif, Mt2 Results and Mt3 in Sp1 site) did not interrupt protein-oligonucleotide in- ␣ Identification of regions within the PP2Ac promoter teraction supports the claim that the mutated region is critical responsible for the control of its activity in normal human for the binding of these proteins (Fig. 2A, lanes 4 and 5 and Fig. T cells 2B, lanes 3 and 4). The third Sp1 site (downstream of the CRE To investigate the transcriptional mechanisms responsible for reg- site, defined by the W2 probe) defined yet another GC region ulating PP2Ac␣ expression, we first identified the PP2Ac␣ pro- that could also bind Sp1. We generated another mutated oligo- moter sequence via a basic local alignment search tool search of nucleotide (Mt4), which, when used in competitive EMSA, in- the human genome against the cDNA sequence of the PP2Ac␣ hibited the binding of Sp1, suggesting that it is not important gene. Using human genomic DNA as a template, we amplified a for the binding (Fig. 2B, lane 5). Supershift assays were per- 1023-bp fragment containing the PP2Ac␣ 5Ј-flanking region, up- formed at the same time to determine which transcription fac- stream of the translation initiation start site (ATG), defined as ϩ0. tors bind this sequence in a specific manner. CREB and its As shown in Fig. 1, A and B, the region was characterized by a activated form, pCREB, could bind to the CRE motif (Fig. 2A, The Journal of Immunology 1503

FIGURE 1. Functional analysis and nucleotide sequence of the PP2Ac␣ promoter. A, Nucleotide sequence and putative regulatory elements within bp Ϫ1104 and ϩ2 of the 5Ј-flanking region of the human PP2Ac␣ gene. The translation initiation codon, ATG, is indicated as ϩ0. The major transcription factor is located at the Ϫ211 position. The potential Sp1 transcription factor sites are highlighted by underlining, and a complete CRE motif is boxed. The starting points of each primer used for the amplification of the fragments that were cloned into the pGL3-basic vector are indicated with arrows. The DNA sequence comparison between rat and human revealed that the genomic DNA is almost identical and contained a set of CRE and GC box immediately preceding each major transcription start site. B, Schematic representation of 5Ј-flanking region of PP2Ac␣ and its CpG islands. Prediction of putative CpG islands was performed using CpG finder at the European Bioinformatics Institute (EMBL; www.ebi.ac.uk/). The parameters defined were: CpG length Ͼ200 bp, GϩC Ͼ50%, and a “CpG value” (the ratio of observed to expected frequencies of the CpG dinucleotide) of at least 0.6. With the exception of the area between Ϫ630 and Ϫ592, the whole region qualifies as a CpG island. C, Activity of different parts of PP2Ac␣ promoter in normal human T cells. Each construct or empty vector (5 ␮g) was transfected into normal human T cells with pRL-TK plasmid as an intrinsic control of transfection efficacy. Luciferase activity was normalized to Renilla activity and is relative to pGL3-basic (empty vector), which was set as 1.0. Compared with the basal activity of empty vector (open bar), the promoter activity of all constructs was increased at statistically significant levels (p value of ⌬-218 ϭ 0.0232; p value of all other constructs is Ͻ0.01). Compared with the activity of the shortest length construct (hatched bar), all constructs showed statististically significant higher activity (p Ͻ 0.02). The results represent the mean Ϯ SD of six independent experiments. lanes 6–8), but anti-CREM Ab did not affect the location and stream of the luciferase gene and compared their activity to that density of the band even though CREM can bind to the CRE driven by the wild-type promoter in human T cells. We chose the motif (Fig. 2A, lane 9). The presence of anti-Sp1 Ab in the ⌬-468 construct as a wild type because it displayed the most potent sample containing nuclear protein and labeled W2 probe re- promoter activity (shown in Fig. 1C) and the GC box was highly sulted in a clear shift of the band (Fig. 2B, lane 6). Abs against represented within this region. The mutant constructs defined the CREM and transcription factor IIB (TFIIB) did not result in a same pattern of mutations as those designed for the mutant oligo- similar band shift (data not shown). nuclotides that we used in EMSA (Table I). As shown in Fig. 3A, the promoter activity of each of the mutant constructs was sup- ␣ CREB and Sp1 regulate the PP2Ac promoter activity pressed compared with the activity of the wild-type construct. Because the competition shift assay shown in Fig. 2 suggested the Next, we designed experiments to determine the effect of the presence of critical binding sites for CREB and Sp1, we cloned the identified transcription factors on the PP2Ac␣ promoter activity. T PP2Ac␣ promoter with the CRE motif or Sp1 site mutated up- cells were cotransfected with 2 ␮g of pCMV-CREB, pORF9-Sp1, 1504 METHYLATION DETERMINES PP2Ac␣ PROMOTER ACTIVITY

FIGURE 3. CREB and Sp1 are essential for the PP2Ac␣ promoter activity. A, Mutagenesis within CRE or Sp1 sites of the PP2Ac␣ core promoter region suppressed the promoter activity. Five micrograms of wild-type construct (⌬-468; nt Ϫ468 50 Ϫ83) or its mutant constructs (⌬-468-MtCREB or ⌬-468-MtSp1) was transfected into normal human T FIGURE 2. EMSA analysis of the core promoter region of the PP2Ac␣ 32 cells. Each sequence of mutant construct is shown in Table I. Luciferase promoter. A, Wild-type probe 1 (W1), that is, P-end-labeled double- activity was normalized to Renilla activity, and normalized luciferase ac- Ϫ Ϫ stranded oligonucleotides including the CRE (nt 250 to 226), was in- tivity for the wild-type promoter was set as 100%. Compared with the basal ␮ cubated with 1 g of nuclear protein from normal human T cells. For activity of the wild-type construct (open bar), the promoter activities of the competition EMSA or supershift assays, excess amounts of unlabeled W1 mutated constructs were decreased significantly. The results represent the ␮ or mutant probe (Mt1) or 1 g of each Ab was preincubated with nuclear mean Ϯ SD of five independent experiments. B, CREB and Sp1 overex- protein. The putative CREB/pCREB and DNA complexes is marked with pression increases the activity of the PP2Ac␣ promoter. The pCMV- an asterisk, whereas the arrow indicates the supershifted complexes. B,A CREB, pORF9-Sp1, or its corresponding empty vector (2 ␮g) was cotrans- similar experiment was conducted with wild-type probe 2 (W2) probe, fected with the ⌬-468 construct into normal human T cells. The normalized Ϫ Ϫ including the GC box in the center (nt 240 to 216). Three different luciferase activity of the sample cotransfected with empty vector was set as mutant oligonucleotides (Mt2–4) were used as competitors to determine 100% (open bar). The promoter activity increased in the presence of co- the critical site for Sp1 binding. The sequences of all probes used in these transfected plasmids encoding CREB or the Sp1 gene. The results represent experiments are shown in Table I. The putative Sp1 and DNA complex is the mean Ϯ SD of three independent experiments. C, CREB and Sp1 shown as an asterisk, whereas the arrow indicates the supershifted overexpression induced increased PP2Ac␣ mRNA expression levels in complex. normal human T cells. Three micrograms of pCMV-CREB, pORF9-Sp1, or its corresponding empty vector was transfected into normal human T cells and cells were harvested 18 h later. PP2Ac␣ transcripts were mea- or the empty vector (pCMV or pORF9) with 3 ␮gof⌬-468 sured and normalized to GAPDH by real-time RT-PCR. The relative ratio ␣ PP2Ac␣ promoter-luciferase fusion construct. Similar to other of PP2Ac RNA in T cells transfected with empty vector was set as 1.0 for a control (open bar). The relative expression levels of PP2Ac␣ mRNA transfection experiments, 0.25 ␮g of pRL-TK was also cotrans- were also increased following overexpression of each transcription factor fected as an internal control for transfection efficiency in all sam- (filled bars). The results represent the mean Ϯ SD of four independent ples. Compared with transfected samples with empty vector, the experiments. level of relative promoter activity was increased in the samples in which pCMV-CREB or pORF9-Sp1 had been cotransfected (Fig. 3B). We also quantified the PP2Ac␣ mRNA expression levels of factors up-regulated the expression of PP2Ac␣ (Fig. 3C). Taken primary T cells transfected with CREB or Sp1 encoding plasmids together, CREB and Sp1 bind to the PP2Ac␣ promoter and en- using real-time RT-PCR. Overexpression of these transcription hance its activity. The Journal of Immunology 1505

Methylation of the PP2Ac␣ promoter affects CREB binding to the CRE motif and suppresses its activity The regulation of gene expression is a complex process that is achieved through the action of selective transcription factors, as well as via an epigenetic regulatory mechanism, including DNA methylation and modification. Methylation of dC bases in the CpG dinucleotide promotes a repressive chromatin struc- ture inaccessible to transcription factors, suppressing gene ex- pression. Having detected the presence of concentrated CpG islands with the core PP2Ac␣ promoter, we conducted experi- ments to determine the effect of DNA methylation on the reg- ulation of its activity (Fig. 1B). The CRE motif has one CpG dinucleotide at the center. We constructed an oligonucleotide in which the dC base at Ϫ238 was converted to deoxymethylcytosine (dmC) with the complementary antisense oligonucleotides, which was also methylated at the cor- responding dC as the sense strand (Table I). Unmethylated or methylated pairs of the complementary oligonucleotides were an- nealed and labeled with 32P. Unlabeled double-strand oligonucle- otides were used as competitors. Supershift assays were performed at the same time to prove the specificity of the bound protein. Comparison of the bands present within lanes 1 and 5 of Fig. 4A demonstrated that methylation at Ϫ238 in the CRE motif inhibited protein binding. Usage of the methylated probe in competitive as- says failed to inhibit the interaction between nuclear protein and labeled unmethylated probe (lanes 2 and 3). We synthesized an oligonucleotide in which the Ϫ230 and Ϫ226 dC bases within the Sp1 binding site were converted to dmC to determine the effect of methylation on Sp1 binding. We did not detect any difference in Sp1 binding to methylated or unmethylated oligonucleotides (Fig. 4A, lanes 9 and 13). Both unmethylated and methylated competitors could interrupt the protein binding to the labeled probe. These results revealed that the methylation of CRE motif inhibited the interaction to CREB directly, whereas methyl- ation of the Sp1 site did not affect protein binding. Furthermore, the pGL-PP2Ac␣ promoter construct (⌬-468) and pGL3-basic vector were methylated using M. SssI and its substrate SAM to determine the effect of DNA methylation on the activity of the promoter. As shown in Fig. 4B, the promoter activity dis- played by the methylated construct was significantly suppressed compared with the mock-methylated construct. This result, how- ever, does not completely exclude the possibility that the used methylation process affects other areas of the construct.

DNA methylation inhibitors induced increased pCREB binding to hypomethylated CRE motif in the promoter and higher expression of PP2Ac␣ Next, we induced DNA hypomethylation in primary T cells using well-known DNA methylation inhibitor 5-azaC to determine the biological significance of our findings. Because DNMT inhibitor FIGURE 4. Effect of DNA methylation on the PP2Ac␣ promoter. A, acts during the S phase of cell division and alters the methylation DNA methylation within specific transcription factor binding sites inter- status in daughter cells, we treated human T cells with IL-2 before ␣ fered with the binding of CREB but not of Sp1 to the PP2Ac promoter. treatment with 5-azaC. EMSA analysis was performed to investigate the influence of the ability of At first, we determined the effect within the CREB binding site each transcription factor to bind to the promoter after methylation. Un- ␣ methylated (U1 and U2, similar to W1 and W2 in Fig. 2) and methylated of the PP2Ac promoter following treatment of cells with the probes (Me1 and Me2), which have only one or two bases changed from DNMT inhibitor. DNA was purified from T cells treated with or dC to dmC within each binding motif, were labeled with 32P after anneal- ing. The complete sequences of all oligonucleotides are shown in Table I. For competition EMSA, unlabeled probes were preincubated before the addition of labeled probe. Supershift assays were also conducted at the or without M. SssI and its substrate SAM. The normalized luciferase same time using CREB or Sp1 Ab. The specific transcription factor binding activity for the mock plasmid (unmethylated construct) was set as 100% band is shown by an asterisk and the supershifted band is marked with an (Ⅺ). The promoter activity decreased significantly following DNA arrow. B, Methylation suppresses PP2Ac␣ promoter activity. The PP2Ac␣- methylation. The results represent the mean Ϯ SD of three independent luciferase fusion construct (⌬-468) and its empty vector were treated with experiments. 1506 METHYLATION DETERMINES PP2Ac␣ PROMOTER ACTIVITY

without 10 ␮M of 5-azaC for 48 h and incubated with the meth- ylation-sensitive restriction enzyme AatII, which recognizes only unmethylated CRE motifs. The product was subjected to PCR us- ing primers as described in Materials and Methods. The presence of dmC in the CRE motif prevents digestion by AatII, and a strong band can be detected using PCR. As shown in Fig. 5A, treatment of T cells with 5-azaC reduced the amount of methylated DNA within the CRE motif of the PP2Ac␣ promoter as the intensity of the PCR bands was decreased. In contrast, the intensity of the PCR products of an area of the promoter that does not define AatII- sensitive motifs, referred to as control band, did not change. Subsequently, we investigated the effect of 5-azaC on pCREB binding to the PP2Ac␣ promoter. ChIP assays revealed that pCREB bound to PP2Ac␣ promoter more intensely when T cells had been treated with 5-azaC (Fig. 5B). Sp1 binding was not af- fected by 5-azaC treatment (data not shown). Finally, PP2Ac␣ transcripts were quantified by real-time RT-PCR after 5-azaC treatment for 48 h. The mRNA expression levels of PP2Ac␣ were increased in a dose-dependent manner (Fig. 5C). These results indicate that the binding of CREB/pCREB to hy- pomethylated CRE motif in the PP2Ac␣ promoter plays an im- portant role in the regulation of its promoter activity. Sp1 may also play an important role in the regulation of the PP2Ac␣ promoter activity, but its effect does not depend on the methylation status of the promoter.

Discussion In this paper we present first evidence on the transcriptional reg- ulation of PP2〈c␣. A core area around the Ϫ240 site that defines both CRE and Sp1 binding sites is sufficient for the complete pro- moter activity. More importantly, whereas methylation excludes the binding of CREB to the CRE site, it does not affect the binding of Sp1 to its cis site. As methylation of some gene promoters that code for proteins involved in human and murine SLE (21, 24, 28, 29, 31) has been proposed to be of pathogenic importance, our data provide further evidence on its role in the expression of PP2Ac, which is involved in the control of the expression of IL-2 (12). PP2A is a highly abundant and ubiquitously expressed serine/ threonine protein phosphatase in eukaryotic cells with various im- portant roles in embryonic development, cell cycle progression, differentiation, oncogenic transformation, and signal transduction (1–3). In general, the expression and activity of PP2A are tightly controlled by associating with regulatory subunits, posttransla- tional modification, or through the interaction with cellular pro- FIGURE 5. Treatment of T cells with a DNA methylation inhibitor in- teins (32, 33). Abnormalities in the expression and function of duced increased pCREB binding to hypomethylated CRE motif in the pro- PP2A have been implicated in several human diseases such as ␣ moter and higher expression of PP2Ac . A, Normal human T cells prein- cancer, Alzheimer disease, and SLE (8–12, 32). cubated with IL-2 were treated with or without 10 ␮M of 5-azaC, a DNA Mammalian cDNA clones encoding two different subunits, methylation inhibitor, for 48 h, and DNA was purified to determine its ␣ ␤ ␣ effect on the CREB binding site of the PP2Ac␣ promoter. We used the termed PP2Ac and PP2Ac , have been identified. PP2Ac and ␤ methylation-sensitive restriction enzyme AatII, which recognizes only un- PP2Ac differ only by 8 amino acids, with all being found within methylated CRE motifs, and then PCR was conducted using two sets of primers to distinguish unmethylated or methylated status within this re- gion. The band can be detected only when dmC remains in CRE motif anti-pCREB Ab and its control Ab precipitates were subjected to PCR because it cannot be digested by AatII. The control bands were generated using PP2Ac␣ promoter-specific primers and visualized on a 1% agarose by another set of primers defining an area of the PP2Ac␣ promoter that did gel. The corresponding nonimmunoprecipitated DNA (input DNA) was not contain any AatII-sensitive motifs. Each OD value of the bands was also analyzed using the same method. Three independent experiments were measured by densitometry. The ratio of the methylation-sensitive product performed, and the results represent the mean Ϯ SD normalized to the to the control was calculated for the semiquantification of DNA methyl- input DNA. C, The expression of PP2Ac␣ mRNA was calculated follow- ation status within CRE of the promoter, and the relative ratio of the sample ing inhibition of DNA methylation in human normal T cells. RNA from T from untreated T cells was set as 1.0. 5-azaC reduced the amount of meth- cells treated with or without 5-azaC for 48 h at the indicated concentration ylated DNA within CRE in PP2Ac␣ promoter. The results represent the was purified, and PP2Ac␣ transcripts were measured using real-time RT- mean Ϯ SD of four independent experiments. B, DNA binding of pCREB PCR in untreated cells (open bar) and treated cells (filled bars) (normalized to the PP2Ac␣ promoter was examined using ChIP assays. T cells were against GAPDH). The results represent the mean Ϯ SD of six independent treated with or without 10 ␮M of 5-azaC for 24 or 48 h and harvested to experiments and were normalized against the untreated control, which was perform ChIP assays. Cells were fixed with formalin and sonicated. The set as 1.0. The Journal of Immunology 1507 the first 30 amino acids encoded by the first exon (6). The specific Furthermore, additional studies may be required to determine function of these two subunits is still unknown, but PP2Ac␣ is whether similar methylation-sensitive regulatory processes affect assumed to the predominant type because of the embryonic lethal- the expression of other components of the PP2A holoenzyme. It ity of PP2AC␣Ϫ/Ϫ knockout mice and because PP2Ac␣ is ex- has been reported that hypomethylation of the regulatory sub- pressed at higher levels than PP2Ac␤ (32–35). unit B (PR55, ␤) induced its expression and was found to be Having established that PP2Ac mRNA is elevated in T cells involved in the expression of estrogen receptors in breast cancer from patients with SLE, we sought to determine how the activity cell lines (44, 45). of the promoter is regulated. After cloning the proximal area, we Several studies have suggested that DNA hypomethylation may conducted experiments aimed at the functional analysis of the account for several T cell abnormalities in patients with SLE and PP2Ac␣ promoter. We identified an area around the Ϫ240 site to to be involved in the pathogenesis of the disease (21, 22, 24, 25). be the essential region of this promoter. It included a complete UV light and certain drugs known to be associated with lupus-like CRE motif, flanked by three GC-rich areas. Activated CREB and symptoms (drug-induced lupus), such as procainamide and hydral- Sp1 could bind to the PP2Ac␣ promoter and significantly up-reg- azine, inhibit DNA methylation in a manner similar to that of ulate its activity (Fig. 3B). The fact that the sequence around these 5-azaC. It has been shown that the levels of DNMT-1, which is binding sites preceding the transcription start site is completely responsible for the methylation for newly replicated daughter conserved in the different species also supports our claim that this DNA strands during mitosis, were decreased in SLE T cells com- region is important in the regulation of the PP2Ac␣ expression pared with normal T cells (26, 46). This abnormality may account (36). In general, TATA-less promoters with CRE elements tend to for the decreased methylation of promoter regions of LFA-1 and show less induction following stimulation with forskolin, a CD70 and the increased expression of these genes in SLE patients pCREB , compared with promoters which contain a TATA (27, 28). Also, high levels of hypomethlated CpGs are found in the box (37). However, several reports have revealed that forskolin circulation of SLE patients, and they have been implicated in the can induce stronger promoter activity or higher expression of induction of autoreactive T and B cells in these patients (25, 46). PP2Ac␣, suggesting that CREB is important in the expression of Taken together, our finding that CREB could bind only to hypom- PP2Ac␣ (31, 36). A previous report by Heim and colleagues also ethylated CRE motifs within the PP2Ac␣ core promoter identifies demonstrated that a CREB-specific small interfering RNA knock- a mechanism whereby PP2Ac is expressed in high levels in SLE T down prevented the induction of PP2Ac by hepatitis virus C pro- cells. tein, and this finding is in perfect agreement with our findings In conclusion, we have demonstrated that the activity of the because it postulates that CREB is required for the up-regulation of PP2Ac␣ promoter is controlled by the binding of pCREB and Sp1 PP2Ac (31). Note that a complete CRE motif is defined by the around a CRE motif flanked by GC boxes, and we have shown that PP2Ac␣ promoter and not by the PP2Ac␤ promoter. This, among pCREB can bind only if the motif is demethylated. Our findings other factors, could explain the differential expression of the two provide novel information on the regulation of the PP2Ac pro- isoforms of PP2Ac. moter activity. Given that hypomethylation has been claimed to be The fact that the CRE site is flanked by CpG islands strongly involved in carcinogenesis (37, 47) and the pathogenesis of SLE suggested to us that epigenetic mechanisms are involved in the (21–25), our studies bring PP2Ac to the forefront of effector regulation of the expression of the PP2Ac␣ promoter (38). Meth- molecules. ylation of DNA represents one of the major epigenetic mecha- nisms involved in the regulation of gene expression. It is known to Disclosures stabilize chromatin in an inactive configuration and thus inhibits The authors have no financial conflicts of interest. gene transcription. In mammalian cells, the term DNA methylation usually refers to the postsynthetic methylation of dC residues at the References 5Ј position to form dmC. Nearly all dmC residues are found in the 1. Virshup, D. M. 2000. Protein phosphatase 2A: a panoply of enzymes. Curr. Opin. 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