of February 17, 2018.This information is current as

Interacting with and Attenuating GATA3byTh1 and Down-Modulates Th2 Phenotypes

The Runx3 Transcription Factor Augments

Masanobu SatakeYamashita, Toshinori Nakayama, Masato Kubo andDaisuke Onda, Takeshi Wakoh, Shunsuke Kon, Masakatsu Kazuyoshi Kohu, Hidetaka Ohmori, Won Fen Wong,

http://www.jimmunol.org/content/183/12/7817doi: 10.4049/jimmunol.0802527November 2009;

2009; 183:7817-7824; Prepublished online 23J Immunol 

MaterialSupplementary

7.DC1http://www.jimmunol.org/content/suppl/2009/11/23/jimmunol.080252

Referenceshttp://www.jimmunol.org/content/183/12/7817.full#ref-list-1

, 14 of which you can access for free at: cites 33 articlesThis article

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Runx3 Transcription Factor Augments Th1 andDown-Modulates Th2 Phenotypes by Interacting withand Attenuating GATA31

Kazuyoshi Kohu,2*‡ Hidetaka Ohmori,2* Won Fen Wong,* Daisuke Onda,* Takeshi Wakoh,*Shunsuke Kon,* Masakatsu Yamashita,† Toshinori Nakayama,† Masato Kubo,‡

and Masanobu Satake3*

Recently, it was reported that the expression of Runt-related transcription factor 3 (Runx3) is up-regulated in CD4� helper T cellsduring Th1 cell differentiation, and that Runx3 functions in a positive feed-forward manner with the T-box family transcriptionfactor, T-bet, which is a master regulator of Th1 cell differentiation. The relative expression levels of IFN-� and IL-4 are alsoregulated by the Th2-associated transcription factor, GATA3. Here, we demonstrate that Runx3 was induced in Th2 as well asTh1 cells and that Runx3 interacted with GATA3 and attenuated GATA3 transcriptional activity. Ectopic expression of Runx3in vitro in cultured cells or transgenic expression of Runx3 in mice accelerated CD4� cells to a Th1-biased population or down-modulated Th2 responses, in part by neutralizing GATA3. Our results suggest that the balance of Runx3 and GATA3 is one factorthat influences the manifestation of CD4� cells as the Th1 or Th2 phenotypes. The Journal of Immunology, 2009, 183: 7817–7824.

T he CD4� helper T cells play a central role in acquiredimmunity. Naive CD4� T cells in peripheral lymphoidtissues are activated by Ag and induced to differentiate

into effector Th cells, which secrete large amounts of effector cy-tokines. Distinct subsets of effector Th cells can be functionallydefined by their discrete cytokine expression profiles. Th1 cellssecrete IFN-�, IL-2, and TNF-� and mediate defense against in-fection with intracellular microbes and isotype switching to IgG2aand IgG2b, whereas Th2 cells secrete IL-4, IL-5, and IL-13 andpromote humoral response to extracellular pathogens and isotypeswitching to IgG1 and IgE (1). Differentiation of Th subsets isregulated and coordinated by a complex network of transcriptionalregulators, including T-box family transcription factor (T-bet)4 andGATA3 (2, 3). GATA3 is a master regulator of Th2 differentiationand activates Th2-specific cytokine genes primarily through epi-genetic modification of the IL-4 locus (4). Ectopic expression ofGATA3 can induce Th2 cell differentiation even under conditionsin which STAT6, a transducer of IL-4 signals, is absent, a situation

that otherwise promotes Th1 cell differentiation (5). Furthermore,GATA3 is involved in the autoactivation of GATA3 gene tran-scription (5). GATA3 is necessary for Th2-mediated immune re-sponses in vivo, as demonstrated by the analysis of conditionalGATA3 knockout mice (6, 7). T-bet is a master regulator of Th1cell differentiation, and activates Th1-specific cytokine geneswhile repressing Th2-related cellular programs (8). When naiveCD4� T cells encounter Ag, GATA3 is transiently transcribed dur-ing the early response phase, even under Th1 conditions. However,at the protein level, T-bet negatively regulates GATA3 activitythrough a physical interaction with GATA3, and it functions as anantagonist of Th2 differentiation (9).

The Runt-related transcription factors (Runx) harbor an evo-lutionarily conserved 128-aa region termed the Runt domain,which is responsible for DNA binding (10 –12). Runx proteinsinteract with coactivators or corepressors to activate or represstarget genes in a context-dependent manner (13). In thymocytedevelopment, Runx1 and Runx3 are essential factors in the gen-eration of CD8� cells. Runx1 and Runx3 have been shown torepress the expression of CD4 and to activate the expression ofCD8 (14 –16). Runx transcription factors are also involved inthe regulation of Th cell differentiation. We previously reportedthat the forced expression of Runx1 in naive CD4� T cellspromotes Th1 cell differentiation even under conditions thatpromote Th2 differentiation. Conversely, introduction of a dom-inant negative form of Runx1 biases cytokine secretion towardthe Th2 phenotype (17). Recently, Djuretic et al. (18) demon-strated that Runx3 expression is up-regulated during Th1 dif-ferentiation and functions in a positive feed-forward manner inTh1 differentiation. They also demonstrated that in Th1 cells,Runx3 interacts with T-bet and simultaneously attenuates IL-4expression and augments IFN-� expression. These effects areachieved by the binding of a Runx3/T-bet complex to the IL-4silencer and IFN-� promoter, respectively. Thus, the interactionof Runx3 and T-bet appears to augment the activity of eachbinding partner, which explains why Runx3-deficient T cells areprone to Th2 differentiation.

*Institute of Development, Aging and Cancer, Graduate School of Life Sciences,Tohoku University, Sendai, Japan; †Department of Immunology, Graduate School ofMedicine, Chiba University, Chiba, Japan; and ‡Laboratory for Signal Network, Re-search Center for Allergy and Immunology, The Institute of Physical and ChemicalResearch, Yokohama Institute, Yokohama, Japan

Received for publication August 1, 2008. Accepted for publication October 8, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by research grants from the Ministry of Education,Science, Sports, Culture and Technology, Japan. M.S. is a participant in the GlobalCOE Program Network Medicine at Tohoku University.2 These authors contributed equally to this work.3 Address correspondence and reprint requests to Dr. Masanobu Satake, Institute ofDevelopment, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku,Sendai 980-8575, Japan. E-mail address: satake@idac.tohoku.ac.jp4 Abbreviations used in this paper: T-bet, T-box family transcription factor; HA,hemagglutinin; Runx, Runt-related transcription factor; ThN, Th cells under non-skewed conditions.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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The relative levels of IFN-� and IL-4 expression are also reg-ulated by the Th2-associated transcription factor GATA3. In thisstudy, we present evidence that Runx3 associates with GATA3 andattenuates GATA3 transcriptional activity, thereby affecting cellphenotypes. The overexpression of Runx3 converted CD4� cellsto a Th1-biased cell population and/or down-modulated Th2 re-sponses, in part by neutralizing the activity of GATA3.

Materials and MethodsCytokines and Abs

Mouse rIL-2, rIL-4, and rIL-12 were purchased from PeproTech. For T cellculture, anti-TCR� (H57-597), anti-CD28 (37.51), anti-IL-4 (11B11), anti-IFN-� (XMG1.2), and anti-IL-12 (C17.8) Abs were purchased fromeBioscience. For intracellular cytokine staining, allophycocyanin-conju-gated anti-IFN-� (XMG1.2) and PE-conjugated anti-IL-4 (11B11) werepurchased from BioLegend. The anti-Runx Ab was described previously(19). Anti-GATA3 (HG3-31), anti-�-tubulin (Ab-1), and anti-�-actin Abswere purchased from Santa Cruz Biotechnology and Calbiochem.

Cell culture and retrovirus infection

CD4� T cells were purified from the spleens of C57BL/6J (B6) mice usingan IMag Cell Separation System (BD Biosciences). The purity of theCD4� fraction was �95% based on flow cytometry. Cells (2 � 105) werefirst stimulated with immobilized anti-TCR� mAb (30 �g/ml) and solubleanti-CD28 mAb (10 �g/ml) in RPMI 1640 containing 10% (v/v) FBS for2 days. To maintain cells under nonskewed (ThN) conditions, cells werecultured in the presence of IL-2 (30 U/ml) for an additional 7 days. For Th2polarization, cells were cultured for 4 days in the presence of murine IL-4(100 U/ml), anti-IL-12 Ab (10 �g/ml), and anti-IFN-� Ab (10 �g/ml); forTh1 polarization, cells were cultured for 4 days in the presence of murineIL-12 (5 ng/ml) and anti-IL-4 Ab (10 �g/ml). Intracellular staining ofIFN-� and IL-4 was performed as described previously (17).

The murine Runx3 cDNA was inserted into the retroviral expressionvector pMX-GFP (17) to generate pMX-Runx3-IRES-GFP. Mutated ver-sions of Runx3 were also inserted into pMX-GFP, and they were Runx3/1–404, Runx3/R178Q, and Runt each. Runx3/1–404 was constructed byPCR, whereas Runx3/R178Q (20) was provided by Drs. K. Ito and Y. Ito(National University of Singapore, Singapore). Runt represents a Runt do-main derived from murine Runx1 (10). Both Runx3/R178Q and Runt func-tion dominant negatively against intact Runx1/3 proteins. The GATA3cDNA was inserted into pMX-ratCD2 to generate pMX-GATA3-IRES-ratCD2. The packaging cell line PLAT-E was transfected with each plas-mid using FugeneHD (Roche). After incubation for 24 h, the culture su-pernatant was harvested, concentrated, and used as a viral stock (21).CD4� T cells that had been stimulated as described for 24 h were infectedwith retroviruses using the DOTAP Liposomal Transfection Reagent(Roche) and then incubated under ThN-, Th1-, or Th2-permissive condi-tions for an additional 6 days. Rat CD2� cells were purified using a biotin-conjugated anti-rat CD2 Ab and an IMag Cell Separation System.

Immunoprecipitation and immunoblot analysis

293T cells were transfected with an expression vector, pcDNA3, encodinghemagglutinin (HA)-tagged Runx3 or GATA3 using the FugeneHD re-agent (Roche). The Runt domain-deleted Runx3 and the Runt domain itselfof Runx3 were also transfected into 293T cells. After incubation for 48 h,the cells were lysed in buffer containing 50 mM Tris-HCl (pH 7.4), 150mM NaCl, 1% (v/v) Triton X-100, 1 mM NaVO4, 1 mM NaF, and 1 �g/mlaprotinin. The lysates were also prepared from splenic CD4� T cells thathad been cultured under Th2 conditions described previously. Each lysatewas incubated with anti-HA (3F10; Roche) or anti-GATA3 (HG3-31)mAb, and the immunoprecipitates were adsorbed to protein G-Sepharosebeads (GE Healthcare). The beads were washed five times with lysis buffer,and then proteins were eluted by boiling the beads in SDS sample buffer.As a control, whole-cell lysate was prepared by sonicating cells in SDSsample buffer. Proteins were resolved by SDS-PAGE and transferred to anImmobilon-P membrane (Millipore). The membrane was probed with anappropriate primary Ab, followed by an alkaline phosphatase-conjugatedsecondary Ab (Promega). Immune complexes were detected using NBT-5-bromo-4-chloro-3-indolyl phosphate reagents (Promega).

Semiquantitative RT-PCR analysis

RNA was isolated from cells using the ISOGEN reagent (Nippon Gene).First-strand cDNA was synthesized from RNA using Superscript II reversetranscriptase (Invitrogen Life Technologies). PCR amplification was per-

formed under nonsaturating conditions using cDNA as the template andLA-Taq polymerase (Takara). The sequences of the sense and antisenseprimers were as follows: for T-bet, 5�-CTAAGCAAGGACGGCGAATGT-3� and 5�-GGCTGGGAACAGGATACTGG-3�; for IFN-�, 5�-GGTGACATGAAAATCCTGCAGAGC-3�and 5�-TCAGCAGCGACTCCTTTTCCGCTT-3�; for TNF-�, 5�-ATCAGTTCTATGGCCCAGACCCT-3� and 5�-TCACAGAGCAATGACTCCAAAGTA-3�; for IL-12, 5�-GGGACATCATCAAACCAGACC-3� and 5�-GCCAACCAAGCAGAAGACAGC-3�; for IL-18, 5�-ACTGTACAACCGCAGTAATAC-3� and 5�-AGTGAACATTACAGATTTATCCC-3�; for GATA3, 5�-CTCCTTTTTGCTCTCCTTTTC-3� and 5�-AAGAGATGAGGACTGGAGTG-3�; for IL-4,5�-TCCACGGATGCGACAAAAAT-3� and 5�-TTCTTCTTCAAGCATGGAGT-3�; for IL-5, 5�-GAGCACAGTGGTGAAAGAGACCTT-3� and5�-ATGACAGGTTTGGATAGCATTT-3�; for IL-13, 5�-AGTTCTACAGCTCCCTGGTTCTCT-3� and 5�-CTTTGTGTAGCTGAGCAGTTTTGT-3�; for Runx1, 5�-ACTCTGCCGTCCATCTCCGACCCGC-3� and 5�-CGTCGCTCTGGCTGGGGAGGCTGGG-3�; for Runx3, 5�-GCTGCAGAGCCTCACAGAGAGCCGC-3� and 5�-GTCGGCTTCCACGCCATCAGGCTGG-3�; for �-actin, 5�-GATGACGATATCGCTGCGCTG-3� and5�-GTACGACCAGAGGCATACAGG-3�.

Luciferase reporter assay

M12 cells were cotransfected with a luciferase reporter plasmid and anexpression plasmid for Runx3 or GATA3 using FugeneHD (Roche). As aninternal control, cells were also transfected with pRL-TK (Promega), andthe activity of Renilla luciferase was used to normalize for transfectionefficiency. The cells were incubated for 24 h after transfection and thenlysed in a passive lysis buffer (Promega). Luciferase activity was measuredusing a luciferase substrate (Promega) and a LumatLB9507 (BertholdTechnologies).

EMSA

The procedure for the EMSA was described previously (15). The GATAbinding sequence from the IL-5 promoter, 5�-GGTGTCCTCTATCTGATTGTT-3�, was used as a probe to detect GATA DNA-binding activity.GATA3, Runx3, and CBF� polypeptides were synthesized in vitro usingthe TnT T7 Coupled Reticulocyte Lysate System (Promega) and eachcDNA template in a pcDNA3 vector.

Mice

The transgenic mouse line expressing a murine Runx3 transgene was pre-viously described (15). Litters of transgenic mice were backcrossed withB6 mice for 10 generations. B6 mice were purchased from Clea Japan.Stat6-deficient mice were obtained from Dr. S. Akira (Osaka University,Osaka, Japan) (22). Experiments were performed using 8- to 12-wk-old,age-matched mice. Mice were maintained and bred according to the guide-lines defined by the Animal Facility of the Institute of Development, Agingand Cancer at Tohoku University (Sendai, Japan). All animal protocolsused in this study were approved by our Institutional Animal Care and UseCommittees.

Immunization, ELISA, and histology

Mice were injected s.c. with 10 �g of OVA (Sigma-Aldrich) in CFA or i.p.with 10 �g of OVA in aluminum hydroxide. Boosting injections wereconducted at days 14 and 28. The titer of serum Ig was measured byELISA. HRP-conjugated anti-mouse IgG1, anti-mouse IgG2a, anti-mouseIgG2b, and anti-mouse IgE were purchased from Bethyl. Spleens wereisolated and fixed in 4% (w/v) paraformaldehyde in PBS for 18 h and thenembedded in Tissue-Tek OCT compound (Sakura Finetechnical). Micro-sections of tissues on slides were stained by HRP-conjugated peanut ag-glutinin and counterstained with hematoxylin.

ResultsExpression profiles of Runx proteins in nonskewed anddifferentiated Th cells

To examine the expression of Runx proteins during Th cell dif-ferentiation, naive CD4� cells were isolated from the spleens ofmice and subjected to immunoblot analysis using an anti-pan-Runx Ab. The advantage of this Ab was that we could visu-alize the levels of each Runx family member simultaneously (theauthenticity of Runx bands detected by the Ab had been confirmedpreviously in Refs. 15 and 19). As seen in Fig. 1, Runx1 was thesole major Runx component expressed in naive CD4� cells (Fig.

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1, lane 2). In contrast, in Th1-differentiated cells, Runx1 was un-detectable, and Runx3 emerged as the principal Runx component(Fig. 1, lane 3). Th2-differentiated cells expressed equivalentamounts of Runx1 and Runx3 (Fig. 1, lane 6). In Stat6-deficientcells under Th2-promoting conditions, Runx expression was con-verted to a Th1-specific phenotype (Fig. 1, lane 5), which indicatedthat the expression of Runx proteins in wild-type Th2 cells is de-pendent on Stat6. When wild-type CD4� cells were stimulatedthrough the TCR using an anti-TCR� Ab and then cultured undernonskewed conditions, the expression patterns of Runx proteinswere similar to those of Th2 cells (Fig. 1, compare lanes 7 and 9).These results indicated that Runx3 is not a Th1-specific factor butis expressed in TCR-stimulated CD4� cells as well as in Th2-differentiated cells.

Overexpression of Runx3 can activate IFN-� and silence IL-4expression even under conditions that promote Th2differentiation

Previously, we reported that the expression of Runx1 in Th2 cellsattenuates the exacerbation of the Th2 response (17). To determinewhether Runx3 functioned in a similar manner, CD4� cells werecultured under conditions that promoted Th2 differentiation, and

then infected by a retrovirus that was engineered to express Runx3.The overexpression of exogenous Runx3 in Th2 cells was con-firmed by RT-PCR and immunoblot analysis (Fig. 2, A and B). Theexpression of Runx3 protein in infected cells was �3.5-fold higherthan in control Th2 cells (Fig. 2B, compare lanes 1 and 2). Fig. 1Cshows the results of the analysis of intracellular IFN-� and IL-4levels in Th2 cells by flow cytometry. In control, pMX-infectedcells, 45% of the cells were IL-4 positive, whereas only 1% wereIFN-� positive. In cells that overexpressed Runx3, this ratio was re-versed, and 56% of the cells were IFN-� positive whereas 8% wereIL-4 positive. These results indicated that the overexpression ofRunx3 can activate IFN-� expression and silence IL-4 expression,even under conditions that promote Th2 differentiation. The level of

FIGURE 1. Expression of Runx proteins during Th cell differentiation.Immunoblot (IB) analysis of Runx proteins in naive CD4� cells and inCD4�-derived Th cells cultured in vitro under ThN, Th1, or Th2 conditionsfor 7 days. CD4� T cells were isolated from the spleens of wild-type(�/�), Stat6-deficient (Stat6�/�) and Runx3-transgenic mice, as indi-cated. Extracts were probed with an anti-pan-Runx Ab. Arrows, Runx1 andRunx3. In the third panel from the top, extracts were also probed with ananti-GATA3 Ab. �-Actin served as a loading control. Relative densities ofRunx3 and GATA3 bands are indicated below the gel. The identity of theprotein indicated by the asterisk is unknown; however, based on its appar-ent m.w., it might represent Runx2.

FIGURE 2. Effect of Runx3 expression on the expression of GATA3,and the relative levels of IFN-� and IL-4. A, Semiquantitative RT-PCRanalysis of Runx3 and GATA3 transcripts. Naive CD4� cells were isolatedfrom the spleens of wild-type (WT) mice, stimulated with TCR, and theninfected by a retrovirus carrying pMX-IRES-rCD2, pMX-Runx3-IRES-rCD2, or pMX-GATA3-IRES-rCD2, as indicated. After 7 days in cultureunder Th2 conditions, rat CD2-positive cells were isolated by cell sorting,and RNA was isolated and converted to cDNA. Increasing amounts ofcDNA were used as the template for the detection of Runx3 and GATA3transcripts by PCR. �-Actin was analyzed as a control. B, Immunoblotanalysis of Runx1, Runx3, and GATA3 in Runx3- or GATA3-expressingcells. Lanes are as described for A, with the exception that �-tubulin wasanalyzed as a loading control. C, Naive CD4� cells were isolated from thespleens of wild-type mice, stimulated with TCR, and infected by a retro-virus carrying pMX-IRES-GFP, pMX-Runx3-IRES-GFP, or pMX-Runx3/1–404-IRES-GFP, as indicated. After 7 days in culture under Th2 condi-tions, the cells were restimulated and analyzed by intracellular staining forIFN-� and IL-4, followed by flow cytometry. The fluorescence intensity ofIFN-� and IL-4 in the GFP-positive gated cell population is displayed. Thenumbers represent the percentage of cells in each quadrant. D, Effect ofreducing endogenous Runx activity on IL-4 and IFN-� expression. Exper-imental conditions were similar to those in C, except that cells were in-fected by a retrovirus carrying pMX-Runt-IRES-GFP and pMX-Runx3/R178Q-IRES-GFP and that the Th2 bias was far less prominent than in C(compare pMX-infected cells in C and D).

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GATA3 was not affected by the overexpression of Runx3 (Fig. 2B,compare lanes 1 and 2). Thus, the observed effect of Runx3 on IFN-�and IL-4 expression was not likely to be mediated through the mod-ulation of GATA3 expression (however, the T-bet transcript was in-duced by the overexpressed Runx3; supplemental Fig. 1).5

The C-terminal five amino acid residues of Runx proteins com-prise a characteristic VWRPY motif, with which a Groucho/TLErepressor interacts. To determine whether this motif contributed toRunx3-mediated IL-4 repression, we expressed a mutant of Runx3in which this motif was deleted (Runx3/1–404). As seen in Fig.2C, 34% of cells that expressed Runx3/1–404 were IL-4 positive,whereas 7% were IFN-� positive.

Reduction of endogenous Runx3 activity augments Th2responses

Endogenous Runx3 activity was then artificially reduced to see itseffect on IL-4 and IFN-� expression. In Fig. 2D, CD4� cells werecultured under Th2 conditions but with less efficacy (the ratio ofIL-4� to IFN-�� cells in pMX of Fig. 2D was 2.1 as comparedwith 32 in pMX of Fig. 2C) and were infected by a retroviruscarrying pMX-Runt-IRES-GFP and pMX-Runx3/R178Q-IRES-GFP. The Runt domain and Runx3/R178Q are known to functiondominant negatively against endogenous Runx protein, thereby re-ducing Runx activity inside the cells (10, 20). Ratios of IL-4� toIFN-�� cells were 8.0 for both Runt- and Runx3/R178Q-infectedcells. Compared with pMX-infected control cells, reduction ofRunx activity resulted in a significant increase of IL-4� cells.

Runx3 interacts with GATA3 and attenuates GATA3transcriptional activity

Runx1 has been shown to directly interact with GATA1 duringmegakaryocyte differentiation (23). As shown above, Runx3 wasdetected not only in Th1 but also in ThN and Th2 cells. To deter-mine whether Runx3 interacted with GATA3, 293T cells werecotransfected with expression vectors for GATA3 and HA-taggedRunx3 and then subjected to immunoprecipitation using an anti-HA Ab, followed by immunoblot analysis using an anti-GATA3Ab. As seen in Fig. 3, Runx3 and GATA3 coimmunoprecipitatedwith each other (Fig. 3A, lane 4). The Runt domain of Runx1 iscritical for its association with GATA1 (23). When we transfectedcells with a mutant of Runx3 in which the Runt domain was de-leted, Runx3 and GATA3 failed to coimmunoprecipitate (Fig. 3B,lane 9). The Runx3 Runt domain alone, however, coimmunopre-cipitated with GATA3 (Fig. 3B, lane 10).

Next, to determine whether endogenous GATA3 interacted withRunx3, cell lysates were prepared from CD4� T cells that werecultured under Th2 conditions and subjected to immunoprecipita-tion using anti-GATA3 Ab (Fig. 3C). Endogenous Runx3 coim-munoprecipitated together with endogenous GATA3 (see the bandindicated by the arrowhead in Fig. 3C, lane 13). These resultsindicated that Runx3 interacts with GATA3.

To examine the functional significance of the Runx3-GATA3interaction, we conducted a luciferase reporter gene assay in whichluciferase expression was driven by the IL-5 promoter. IL-5 is awell-characterized GATA3-responsive Th2-specific gene in re-porter gene assays (24). A fragment of the IL-5 promoter spanning1200 nt upstream of the transcription initiation site was ligated tothe luciferase coding sequence (Fig. 4A). The promoter fragmentcontained 2 Runx sites (at positions �1030 and �470 relative tothe transcriptional start site) and 1 GATA site (�20 nt). M12 Bcells were cotransfected with the IL-5-luciferase reporter constructand expression vectors for Runx3 and/or GATA3. As indicated in

Fig. 4B, the expression of GATA3 alone enhanced luciferaseactivity through the IL-5 promoter 4-fold as compared with mock,whereas Runx3 had only a marginal effect. However, coexpressionof Runx3 and GATA3 completely attenuated the enhancement ofluciferase activity by GATA3. Mutation of the GATA site of the1200-nt IL-5 promoter fragment abolished its responsiveness toGATA3. A shorter fragment of the IL-5 promoter (460 nt), whichdid not contain either of the Runx sites, was similar to the longer5 The online version of this article contains supplemental material.

FIGURE 3. Physical interaction of Runx3 and GATA3. A and B, 293Tcells were transfected with the indicated expression vectors for GATA3and HA-tagged Runx3 or Runx3 mutants (Runt domain-deleted Runx3 orthe Runx3 Runt domain alone). Lysates were prepared and subjected toimmunoprecipitation (IP) using anti-HA Ab-conjugated protein G-Sepha-rose, followed by immunoblot (IB) analysis using an anti-GATA3 Ab.Arrowhead, GATA3. C, Interaction of endogenous GATA3 and Runx3proteins. CD4� T cells were isolated from the spleen of wild-type mice,stimulated with TCR, and cultured under Th2 conditions for 7 days. Thelysates were prepared and subjected to immunoprecipitation using anti-GATA3 mAb or control IgG. The precipitates were immunoblotted byanti-pan-Runx Ab. Arrowhead, Runx3.

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1200-nt promoter fragment in its responsiveness to GATA3 and/orRunx3.

EMSA was then performed using a GATA binding sequencefrom the IL-5 promoter as a probe (Fig. 4C). GATA3 and Runx3/CBF� proteins were synthesized in vitro from the respectivecDNAs and used. As expected, GATA3 but not Runx3 bound tothe IL-5 promoter (Fig. 4C, lanes 2 and 3). Coexistence of Runx3/CBF� abolished DNA binding activity of GATA3 in a dose de-pendent manner (Fig. 4C, lanes 4 and 5). These results indicated

that Runx3 can interact with GATA3 and attenuate GATA3 tran-scriptional activity by inhibiting DNA-binding activity of GATA3.Thus, the mechanism of action of Runx3 and GATA3 is quitedistinct from that of Runx3 and T-bet, in which the two factorsinteract with each other and augment the transcriptional activity oftheir binding partner (18).

The balance of Runx3 and GATA3 regulates the relativeexpression levels of IFN-� and IL-4 in TCR-stimulated CD4�

cells

To determine the effect of modulating the relative amounts ofRunx3 and GATA3 on IFN-� and IL-4 expression, isolated splenicCD4� cells were stimulated with TCR and cultured under non-skewed conditions. The cells were then doubly infected by tworetroviruses (Fig. 5), one encoding bicistronic Runx3 and GFP andthe second encoding GATA3 and rat CD2. GFP and rat CD2 wereused as markers for monitoring Runx3 and GATA3 expression,respectively. In noninfected cells, the IFN-�- and IL-4-positivefractions were present in a 1:1 ratio (11 and 10%, respectively; Fig.5, lower left). The overexpression of GATA3 decreased the ratio ofIFN-� and IL-4 to 0.4 (9 and 23%, respectively; upper left),whereas the overexpression of Runx3 increased the ratio of IFN-�and IL-4 to 4.2 (23 and 5%, respectively; lower right). In doublyinfected cells, the ratio of IFN-� to IL-4 reverted to 1.1 (17 and15%, respectively; Fig. 5, upper right). These results indicated thatin TCR-stimulated cells, Runx3 and GATA3 can attenuate eachother’s effect on the expression of IFN-� and IL-4 (See supple-mental Fig. 2 in which similar experiments as Fig. 5 was per-formed but under Th1 and Th2 conditions as well. Mutual coun-tering effects of Runx3 and GATA3 were confirmed again.).

Transgenic expression of Runx3 can activate IFN-� and silenceIL-4 in cultured CD4� cells

To determine whether the effects we were observing in vitro incultured CD4� cells were also true in vivo, we analyzed cells frommice that expressed an Lck-driven Runx3 transgene (15). Fig. 1

FIGURE 5. Effect of coexpression of Runx3 and GATA3 on IFN-� andIL-4 expression. Naive CD4� cells were isolated from the spleens of wild-typemice, stimulated with TCR, and then doubly infected by retroviruses carryingpMX-Runx3-IRES-GFP and pMX-GATA3-IRES-ratCD2. After 7 days inculture under nonskewing conditions, the cells were restimulated and thenanalyzed by intracellular staining for IFN-� and IL-4, followed by flow cy-tometry. The cells were gated into four subpopulations based on GFP- and ratCD2-specific fluorescence (left): GFP-negative/rat CD2-negative (bottom left);GFP-positive/rat CD2-negative (bottom right); GFP-negative/rat CD2-positive(top left); and GFP-positive/rat CD2-positive (top right). The fluorescence in-tensity of IFN-� and IL-4 is shown for each subpopulation. Numbers representthe percentages of cells in each quadrant.

FIGURE 4. Effect of Runx3 and GATA3 on the activity of the IL-5promoter. A, Schematic illustration of the luciferase (LUC) reporter plas-mid, in which luciferase expression is driven by the murine IL-5 promoter.The 1200-nt region upstream of the transcriptional initiation site of IL-5harbors 2 Runx sites (at nt �1030 and �470 relative to the transcriptionalstart site) and 1 GATA site (at nt �20). This 1200-nt fragment was ligatedto a luciferase reporter gene. The IL-5 promoter in which the GATA sitewas mutated (mut), and a shorter 460-nt fragment of the IL-5 promoterwere also ligated to the luciferase reporter gene. B, M12 cells were trans-fected with the indicated luciferase reporter plasmid together withpcDNA3, pcDNA3-GATA3, and/or pcDNA3-Runx3. Data are themeans � SD of three independent experiments. Statistically significantdifferences were determined by the t test and are indicated by brackets (pvalues are indicated for each panel). C, EMSA of GATA3 and Runx3proteins to the IL-5 promoter sequence. GATA3, Runx3, and CBF� pro-teins were synthesized from the respective cDNA in vitro, incubated witha radiolabeled oligonucleotide probe spanning the GATA-binding se-quence of the IL-5 promoter, and processed to EMSA. Proteins were addedas in the indicated combinations, whereas a cold probe means an excessiveamount of nonlabeled oligonucleotide.

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shows the Runx expression profile of isolated Runx3-transgenicCD4� cells that were stimulated through the TCR with anti-TCR�Abs. The level of Runx3 in transgenic ThN cells was 1.5-foldhigher than that in nontransgenic ThN cells and rather equivalentto that in nontransgenic Th1 cells (Fig. 1, compare lanes 7, 8, and10). To determine whether Runx3-transgenic cells under non-skewed conditions also mimicked nontransgenic Th1 cells in termsof IFN-� and IL-4 expression, we analyzed intracellular IFN-� andIL-4 levels by flow cytometry. As seen in Fig. 6A, under non-skewed conditions, Runx3-transgenic cells exhibited enhancedIFN-� expression (73%) and marked repression of IL-4 expression(2%) as compared with nontransgenic cells (Transcription of Th2-type cytokines including IL-4, IL-5, and IL-13 were substantiallyreduced in Runx3-transgenic ThN cells. See supplemental Fig. 3.).

We next examined whether the magnitude of IFN-� and IL-4expression in Runx3-transgenic cells was also responsive to thebalance of Runx3 and GATA3, as observed in wild-type CD4�

cells. The introduction of GATA3 into Runx3-transgenic cells re-sulted in a decrease in the percentage of IFN-�-positive cells(37%), and an increase in the percentage of IL-4-positive cells(16%) as compared with the nonintroduction of GATA3 (Fig. 6A).However, Runx3-transgenic cells remained to exhibit a Th2-likeexpression profile of IFN-� and IL-4 even when cultured underTh2-permissive conditions (Fig. 6B; there was no virus infection inthis panel). The result in Fig. 6B was in agreement with the relative

expression levels of Runx3 and GATA3 proteins as detected inFig. 1 (see the legend of Fig. 6 for detailed discussion on theRunx3:GATA3 or GATA3:Runx3 ratios).

These results collectively indicated that transgenic expression ofRunx3 can activate IFN-� expression and silence IL-4 expression,similar to its effects in vitro in cultured CD4� cells.

Runx3-transgenic naive CD4� cells are biased toward a Th1phenotype

When CD4� cells were isolated from Runx3-transgenic spleensand analyzed immediately by immunoblot, an observed Runx ex-pression profile was the same as that seen in Fig. 1, lane 10 (datanot shown). This suggests that Runx3-transgenic naive CD4� cellshave already acquired a Th1 phenotype. To test this hypothesis, wecompared the expression profiles of various Th1- and Th2-specificgenes in naive CD4� cells that were isolated from Runx3-trans-genic and nontransgenic mice by semiquantitative RT-PCR anal-ysis (Fig. 7). As a control, we also examined gene expression inCD8� cells in parallel. As compared with nontransgenic cells,Runx3-transgenic cells expressed an excessive amount of Runx3transcript, however, the levels of T-bet and GATA3 transcriptswere unaffected. The levels of several Th1-type cytokine tran-scripts (IFN-�, �F�, IL-12, and IL-18) were uniformly increasedin Runx3-transgenic cells. In contrast, the levels of Th2-type cy-tokine transcripts (IL-4 and IL-13) were decreased in transgeniccells. These results indicated that Runx3-transgenic CD4� cells areprone to a Th1 phenotype, even in the absence of TCR-stimulation.

Th1-specific immunological responses in Runx3-transgenic mice

We next examined the Th1- and Th2-mediated immunological re-sponses of Runx3-transgenic mice. Mice were immunized withOVA mixed with CFA, and serum Ab titers were measured (Fig.8A). After a second boosting injection (day 28), the titers of IgG2aand IgG2b were significantly higher in Runx3-transgenic mice thanin nontransgenic mice. IgG2a and IgG2b are the Ig subclasses theproduction of which depends on a Th1 response. We also exam-ined mice that were immunized with OVA mixed with aluminumhydroxide (Fig. 8B). OVA mixed with aluminum hydroxide is aform of Ag that tends to trigger the Th2-dependent production of

FIGURE 6. Effect of transgenic (tg) Runx3 expression on IFN-� andIL-4 expression. CD4� cells were isolated from the spleens of nontrans-genic (non-tg) and Runx3-transgenic mice, stimulated with TCR, and theninfected by a retrovirus carrying pMX-GFP or pMX-GATA3-IRES-GFP.After 7 days in culture under nonskewing (A) or Th2 (B) conditions, thecells were re-stimulated and then analyzed by intracellular staining forIFN-� and IL-4, followed by flow cytometry. The fluorescence intensity ofIFN-� and IL-4 in the GFP-positive population is displayed. Numbers rep-resent the percentages of cells in each quadrant. The GATA3:Runx3 pro-tein ratio in Th2-conditioned Runx3-transgenic cells was �1.0 (actually, itwas 0.7:0.3 2.3 in Fig. 1, lane 12) as in the case of Th2-conditionednontransgenic cells (1.7:0.5 3.4 in Fig. 1, lane 9). Therefore, it may notbe unreasonable that Th2-conditioned Runx3-transgenic cells showed aTh2-like phenotype (B). In contrast, in Fig. 2, retrovirally introducedRunx3 overexpressing cells for which the Runx3:GATA3 ratio was 3.5:1.0 3.5 exhibited a Th1 phenotype even under a Th2 condition.

FIGURE 7. Cytokine expression profiles in naive Runx3-transgenic (tg)CD4� cells. RNA was prepared from naive CD4� and CD8� cells from non-transgenic (non-tg) and Runx3-transgenic mouse spleens. Increasing amountsof cDNA were used for semiquantitative RT-PCR of cytokine gene transcriptsunder nonsaturating conditions. �-Actin was analyzed as a control.

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IgG1 and IgE. However, the production of these two subclasseswas poor in Runx3-transgenic mice as compared with nontrans-genic mice. These results demonstrated that Runx3-transgenicmice respond efficiently in a Th1-dependent manner, but that theirTh2-mediated response is poor.

B cell activation is impaired in Runx3-transgenic mice

During immunization, we noticed a striking feature of Runx3-transgenic mice. When spleens were isolated after a primary chal-lenge of OVA-aluminum, the weight of the tissue was on average135 mg in nontransgenic mice (Fig. 9, A and B), whereas thespleen weight in Runx3-transgenic mice was 80 mg. This corre-sponded to �60% of weight of the nontransgenic spleens. Weprepared histological sections of spleens from transgenic and non-transgenic mice, and stained them using peanut agglutinin (Fig.9C). In nontransgenic spleens, a number of lectin-stained clusterswere detected, but these clusters were scarcely detectable inRunx3-transgenic spleens. Because peanut agglutinin stains B cellsin activated germinal centers, these results suggested that B cellswere not sufficiently activated in Runx3-transgenic spleens follow-ing immunization.

DiscussionRunx3 was originally identified as a critical transcription factor inCD8 single-positive thymocyte differentiation that induced CD8and repressed CD4 gene expression (14–16, 25). Recently, a novelfunction of Runx3 as a feed-forward regulator of T-bet during Th1cell differentiation of peripheral CD4� Th cells was described(18). Runx3 is barely detectable in naive CD4� T cells, but itsexpression is markedly up-regulated under Th1-skewing condi-tions. Runx3 induction is dependent on T-bet, and T-bet andRunx3 form a regulatory complex that activates the IFN-� pro-moter and suppresses IL-4 expression, at least in part, by bindingto an IL-4 silencer. In agreement with this novel function ofRunx3, the derepression of IL-4 expression is observed in Th1cells that are deficient in CBF� (26), which has been shown to benecessary for Runx function (27).

Under nonskewed culture conditions, TCR-stimulated CD4�

cells express both T-bet and GATA3. The cells are more or lessdifferentiated along a Th1- or Th2-specific lineage, and the twosubpopulations are maintained in a kind of equilibrium. As forGATA3, it is detected even under Th1 conditions, although to asubstantially lower extent than Th2. Here, we demonstrated thatRunx3 physically interacts with GATA3, and suppresses the tran-scriptional activity of GATA3. Thus, under nonskewed as well asTh1 conditions, Runx3 appears to negatively regulate Th2 cyto-kine expression by antagonizing GATA3 activity, contributing tothe augmentation or stabilization of Th1 responses. In agreementwith this hypothesis, double infection of nonskewed CD4� cellswith retroviruses that expressed Runx3 and GATA3 polarized thecells toward IFN-�-only or IL-4-only expression, depending on thebalance of the two factors. We also demonstrated that Runx3-trans-genic CD4� cells were prone to the Th1 phenotype, even in theabsence of TCR stimulation. However, Runx3-transgenic cellswere still able to differentiate along either lineage, given that theintroduction of GATA3 or culturing of cells under Th2 conditionscould redirect the cells to be IL-4 producers. In Runx3-transgeniccells, the protein levels of Runx3 and GATA3 were 1.5-fold higherand 0.7-fold lower, respectively, than in nontransgenic cells. Incontrast, in cells in which Runx3 was ectopically overexpressed upto 3.5-fold over wild-type cells, culturing under Th2 conditionsfailed to restore IL-4 production, which suggests that this level ofRunx3 might be above a certain threshold level that can be neu-tralized by IL-4-induced GATA3. We propose that one mechanism

FIGURE 8. Ig titers in the sera of immunized Runx3-transgenic (tg)mice. Nontransgenic (non-tg) and Runx3-transgenic mice were injected s.c.or i.p. with OVA mixed with CFA (A) or aluminum hydroxide (B), re-spectively. After the second boosting injection, sera were collected, and thetiters of IgG1, IgE, IgG2a, and IgG2b were measured by ELISA. Horizon-tal lines represent the average OD450 for each group. A statistically sig-nificant difference was detected between nontransgenic and Runx3-trans-genic mice (p values are indicated).

FIGURE 9. Impaired activation of B cells in Runx3-transgenic (tg)mice. A, Nontransgenic (non-tg) and Runx3-transgenic mice were immu-nized using OVA mixed with aluminum hydroxide, as described for Fig. 8.Animals were sacrificed after the second boosting injection, and the weightof the spleens was measured. Horizontal lines represent the averages ofeach group. B, Representative images of spleens. Bar, 1 cm. C, Nontrans-genic and Runx3-transgenic mice were immunized once with OVA mixedwith aluminum hydroxide, and the spleen were removed 1 wk after im-munization. Spleen tissue sections were stained using HRP-conjugatedpeanut agglutinin (brown color).

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of regulating the Th1/Th2 preference of TCR-stimulated cells in-volves the balance of Runx3 and GATA3.

In one sense, the role of Runx3 resembles that of T-bet, whichrepresses IL-4 expression through a direct association with GATA3and inhibition of the DNA binding property of GATA3 (9). We dem-onstrated that Runx3/1–404, a Runx3 mutant that is devoid of theGroucho-binding domain, failed to repress IL-4 expression. Groucho/TLE is an evolutionarily conserved corepressor of Runx family pro-teins, and functions by recruiting histone deacetylases (28–30). Ourresults suggest that the repression of IL-4 expression by the GATA3/Runx3 complex may be mediated through the recruitment of histonedeacetylase activity by Groucho.

In nontransgenic Th1 cells, Runx3 was the principal Runx compo-nent, and Runx1 was barely detectable. Similarly, naive CD4� cellsisolated from Runx3-transgenic mice contained only a trace amount ofRunx1. The low level of Runx1 seen in these cells probably reflectsthe transcriptional repression of the Runx1 promoter by an excess ofthe Runx3 transcription factor (31). In contrast, in nontransgenic Th2cells, equivalent amounts of Runx3 and Runx1 were present. Runx3in this cell population might prevent the exacerbation of the Th2 re-sponse, as was previously suggested for Runx1. We actually obtainedresults that are in agreement with this notion. When endogenous Runxactivity was reduced by introducing dominant negative forms ofRunx3, relative expression ratios of IL-4 to IFN-� under Th2 condi-tions were substantially enhanced. Runx1 also interacted withGATA3, similar to Runx3 (data not shown), which suggests that theRunx1-GATA3 complex likely exerts a similar biological effect as theRunx3-GATA3 complex.

T cell-specific Runx3-deficient mice spontaneously developasthma-related symptoms, including elevated serum IgE, which isa hallmark of a Th2 bias (26, 32). We showed that Runx3-trans-genic mice exhibit a Th1-biased phenotype, including elevated ti-ters of serum IgG2a and IgG2b following immunization. Theseresults support the hypothesis that Runx3 is a critical regulator ofTh1 and Th2 responses in vivo. A rather unexpected observation inthe present study was that B cells failed to respond quickly toimmunization in Runx3-transgenic mice, as revealed by the poorstaining of B cells by peanut agglutinin in Runx3-transgenicspleens. Impairment of B cell activation may be due either to thereduction of IL-4 production or an insufficiency of follicular Tcells, both of which are crucial factors for B cell activation (33).

AcknowledgmentsWe thank Dr. S. Akira for Stat6-targeted mice, Drs. K. Ito and Y. Ito forRunx3/R178Q plasmid, and M. Kuji for secretarial assistance.

DisclosuresThe authors have no financial conflict of interest.

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