Inhibition of Suppressive T Cell Factor 1 (TCF-1) Isoforms in Naive CD4+ T Cells Is Mediated by...

INHIBITION OF SUPPRESSIVE T CELL FACTOR 1 (TCF-1)ISOFORMS IN NAÏVE CD4+ T CELLS IS MEDIATED BY IL-4/STAT6 SIGNALING1

Elisabeth Maier*,2, Daniel Hebenstreit†, Gernot Posselt*, Peter Hammerl*, Albert Duschl*,and Jutta Horejs-Hoeck**Department of Molecular Biology, University of Salzburg, Salzburg, Austria†Department of Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, UK

AbstractThe Wnt pathway transcription factor T cell factor 1 (TCF-1) plays essential roles in the control ofseveral developmental processes, including T cell development in the thymus. Althoughpreviously regarded as being required only during early T cell development, recent studiesdemonstrate an important role for TCF-1 in T helper (Th) 2 cell polarization. TCF-1 was shown toactivate expression of the Th2 transcription factor GATA-binding protein 3 (GATA3) and thus topromote the development of Interleukin (IL)-4-producing Th2 cells independent of signaltranducer and activator of transcription 6 (STAT6) signaling. In this study we show that TCF-1 isdown-regulated in human naïve CD4+ T cells cultured under Th2-polarizing conditions. Thedown-regulation is largely due to the polarizing cytokine IL-4, as IL-4 alone is sufficient tosubstantially inhibit TCF-1 expression. The IL-4-induced suppression of TCF-1 is mediated bySTAT 6, as shown by electrophoretic mobility shift assays, chromatin immuno-precipitation andSTAT6 knock-down experiments. Moreover, we found that IL-4/STAT6 predominantly inhibitsthe shorter, dominant-negative TCF-1 isoforms, which were reported to inhibit IL-4 transcription.Thus, this study provides a model for an IL-4/STAT6-dependent fine-tuning mechanism ofTCF-1-driven T helper cell polarization.

The transcription factors T cell factor 1 (TCF-1)3 and lymphoid enhancer-binding factor 1(LEF-1) are downstream effectors of the canonical Wnt pathway, also known as the Wnt-β-catenin signaling pathway (1-2). TCF-1 and LEF-1 have a dual function in gene regulationthat is determined by their associated co-factors. In the absence of Wnt-ligand the co-factorβ-catenin is targeted for proteasomal degradation and the Wnt pathway transcription factorsTCF-1 and LEF-1 repress target gene expression by interacting with repressors that belongto the groucho-related family (3-4). Transient activation of the Wnt cascade allows β-cateninto escape proteasomal degradation. Stabilized β-catenin then translocates to the nucleus

1This work was supported by the University of Salzburg priority program ‘BioScience and Health’ and the Austrian ‘Fond zurFörderung der Wissenschaftlichen Forschung’ Grant P18409.Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.Correspondence should be addressed to: Jutta Horejs-Hoeck, Department of Molecular Biology, Paris Lodron University of Salzburg(PLUS), Hellbrunner Str. 34, A-5020 Salzburg, Phone: +43-662-8044/5736, Fax: +43-662-8044/5751, [email protected]. is a recipient of a DOC-fFORTE-fellowship of the Austrian Academy of Sciences at the Department of Molecular Biology,University of SalzburgDisclosuresThe Authors have no financial conflicts of interest.

UKPMC Funders GroupAuthor ManuscriptJ Biol Chem. Author manuscript; available in PMC 2011 January 11.

Published in final edited form as:J Biol Chem. 2011 January 14; 286(2): 919–928. doi:10.1074/jbc.M110.144949.

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where it displaces the co-repressors from LEF-1 and TCF-1, resulting in the formation of atranscription-complex which activates the expression of Wnt target genes (4-7).

Wnt signaling is pivotal to numerous aspects of embryonic development and remainsessential in some self-renewing tissues throughout life. In hematopoiesis the canonical Wntsignaling pathway controls the maintenance and self-renewal of hematopoietic stem cells(3,8-9), regulates B cell development in fetal liver and neonatal bone marrow (10-11) and iscritically involved in early T cell development in the thymus (12-15). Moreover,components of the Wnt pathway were shown to be expressed in mature, activated T cells,including CD4+ T helper (Th) cells (16-18). Several recent studies indicate that LEF-1 andTCF-1 are central in Th2 cell development. LEF-1, introduced into developing Th2 cells,was shown to suppress the transcription of Th2-specific cytokines by interacting withGATA-binding-protein 3 (GATA3), a key factor for Th2 differentiation. The observedinhibition of cytokines is thought to be a consequence of LEF-1/GATA3 interactions,resulting in reduced binding of GATA3 to the cytokine gene promoters (19). Recently, weidentified a high-affinity LEF-1-binding site in the negative regulatory element of the IL4promoter and showed that LEF-1 binds to this element with significantly higher affinity thandoes TCF-1. Silencing of LEF-1 results in an increase of IL-4-mRNA expression induced inresponse to stimulation by phorbol 12-myristate 13-acetate (PMA)/ionomycin, indicatingthat LEF-1 contributes to the negative regulation of the IL4 gene through transcriptionalrepression of the IL4 locus (18). Although these studies suggest that LEF-1 is involved inthe negative regulation of Th2-specific cytokine production, a very recent studydemonstrates that TCF-1 and its co-factor β-catenin promote the differentiation of TCR-activated CD4+ T cells into Th2 cells by inducing early GATA3 expression (20). Thisindicates that LEF-1 and TCF-1 contribute to Th2 cell development in rather different ways.In the present study we show that the main Th2 cytokine IL-4 is a potent suppressor of

3The abbreviations used are:

ChIP chromatin immuno-precipitation

CD62L L-selectin

EMSA electrophoretic mobility shift assay

GATA3 GATA-binding protein 3

H3 histone 3

IL interleukin

LEF-1 lymphoid enhancer-binding factor-1

PBMC peripheral blood mononuclear cell

PBS phosphate-buffered saline

PMA phorbol 12-myristate 13-acetate

qRT-PCR quantitative RT-PCR

siRNA small interfering RNA

STAT signal transducer and activator of transcription

TCF-1 T cell factor 1

TCR T cell receptor

Th T helper cell

WT wild type

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J Biol Chem. Author manuscript; available in PMC 2011 January 11.

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TCF-1 in naïve human CD4+ T cells. Analyses of TCF-1 protein and mRNA levels revealedthat IL-4 signaling preferentially targets the short TCF-1 isoforms, which are constitutivetranscriptional repressors. To investigate the molecular mechanisms underlying the IL-4-mediated suppression of TCF-1, we analyzed signaling pathways downstream of the IL-4receptor and found STAT6 to be crucially involved in the down-regulation of TCF-1.

EXPERIMENTAL PROCEDURESIsolation of human peripheral blood mononuclear cells (PBMCs) and preparation of naïveand memory CD4+ T cells

All studies involving human cells were conducted in accordance with the guidelines of theWorld Medical Association’s Declaration of Helsinki. Human PBMCs were isolated frombuffy coats of healthy donors by means of Ficoll-Paque Plus® (Amersham,Buckinghamshire, UK) density gradient centrifugation and washed twice with PBS (PAA,Pasching, Austria). Naïve and memory CD4+ T cells were purified from the preparedPBMCs by using the Human Naïve CD4+ T cell isolation kit or the Human Memory CD4+ Tcell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to themanufacturer’s protocols. Cells were cultured in x-vivo 15 medium (BioWhittaker, Lonza,Cologne, Germany), supplemented with 5% heat-inactivated human serum AB(BioWhittaker, Lonza, Cologne, Germany), 2mM L-glutamine, 100U/ml penicillin and100μg/ml streptomycin (all purchased from PAA, Pasching, Austria) in plastic tissue culturedishes at 37°C in a humidified atmosphere containing 5% CO2. T cells were stimulated withplate-bound αCD3 at a coating concentration of 10μg/ml (clone OKT3, eBioscience,Vienna, Austria) and 1μg/ml soluble αCD28 (BD Pharmingen, Schwechat, Austria).Recombinant human IL-12 (25ng/ml) (Immunotools, Friesoythe, Germany) or 50ng/ml IL-4(kind gift from Novartis, Vienna) were added.

Mouse T cell cultureBALB/c mice were purchased from Charles River Laboratories (Sulzfeld, Germany),STAT6−/− mice (21) were a kind gift from Dr. A. Gessner (University of Erlangen,Germany). Mouse naïve T helper cells were isolated from splenocytes by using the mouseCD4+CD62L+ T cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) andcultured in MEM medium supplemented with 2mM L-glutamine, 100U/ml penicillin and100μg/ml streptomycin, 1mM sodium pyruvate, 2mM Hepes, 1x non-essential amino acids,20μM β-mercaptoethanol (all purchased from PAA, Pasching, Austria) and 5% heat-inactivated fetal calf serum (Gibco, Invitrogen, Lofer, Austria). Cells either remaineduntreated or were induced with 25ng/ml recombinant murine IL-12 (Peprotech, Eubio,Vienna, Austria) or 50ng/ml recombinant mouse IL-4 (Immunotools, Friesoythe, Germany)in plastic tissue culture dishes at 37°C in a humidified atmosphere containing 5% CO2.

SDS-PAGE and Immuno-BlottingNaïve CD4+ T cells were stimulated with IL-4 for the indicated times or left untreated. Cellswere harvested by centrifugation, lysed in 2x Laemmli Sample Buffer (Bio-Rad, Vienna,Austria) and frozen at −75°C. After thawing, lysates were denaturated by 7 minutes’incubation at 95°C and afterwards centrifuged to remove the cell debris. Protein lysates wereseparated on a precast NuPAGE 12% or a 4-12% gradient gel (Invitrogen, Lofer, Austria)and blotted onto nitrocellulose membranes in a Trans-blot semi-dry blotting chamber (bothBio-Rad, Vienna, Austria). The membrane was blocked by incubation in Tris-bufferedsaline containing 0.1% Tween 20 and 5% non-fat dry milk for 1 hour. The primaryantibodies and HRP-linked secondary antibodies were purchased from Cell SignalingTechnology (Danvers, MA) and used according to the manufacturer’s instructions.Detection was carried out using Supersignal enhanced chemiluminescence substrate (West

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Pico, Pierce, Rockford, IL). For stripping, the membrane was incubated in 50mM Tris-HCl,pH 6.8, 2% SDS, 10mM β-mercaptoethanol for 20 minutes at 50°C.

RNA isolation and quantitative real-time PCRTotal RNA from cells was isolated using the TRIzol reagent (Invitrogen, Lofer, Austria) andreverse-transcribed with RevertAid H Minus M-MulV reverse transcriptase (MBIFermentas, St. Leon-Roth, Germany) according to the manufacturer’s instructions.Quantitative real-time PCR was carried out on a Rotorgene 3000 (Corbett Research) usingthe iQ SYBR Green Supermix (Bio-Rad, Vienna, Austria) and the primers listed below. Thetranscript for the large ribosomal protein P0 (RPL P0) was used as reference. The specificityof the PCRs was checked by recording a melting curve for the PCR products. RelativemRNA expression levels were calculated using the formula x=2−ΔCt, where Ct representsthe threshold cycle of a given gene and ΔCt signifies the difference between the Ct values ofthe gene in question and the Ct value of the reference gene RPL P0. Induction ratios werecalculated using the formula x=2−ΔΔCt. ΔΔCt represents the difference between the ΔCtvalues of induced and uninduced samples.

Primer sequences are as follows: human TCF-1 sense 5′-CGGGACAGAGGACCATTACAACTAGA TCAAGGAG-3′ and anti-sense 5′-CCACCTGCCTCGGCCTGCCAAAGT-3′, human LEF-1 sense 5′-CGACGCCAAAGGAACACTGACATC-3′ and anti-sense 5′-GCACGCAGATATGGGGGGAGAAA-3′, human β-catenin sense 5′-CCTTTAGCTGTATTGTCTGAACTTGCAT TGTGATTG-3′ and anti-sense 5′-GCTAAATTCCATCTTGTGATCCATTCTT GTGCATTC-3′, mouse TCF-1 sense 5′-AGCCCCCCCACAGCACCCTCCAGAATC-3′ and anti-sense 5′-CCAGGTTCAGGGAGTTGTGCAGCC-3′, mouse LEF-1 sense 5′-AGCCAAGGCAGCGACCCCAGG-3′ and anti-sense 5′-CGGCGCTTGCAGTAGACGACAGA-3′, human RPL P0 sense 5′-GGCACCATTGAAATCCTGAGTGATGTG-3′ and anti-sense 5′-TTGCGGACACCCTCCAGGAAG-3′, mouse RPL P0 sense 5′-TGCACTCTCGCTTTCTGGAGGGTG-3′ and anti-sense 5′-AATGCAGATGGATCAGCCAGGAAGG-3′ , human TCF-1 p30 sense 5′-GGTGACTGACTAATCCGCCGCCTT-3′ and anti-sense 5′-AAATGTTCGTAGAGAGAGAGTTGGGG GAC-3′, human CCL17 sense 5′-CCAGGGATGCCATCGTTTTTGTAACTG TGC-3′ and anti-sense 5′-CTCACTGTGGCTCTTCTTCGTCCCTGGA A-3′.

STAT6 siRNA knockdownNaïve human CD4+ T cells were cultured at a density 10 of 2 6 cells/ml in x-vivo 15medium supplemented as described above. Prior to transfection cells were activated byovernight incubation in the presence of 10ng/ml PMA (Sigma, Vienna, Austria) and 1μMionomycin (Calbiochem, Beeston, Nottinghamshire, UK). Cells were transfected with eitherStealth RNAi siRNA (Invitrogen, Lofer Austria) targeting STAT6 (sense 5′-CCAAAGCCACUAUCCUGUGGGACAA-3′ , anti-sense 5′-UUGUCCCACAGGAUAGUGGCUUUGG-3′) or control siRNA having no target mRNA(sense 5′-CCCAGAATCCGATTACTACTGAACT-3′, anti-sense 5′-AGUUCAGUAGUAAUCGGAUUCUGGG-3′) by using the Amaxa Nucleofector® systemfor primary human T cells (Lonza, Cologne, Germany). Transfection protocols wereperformed according to the manufacturer’s instructions. Best transfection efficiency wasachieved using 510 6 cells and 100pmol siRNA per cuvette and transfection program T-23.Four hours post-transfection the medium was changed to medium containing 100U/ml IL-2

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(Immunotools, Friesoythe, Germany). 72 hours post-transfection, cells were incubated for24 hours in the presence or absence of IL-4.

Preparation of Nuclear extracts and Electrophoretic Mobility Shift Assays (EMSA)Nuclear extracts from IL-4-induced or non-induced CD4+ T cells were prepared accordingto the method of Andrews and Faller (22). EMSAs were carried out as described previously(23-24). Double-stranded oligonucleotide probes corresponding to the sequences −2817 to−2774, −428 to −389, +1673 to +1701, +7108 to +7137 and +20993 to +21022 relative tothe translational start site located in exon 1a of the human TCF-1 gene (25) were generatedby annealing synthetic sense and anti-sense oligonucleotides for 2 hours during which thetemperature was gradually reduced from 95°C to 25°C, followed by radio-active 3′-end-labeling with [32P]dCTP (Hartmann Analytic, Braunschweig, Germany) using Klenowfragment (Fermentas, St. Leon-Roth, Germany). For oligonucleotide competition assays,non-labeled oligonucleotide was added in 50-molar excess to the binding reaction 30minutes prior to the radiolabeled probe. Super-shifting of bands was achieved by adding200ng/μl antibody (αSTAT6 M20x, αSTAT5 N20x, Santa Cruz Biotechnology, Heidelberg,Germany) to the binding reaction. Sequences of oligonucleotides are as follows (STATconsensus nucleotides underlined): −2817/−2774 sense 5′-GGGCCGCACCAACTTCTGGGGAACAA ATCAGAGCTCTTGTT-3′ and anti-sense 5′-GGGAACAAGAGCTCTGATTTGTTCCCCAGAAGTTGGTGCGG-3′, −428/−389 sense5′-GTTAGAGTCTCTGCGTTTCGGGAGAAGGGTGATCATT-3′ and anti-sense 5′-GGGAATGATCACCCTTCTCCCGAAACG CAGAGACTCT-3′, +1673/+1701 sense 5′-GTGGGTTCTTAGGAAACTGGCTTGGT-3′ and anti-sense 5′-GAGACCAAGCCAGTTTCCTAAGAACC-3′, +7108/+7137 sense 5′-TGAATTACCTTTCTTTCCTTGGAAGG-3′ and anti-sense 5′-GCAGCCTTCCAAGGAAAGAAAGGTAA T-3′, +20993/+21022 5′-AGAAACTCTTTCAAGGAAGTGGGAGA AA-3′ and anti-sense 5′-TGGTTTCTCCCACTTCCTTGAAAGAGTT-3′. In mutated oligonucleotides thepalindromic halfsite TTC was exchanged to TAT.

Chromatin Immuno-Precipitation (ChIP)Naïve human CD4+ T cells were isolated as described above and cultured in RPMI 1640medium supplemented with 10% heat-inactivated fetal calf serum, 2mM L-glutamine, 100U/ml penicillin and 100μg/ml streptomycin (all purchased from PAA, Pasching, Austria) at adensity of 106 cells/ml. T cells were stimulated with 50ng/ml IL-4 (kind gift from Novartis,Vienna) for 4 hours. ChIP was performed using the LowCell# ChIP kit (Diagenode, Liège,Belgium) according to the manufacturer’s instructions, except for the following alterations:cells were fixed for 10 minutes with 1% formaldehyde at room temperature and thesonication time was prolonged to 45 minutes. Precipitation reactions contained chromatinfrom 2.5·104 cells and 3μg of antibodies (αSTAT6 M20x, Santa Cruz Biotechnology,Heidelberg, Germany or rabbit negative control IgG, Diagenode, Liège, Belgium).Precipitation of STAT6 binding motifs was analyzed by means of qRT-PCR using iQ SYBRGreen Supermix (Bio-Rad, Vienna, Austria) and the primers listed below. −2804 sense 5′-GCCGCACCAACTTCTGGG-3′ and anti-sense 5′-GATGGCTTAGGAGGGTTTGGAA-3′,−412 sense 5′-TATCTGGTTGTAGGTGAGTAAGCGGG-3′ and anti-sense 5′-AAACACTCAAAAGTTCTACTGGCCACGG C-3′, +1678 sense 5′-CCAAAGAAGGACCCTGAACACC -3′ and anti-sense 5′-TGAGACCAAGCCAGTTTCCTAAG-3′, +7122 sense 5′-TAGGTTTGTGGTGGGCAGTG-3′ and anti-sense 5′-CCAATACTACGCAGCCTTCCA-3′

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Analysis of histone modifications along the human TCF1 locusChIP-Sequencing data of Barski et al. (26) on histone 3 single- and triple-methylation(H3K4me1 and H3K4me3, respectively) in human CD4+ cell were used. UCSC tracks forH3K4me1 and H3K4me3 as provided by the websitehttp://dir.nhlbi.nih.gov/papers/lmi/epigenomes/hgtcell.aspx were uploaded to UCSC genomebrowser (http://genome.ucsc.edu/) and aligned with the genomic sequence of human TCF1.

RESULTSIL-4 enhances suppression of LEF-1 and TCF-1 expression in TCR-stimulated naïve CD4+ Tcells

Upon antigen challenge, naïve CD4+ T cells differentiate into distinct subsets of Th cells.The cytokine milieu in which TCR stimulation takes place is essential to the differentiationof CD4+cells and determination of Th cell functions. In a recent study, we investigatedLEF-1 and TCF-1 expression in a total pool of CD3+ T cells. We found down-regulation ofLEF-1 and TCF-1 upon TCR stimulation, which was even more pronounced upon additionalIL-4 treatment (18). Because IL-4 is the main cytokine driving the differentiation of Th2cells, we analyzed LEF-1 and TCF-1 expression in the course of Th1/Th2 differentiation.Naïve human CD4+ T cells were isolated and cultured under Th1 (αCD3/αCD28, IL-12) orTh2 (αCD3/αCD28, IL-4) polarizing conditions. After 48h, mRNA was isolated and LEF-1and TCF-1 mRNA expression was determined by qRT-PCR analysis. In line with ourprevious data, TCF-1 and LEF-1 were down-regulated upon TCR activation. Similar to theobservations from Yu et al. (20), who demonstrated reduced TCF-1 expression in Th1-skewed mouse CD4+ T cells compared to undifferentiated cells, we found that addition ofthe Th1-polarizing cytokine IL-12 resulted in decreased LEF-1 and TCF-1 expression.Nonetheless, the strongest effect was observed when naïve T cells were cultured under Th2-polarizing conditions (Fig. 1).

IL-4-induced suppression of TCF-1 and LEF-1 is restricted to naïve CD4+ T cellsTo study the effects of Th1- and Th2-polarizing cytokines in the absence of TCR ligation,naïve and memory CD4+ T cells were isolated separately and treated with IL-4 or IL-12 fordefined time periods. LEF-1 and TCF-1 mRNA expression was detected by qRT-PCR.Whereas TCF-1 levels increased in uninduced and IL-12-treated cells over time, the additionof IL-4 resulted in significant down-regulation of TCF-1 mRNA. The down-regulation wasrapid (2h) and persisted for up to 48h. Similar, but time-delayed, effects were observed forLEF-1 (Fig. 2, left panel). In contrast to the results for naïve CD4+ T cells, neither of theculturing conditions induced changes in the mRNA expression of TCF-1 and LEF-1 inmemory CD4+ T cells (Fig. 2, right panel).

Low IL-4 concentrations are sufficient to suppress LEF-1 and TCF-1 expressionTo analyze the IL-4-mediated effects in more detail, a concentration kinetics experiment wascarried out. mRNA expression of LEF-1, TCF-1, β-catenin and the IL-4-inducible geneCCL17 (27) was analyzed by means of qRT-PCR. While β-catenin remained stable, LEF-1and, to a greater extent, TCF-1 were down-regulated by IL-4 in a concentration-dependentway. Interestingly, very low concentrations of IL-4 (≤50pg/ml) produced a significantsuppression on both LEF-1 and TCF-1, and concentrations ≤500pg/ml had a full inhibitoryeffect. In contrast, up-regulation of CCL17 was not observed at IL-4 concentrations below500pg/ml and its expression increased with rising concentrations of IL-4 up to 50ng/ml (Fig.3). As these results are in line with earlier observations on the expression of IL-4-induciblegenes (24,27), we conclude that TCF-1 and LEF-1 are far more sensitive to IL-4 than areother genes known to be activated by IL-4.

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http://dir.nhlbi.nih.gov/papers/lmi/epigenomes/hgtcell.aspx

http://genome.ucsc.edu/

Reduction of TCF-1 expression is restricted to the short, inhibitory isoformsSo far, our data show that IL-4 alone exhibits a potent inhibitory effect on TCF-1expression. For detailed analyses of TCF-1 expression and data interpretation it has to beconsidered that at least 8 isoforms of TCF-1 have been described. These result fromalternative promoter usage, the presence of alternative exons and the usage of three differentC-terminal splice-acceptor sites (25,28) and can be roughly divided into two groups. Thelong isoforms (42-60 kDa) contain a β-catenin interaction domain which is essential fortranscriptional activation (25,29), a Groucho protein-binding domain known to mediatetranscriptional repression (4) and a C-terminal DNA-binding domain. In contrast, the shortTCF-1 isoforms (25-40 kDa) lack the β-catenin interaction motif but contain Groucho- andDNA-binding sites (Fig. 4A). Therefore, the shorter TCF-1 isoforms act as persistenttranscriptional repressors. Transcripts of short and long TCF-1 isoforms can bedistinguished by the presence of exon 1, which is exclusively found in the short isoforms. Todetect the short TCF-1 isoforms by means of qRT-PCR, a forward primer binding to exon 1was designed. Whereas expression of the short isoforms was dramatically inhibited by IL-4(reduction of 92%), the effects observed on total TCF-1 expression were less pronounced,indicating that IL-4 preferentially down-regulates the expression of short TCF-1 isoforms(Fig. 4B).

Next we investigated TCF-1 protein expression by Western blotting. Analysis of TCF-1protein revealed no inhibition of the long TCF-1 isoforms upon IL-4 stimulation. In contrast,shorter isoforms were significantly down-regulated after 24h to 48h of IL-4 treatment. Inline with our mRNA expression results, these data indicate a specific effect of IL-4 on theexpression of short TCF-1 isoforms. TCF-1 isoform expression in a Jurkat cell lysate wasdetected as a control. To verify functional IL-4 signaling and equal loading we showphosphorylation of STAT6 upon IL-4 stimulation and total STAT6 protein expression (Fig.4C).

STAT6 binds to specific binding motifs within the human TCF1 locusEngagement of the IL-4 receptor results in the activation of Janus kinases (Jaks) whichsubsequently phosphorylate tyrosine residues on IL-4Rα, thus providing docking sites forSTAT6. Once bound to the receptor, STAT6 monomers become phosphorylated, dimerizeand translocate to the nucleus, where the active STAT6 dimers bind to cognate responsiveDNA elements and initiate IL-4-mediated transcription (30). To date, more than 35 genesregulated by STAT6 have been described (30). Although the vast majority of these genes arepositively regulated, IL-4-mediated STAT6 activation was also described to havesuppressive effects on gene expression (31-32). Analysis of the TCF-1 locus revealed thepresence of four putative STAT6-binding sites characterized by the sequence TTC(N)4GAA(Fig. 5A). These motifs are located at positions −2804 (site 1), −412 (site 2), +1678 (site 3)and +7122 (site 4) relative to the translational start site in exon 1a described by van deWetering and colleagues (25). Besides the putative STAT6-binding sites an additionalSTAT consensus motif was identified at position +21001 (site 5). This site is not a classicalSTAT6 consensus binding site as it carries only three nucleotides dividing the palindromichalf-sites TTC/GAA and therefore rather resembles the consensus motif for STAT1, STAT3or STAT5. Since it was reported that IL-4 can also activate STAT5 in lymphocytes (33-34),we included this site in our study. The interaction of STATs with putative STAT-bindingsequences within the TCF-1 locus was assessed by EMSAs. Nuclear extracts prepared fromuntreated and IL-4-treated naïve CD4+ T cells were incubated with double-strandedoligonucleotide probes harboring the sequences −2804/−2815 (site 1), −412/−423 (site 2),+1678/1687 (site 3), +7122/+7131 (site 4) and +21001/21009 (site 5), and nucleoproteincomplexes were resolved in native polyacrylamide gels. Incubation of nuclear extracts fromuntreated or IL-4-induced cells with any of the four oligonucleotides harboring a classical

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STAT6-binding sequence resulted in the formation of an IL-4-specific nucleoproteincomplex (Fig. 5B). In contrast, no IL-4-induced complex formation was observed using theTTC(N)3GAA motif at position +21001 (site 5). Mutation of the STAT6 consensus siteTTC(N)4GAA to TAT(N)4GAA was shown to abrogate binding of STAT6 in previousstudies (24,35-37). By introducing this mutation into the STAT6 consensus motifs withinthe oligonucleotides from the human TCF-1 locus, IL-4-induced complex formation wasinhibited (Fig. 5C). For competition assays, 50-fold molar excess of unlabeled wild-typeoligonucleotide was added to the binding reaction. This resulted in the loss of the IL-4-induced formation of the radiolabeled nucleoprotein-complexes. In contrast, addition ofmutated oligonucleotide had no effect. Addition of anti-STAT6 antibodies (M20) prior tothe addition of radiolabeled probes specifically reduced the formation of the IL-4-inducednucleoprotein complexes and partly led to the formation of a super-shifted complex (sites 1,3 and 4). An antibody directed against STAT5 (N20), which was used as a control antibody,did not affect the IL-4-induced complex (Fig. 5C). These data suggest that IL-4 stimulatesthe interaction of STAT6 and specific DNA motifs within the human TCF-1 locus.

For in vivo binding of transcription factors to genomic DNA the chromatin has to be in anopen conformation. Methylation of histone 3 on lysine residue 4 (H3K4) is a known markerfor chromatin that is amenable to transcription factor binding (26). Wei et al. recentlyshowed that functional STAT6-binding sites are often associated with triple-methylatedH3K4 (H3K4me3) islands (38). Barski and colleagues performed Chromatin-immunoprecipitation/sequencing-based profiling of histone modifications in human CD4+ Tcells (26). Alignment of these data with the human TCF1 locus shows the presence of tripleand single methylated H3K4 islands which are co-localized with STAT consensus motifs(Fig. 6A). To evaluate the capacity of each motif to bind STAT6 in living cells weperformed chromatin immuno-precipitation with STAT6 specific antibodies. Theenrichment of specifically precipitated DNA containing the STAT6 binding sites wasanalyzed by qRT-PCR using site-specific primer-pairs. Control immuno-precipitations withnormal rabbit IgG serum resulted in equal amplification of IL-4 and uninduced samples forall four STAT6-binding sites. In contrast, precipitation of chromatin from IL-4 induced cellswith anti-STAT6 resulted in enrichment of the STAT6-binding motifs 2, 3 and 4. For motif1, only slight differences between unstimulated and IL-4-induced cells were detected (Fig6A). These findings indicate that sites 2, 3 and 4 are possibly more relevant for IL-4mediated TCF-1 suppression than site 1. To control for appropriate IL-4 effects, TCF-1expression and STAT6 activation were analysed (Fig. 6B).

STAT6 is critically involved in IL-4-induced suppression of inhibitory TCF-1 isoformsTo further investigate the role of STAT6 in the IL-4-induced inhibition of the short TCF-1isoforms, we analyzed TCF-1 expression in STAT6-deficient, naïve human CD4+ T cells.Silencing of STAT6 was performed via transfection of naïve human CD4+ T cells withsiRNA targeting STAT6. Decreased levels of STAT6 protein, but unaltered STAT1 levels,were detected 96h post-transfection by Western blotting (Fig. 7A). Three days post-transfection, cells were incubated overnight in the presence or absence of IL-4. Real-timePCR analyses showed that stimulation with IL-4 inhibited the expression of TCF-1 –especially the short isoforms – in control cells, whereas silencing of STAT6 almostcompletely rescued the total TCF-1 as well as the short TCF-1 isoforms from IL-4-mediatedsuppression (Fig. 7B). As the putative STAT6-binding motifs within the human TCF-1 locusdescribed in Figure 5 are located in regions highly conserved between mouse and human,CD4+CD62L+ murine T cells isolated from wild-type and STAT6−/− mice were also testedfor IL-4-mediated down-regulation of TCF-1. Consistent with the data obtained from humancells, naïve T cells from Balb/c mice also showed diminished TCF-1 mRNA expressionafter IL-4 stimulation, whereas in T cells derived from STAT6−/− mice, effects on TCF-1

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expression were clearly less pronounced and occurred in a time-delayed fashion (Fig. 6C,upper panel). Similar, but less prominent, effects were observed for LEF-1 expression (Fig.6C, lower panel). These data show that STAT6 mediates the IL-4-induced down-regulationof the transcription factors LEF-1 and TCF-1, in particular the short, suppressive TCF-1isoforms, in naïve CD4+ T cells.

DISCUSSIONWhile the role of Wnt pathway-associated transcription factors in early thymocytedevelopment is well documented, the function of LEF-1 and TCF-1 at later stages of T celldevelopment remained ambiguous for a long time. We and others recently showed thatLEF-1 and TCF-1 play a crucial role in the regulation of Th2 cell development. We reportedthat LEF-1 acts as a transcriptional repressor of IL-4 by binding to a negative responseelement within the IL4 locus. Activation of cells through TCR engagement down-regulatesLEF-1 and thereby releases IL4 of its transcriptional repressor. Once expressed, IL-4enhances the inhibition of LEF-1 and thus further amplifies IL-4 expression via a positivefeedback loop (18). Although LEF-1 regulates IL-4 via a negative feedback loop, TCF-1was reported to promote Th2 differentiation by activating the Th2-specific transcriptionfactor GATA3 (20). These opposing effects of LEF-1 and TCF-1 in mature T cells are ofparticular interest because studies analyzing the functions of LEF-1 and TCF-1 during earlyT cell development revealed a strong functional redundancy between the two transcriptionfactors (39). Our data show that TCF-1 is strongly down-regulated in CD4+ T cells culturedunder Th cell-polarizing conditions (αCD3/αCD28 + polarizing cytokine). TCR-dependentsignals are a common prerequisite for Th cell differentiation. However, the cytokine milieuis the most important factor determining the fate of Th cell differentiation. When wecompared the effects of the Th1-polarizing cytokine IL-12 with the Th2-polarizing cytokineIL-4 in the absence of TCR stimulation we observed a significant suppression of TCF-1 inIL-4-treated, but not in IL-12-treated, naïve CD4+ T cells. This might be explained by theobservation that resting naïve T cells do not express the IL-12 receptor subunit β2, which isessential for IL-12 signaling and up-regulated upon T cell activation (40). Thus, IL-12 mightalso have an inhibitory effect on TCF-1 mRNA expression, but – unlike IL-4 – cannot exertit in absence of TCR stimulation.

Whereas IL-4 down-regulates TCF-1 expression, we observed increasing TCF-1 mRNAlevels in untreated and IL-12-stimulated cells within the first 6 hours of in vitro culture. Thisobservation may be due to the fact that removing T cells from their in vivo environmentleads to the loss of certain stimuli such as IL-7. Yu et al. demonstrated that IL-7 is involvedin the inhibition of TCF-1 in mature peripheral T cells (41). Since IL-7 is not produced by Tcells (42-44) loss of IL-7 signals in the course of in vitro culturing may therefore result inincreasing TCF-1 expression. When we investigated TCF-1 expression in memory CD4+

cells compared to naïve CD4+, we found that the overall TCF-1 expression in memory cellswas rather low and none of the tested cytokines showed any effect. This is in line withseveral other studies showing that TCF-1 is down-regulated upon T cell activation (16,18).Moreover, this finding indicates that TCF-1 plays a crucial role especially during early Thcell development.

STAT proteins are a family of transcription factors involved in signal transduction inducedby various cytokines. STAT6 is the main transcription factor mediating IL-4-inducedsignaling, as demonstrated by the greatly diminished functions of IL-4 in STAT6-deficientmice (21,45). However, STAT5 was shown to be activated by IL-4 in lymphocytes, too(33-34). This activation seems to be independent of tyrosine phosphorylation of IL-4Rα, butmay rather be mediated by the direct interaction of STAT5 and Janus Kinases (46). Despitethe presence of a putative STAT5-binding site identified in the TCF1 locus we did not

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observe IL-4-induced binding of STAT5 to TCF1 gene sequences. In contrast, all fouridentified STAT6 motifs seem to be capable of binding STAT6, as shown by gel shiftassays, indicating that STAT6 is the crucial factor involved in TCF-1 suppression. Thisassumption was confirmed when we performed knock-down experiments targeting STAT6in naïve human CD4+ T cells. IL-4 inhibited TCF-1 – in particular the short isoforms – incontrol cells whereas no significant effect of IL-4 was observed in STAT6-deficient cells.Similar results were obtained when we analyzed naïve T cells from STAT6−/− micecompared to WT mice.

To validate the binding capacity of each STAT6 motif identified in the human TCF1 locusChIP assays were carried out. Analyses of histone modifications along the TCF1 locusrevealed that the chromatin appears to be accessible for transcription factor binding at allfour binding sites as we found an island of H3K4me1 co-localized with site 1 and H3K4me3modifications surrounding sites 2, 3, and 4. ChIPs with STAT6 specific antibodies indicatethat sites 2, 3 and 4 seem to be more relevant for IL-4 mediated TCF-1 suppression than site1. This is in agreement with the study of Wei et al. who showed that STAT6-binding sitesand triple-methylated H3K4 (H3K4me3) islands often overlap (38). Noteworthy, binding ofSTAT6 to specific motifs within the TCF1 locus is transient. Highest occupancies of STAT6binding motifs were observed after 4 hours of IL-4 stimulation, whereas no differencesbetween IL-4 stimulated and untreated samples were detectable after 20 hours of IL-4treatment (data not shown). Thus, we propose that STAT6 is particularly involved in earlyIL-4 mediated effects on TCF-1. This assumption is substantiated by the fact that STAT6deficiency does not entirely rescue TCF-1 from IL-4 mediated suppression as shown inhuman and mouse naïve T cells.

For a long time IL-4 and the IL-4-induced STAT6 signaling pathway were regarded asprincipal factors driving Th2 polarization. Activated STAT6 induces the transcription ofGATA3, which is known to be a master regulator of Th2 differentiation. However, severalstudies showed that IL-4/STAT6 signaling can be bypassed to generate Th2 cells by otherpathways, including Notch (47-48) and IL-2/STAT5 signaling (49-50). The fact that Th2differentiation still occurs in STAT6-deficient mice (51) further indicates that STAT6-independent mechanisms exist which may be sufficient to activate GATA3 transcription. Arecent study provides clear evidence that early after the activation of CD4+ T cells, TCF-1induces GATA3 expression from GATA3-1b transcripts in an IL-4- and STAT6-independent way (20). GATA3 expression is driven by two different promoters, both ofwhich are used to transcribe GATA3 in T cells. Activation of the two distinct promotersresults in mRNA transcripts which can be distinguished by the presence of promoter-specific sequences unique to exon 1a (GATA3-1a) and exon 1b (GATA3-1b) (52). WhereasGATA3-1a expression is increased after 2-4 days of stimulation and clearly depends onSTAT6, GATA3-1b seems to be more active during the early phase of Th2 differentiationand is activated even in the absence of STAT6 (20,53). The fact that TCF-1 promotes theactivation of GATA3-1b transcripts indicates that TCF-1 and STAT6 act through differentmechanisms in the regulation of early and delayed GATA3 expression.

In the present study we demonstrate IL-4/STAT6-dependent suppression of short TCF-1isoforms in naïve CD4+ T cells which results in the exclusive expression of long TCF-1isoforms. Differential regulation of the isoforms, as shown here, is of particular interest,because long and short TCF-1 isoforms have distinct functions during Th2 cell development.Yu et al. reported that TCF-1 induces GATA3-1b transcription in T cells, resulting in a Th2phenotype characterized by enhanced IL-4 production. The same study demonstrated thatover-expression of a mutated TCF-1 form resembling the naturally occurring short TCF-1isoforms showed the opposite effect and resulted in decreased IL-4 production (20),indicating that the short isoforms act as inhibitors of TCF-1 mediated Th2 differentiation.

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Therefore, we suggest that specific down-regulation of inhibitory TCF-1 isoforms – asshown in the present study – contributes to early TCF-1-mediated Th2 differentiation.

Based on our findings we propose an IL-4/STAT6-dependent fine-tuning mechanismemerging at the initial phase of Th2 differentiation. At this stage of Th2 cell developmentvery low amounts of IL-4 are produced. Notably, the suppression of the short, inhibitoryTCF-1 isoforms occurs at an IL-4 concentration of less than 0.5ng/ml which is probably notsufficient to induce STAT6-driven, GATA3-1a-dependent Th2 differentiation.

Although not capable of inducing the STAT6-dependent GATA3-1a transcript, the presentIL-4 still is sufficient to contribute to early TCF-1 mediated GATA3-1b transcription bysuppressing the inhibitory TCF-1 isoforms. As a consequence the developing Th2 cellsgenerate large amounts of IL-4 and the classical IL-4/STAT6-dependent pathway allows forthe manifestation of the Th2 phenotype.

Taken together, STAT6 does not only promote the development of Th2 cells through directinteraction with the GATA3 promoter, but also through down-regulation of the naturalinhibitors of the newly described, TCF-1-dependent, alternative Th2 differentiationpathway.

AcknowledgmentsWe thank Prof. Dr. André Gessner (University Erlangen, Germany) for providing STAT6-deficient mice.

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Fig. 1.TCF-1 and LEF-1 are predominantly down-regulated in naïve CD4+ T cells undergoing Th2cell differentiation.Naïve human CD4+ T cells were isolated from human blood and either left uninduced (ui),stimulated with αCD3/αCD28 (TCR) or cultured under Th1- or Th2-polarizing conditions(αCD3/αCD28 + IL-12, αCD3/αCD28 + IL-4, respectively). Expression of the Wnt pathwaytranscription factors TCF-1 and LEF-1 was analyzed by qRT-PCR after 48 hours ofinduction. Mean values of two separate donors are shown. Error bars indicate standarddeviations.

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Fig. 2.IL-4-mediated inhibition of TCF-1 and LEF-1 is restricted to naïve CD4+ T cells.Naïve (left panel) and memory (right panel) CD4+ T cells were isolated from human bloodand cultured in the absence (ui) or presence of either IL-4 or IL-12 for the indicated times.Expression of LEF-1 and TCF-1 mRNA was analyzed by qRT-PCR. Results arerepresentative for three independent experiments carried out in duplicates. Error barsindicate standard deviations.

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Fig. 3.Wnt transcription factors are highly sensitive to IL-4 treatment.Naïve human CD4+ T cells were isolated and cultured overnight in the presence of IL-4 atthe indicated concentrations. mRNA expression of the Wnt pathway molecules TCF-1,LEF-1, β-catenin and the IL-4-inducible gene CCL17 was examined by means of qRT-PCR.One representative experiment out of three is shown. Error bars indicate standard deviations.

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Fig. 4.IL-4-induced down-regulation of TCF-1 targets the short isoforms.A, Schematic representation of the main TCF-1 isoforms. Long TCF-1 isoforms include aDNA-binding domain (HMG box), a motif that mediates the interaction with co-repressorsof the Groucho family (groucho binding) and an NH2-terminal β-catenin-binding domain.The latter domain is not present in the short TCF-1 isoforms.B, qRT-PCR-based quantification of short (TCF-1 p30) and total isoforms expressed bynaïve human CD4+ T cells after 48 hours of IL-4 stimulation. Mean values of threeindependent experiments are shown. Error bars indicate standard deviations.C, Naïve CD4+ T cells were cultured in the presence or absence (ui) of IL-4 for the indicatedtimes. Total protein was isolated and subjected to polyacrylamide gel electrophoresis. TCF-1protein was detected by Western blotting. To control for efficient IL-4 stimulation and equalloading, phosphorylated STAT6 and total STAT6 were detected. A Jurkat cell lysate wasloaded as control for TCF-1 isoform expression.

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Fig. 5.STAT6 binds to specific DNA motifs within the human TCF1 gene.A, Schematic representation of the human TCF1 genomic locus as described by van deWetering et al. (29). Positions of the five putative STAT-binding motifs are indicatedrelative to the translational start site.B, Electrophoretic Mobility Shift Assays (EMSAs) using radiolabeled probes harboring thesequences −2804/−2815 (site 1), −412/−423 (site 2), +1678/1687 (site 3), +7122/+7131(site 4) and +21001/21009 (site 5) were performed. Nuclear extracts prepared from untreatedand IL-4-treated naïve CD4+ T cells were incubated with double-stranded oligonucleotide,and nucleoprotein complexes were resolved in native polyacrylamide gels.C, EMSAs using radiolabeled probes comprising wild-type STAT6 motifs (WT) or mutatedmotifs (mut) were performed. For competition assays 50-fold molar excess of unlabeledwild-type or mutated oligonucleotide was added to the binding reaction. In supershiftexperiments, nuclear extracts were pre-incubated with an antibody directed against STAT6(M20) or a control antibody (N20, directed against STAT5).

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Fig. 6.Chromatin Immuno-Precipitation (ChIP) demonstrates binding of STAT6 to the TCF1 locusin living cells.A, Distribution of H3K4me1 and H3K4me3 histone modifications along the human TCF1locus. The positions of STAT6 binding motifs are indicated and largely overlap with histonemarks of either kind, indicating open chromatin formations (upper panel). To demonstrateSTAT6 binding to the binding motifs ChIPs using STAT6-specific antibodies were carriedout. Naïve CD4+ T cells were stimulated with IL-4 or left untreated (ui). Samples wereprecipitated using anti-STAT6 antibodies or an equal amount of negative control IgG. Thedata represent the amplification signal obtained by qRT-PCR for each binding site,expressed as percentage of the corresponding input samples (lower panel). Mean values oftwo experiments are shown. Error bars indicate standard deviations.B, Aliquots from cells used in the ChIP assays were taken to isolate mRNA and to preparetotal cell lysates to control for the down-regulation of TCF-1 on mRNA- (left panel) andprotein level (right panel). To ascertain efficient IL-4 stimulation and equal loading,phosphorylated STAT6 and total STAT6 were detected.

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Fig. 7.IL-4-induced suppression of TCF-1 depends on STAT6A, To knock down STAT6 in naïve human CD4+ T cells, cells were either transfected withsiRNA targeting STAT6 or with control siRNA. Decreased levels of STAT6 protein weredetected 96 hours post-transfection by immuno-blotting. STAT1 protein was detected tocontrol for equal loading.B, 72 hours post-transfection cells were incubated in the presence or absence of IL-4 for 24hours. mRNA expression of total TCF-1 and the short TCF-1 isoform (TCF-1 p30) wereanalyzed by qRT-PCR. Results are representative for two independent experiments carriedout in duplicates.C, The putative STAT6-binding motifs within the human TCF1 locus described in Figure 5are located in regions highly conserved between mouse and human. Therefore, splenicCD4+CD62L+ T cells from Balb/c and STAT6−/− mice were isolated and cultured in thepresence of IL-4 or IL-12 for the indicated times. TCF-1 expression (upper panel) andLEF-1 expression (lower panel) were measured by qRT-PCR. Mean values of threeindependent experiments are shown. Error bars indicate standard deviations.

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Inhibition of Suppressive T Cell Factor 1 (TCF-1) Isoforms in Naive CD4+ T Cells Is Mediated by...

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