T-bet expression is regulated by EGR1-mediated signaling in activated T cells

Clinical Immunology (2009) 131, 385–394

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

Clinical Immunology w w w. e l s e v i e r. c o m / l o c a t e / y c l i m

T-bet expression is regulated by EGR1-mediated signaling in activated T cells Hyun-Jin Shin a , Jee-Boong Lee b , Sung-Hwan Park c , Jun Chang b , Chang-Woo Lee a,⁎ a

Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi 440-746, Republic of Korea b Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea c Center for Rheumatic Diseases and Rheumatism Research Center, Catholic Institute of Medical Sciences, The Catholic University of Korea, Seoul 137-701, Republic of Korea

Received 10 February 2009; accepted with revision 13 February 2009 KEYWORDS T-bet; EGR1; Promoter; Transcription; T helper cells; TCR signaling

Abstract T-bet is a Th1-specific transcription factor that is directly involved in three important pathways for Th1 cell differentiation, namely TCR signaling, and the IFN-γ-STAT1 and IL-12-STAT4 pathways. A recent study also showed that T-bet plays a vital role in innate immunity. However, the molecular mechanism responsible for transcriptional activation of T-bet during T cell development is not yet known. Here, we characterize the essential human T-bet promoter elements and show that binding of EGR1 to this promoter induces T-bet transcription. Notably, overexpression of EGR1 transactivates and, synergistically in concert with TCR signaling, induces T-bet expression in activated T cells. In contrast, depletion of EGR1 significantly decreases T-bet induction. Finally, we report a positive correlation between EGR1 and T-bet expression during T helper cell differentiation. Collectively, these findings provide molecular insight into T-bet transcription and suggest that EGR1 is an upstream regulator of T-bet induction. © 2009 Elsevier Inc. All rights reserved.

Introduction Differentiation of naïve CD4+ T cells into T helper type 1 (Th1) effector cells requires both T cell receptor (TCR) signaling and the action of cytokines such as interleukin 12 (IL-12) and interferon-γ (IFN-γ). When naïve CD4+ T cells are initially stimulated, latent transcription factors non-selectively stimulate low-level production of both IFN-γ and IL-4 [1–3]. In the presence of IL-12, Th1 cell development is ⁎ Corresponding author. Fax: +82 31 2996269. E-mail address: [email protected] (C.-W. Lee).

efficiently induced when IFN-γ stimulates the Janus family kinases (Jaks) and Jak-dependent IFN-γ receptor signaling, leading to the activation of STAT1 and subsequent expression of the transcription factor, T-bet [4–7]. Notably, T-bet expression during T cell activation is strongly dependent on IFN-γ signaling and STAT1 activation. T-bet is a Th1-specific transcription factor that directly binds to the IFN-γ promoter and activates the expression of both IFN-γ and the β2 subunit of the IL-12 receptor (IL-12R β2) [5,8,9]. T-bet increases IFNγ production in Th2 cells and represses the expression of Th2 cytokines such as IL-4, IL-5 and IL-13 [10]. T-bet is also known to interact with GATA-3, a Th2-specific transcription factor, and suppresses GATA-mediated production of Th2 cytokines

1521-6616/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2009.02.009

386 [11]. Thus, T-bet appears to act as a master switch for Th1 development. An initial study on T-bet suggested that IL-12 induces Tbet expression through the activation of STAT4. However, another study placed T-bet expression upstream of IL-12 and STAT4 during Th1 development, and found that T-bet is expressed at normal levels in STAT4-deficient T cells [8]. A more recent study showed that ectopic T-bet expression strongly increased IFN-γ production in Th2 cells activated by PMA and ionomycin, but increased IFN-γ production only weakly in Th2 cells stimulated by IL-12, IL-18 or OVA peptideantigen presenting cells [5]. It thus appears that various modes of stimulation (TCR signaling, cytokine treatment or chemical activation) trigger different signaling pathways and mechanisms for T-bet expression and its involvement in IFN-γ production and Th1 development. The physiological effects of T-bet were demonstrated in Tbet-deficient mice, which showed impaired Th1 immunity characterized by a pronounced decrease in IFN-γ production and elevated levels of Th2 cytokines [12]. These mice resisted the development of autoimmune diseases, but spontaneously developed airway eosinophilia, bronchial hyperresponsiveness, and airway remodeling [13]. It has been further reported that T-bet is associated with allergic asthma in children [14], and with pharmacogenetic responses to asthma therapies based on inhaled corticosteroids [15]. T-bet also appears to be required for protective response against infectious pathogens [16,17]. Importantly, a recent study found that mice lacking T-bet expression showed spontaneous and communicable ulcerative colitis in the innate immune system, and increased susceptibility to colitis in immunologically intact hosts, indicating that T-bet also plays a critical role in protecting against inflammatory disease [18]. In addition to its critical role in CD4+ T cell differentiation, T-bet also appears to influence the generation of type 1 immunity by controlling IFN-γ production and effector function in CD8+ T cells, natural killer (NK), and dendritic cells (DCs) [19–21]. Despite our molecular and physiological understanding of T-bet-mediated signaling, however, it is not yet clear how T-bet is transcriptionally activated in response to TCR, cytokine signaling, and chemical activation during T cell development. Early growth response gene 1 (EGR1), a transcriptional regulator that contains a zinc-finger DNA binding domain, is expressed in many cell types and is rapidly induced in response to a number of extracellular stimuli [22–24]. This suggests that EGR1 may be an example of an ‘immediate early gene,’ i.e. one that increases rapidly after stimulation and variously helps the cell transition from resting/quiescent status to the states of growth, division and/or differentiation. However, the precise function of EGR1 still remains to be elucidated. EGR1 is induced in both B and T cells upon antigen receptor cross-linking or in response to mitogenes or TCR signals [23,25,26]. In T lymphocytes, EGR1 is rapidly induced in response to TCR signaling, and activates the transcription of IL-2, IL-2Rβ, TNF-α, FasL and CD154 [27–31]. This seems to indicate that EGR1 forms an important link between the various signaling pathways and the downstream regulators of gene expression. In this study, we characterized the essential transcriptional regulatory elements in the T-bet promoter, and examined whether EGR1 binds to the T-bet promoter for

H.-J. Shin et al. transactivation of T-bet expression. Our results collectively suggest that the expression of T-bet during T helper cell differentiation appears to be required for EGR1-mediated transcriptional activation, indicating that EGR1 is a critical transcriptional regulator of T-bet expression.

Materials and methods Plasmids The putative T-bet promoter region (spanning nt − 1029 to + 171) was amplified from genomic DNA isolated from Jurkat T cells and cloned into the pXP2 vector, which contains a promoterless luciferase gene (Promega). To identify the essential promoter region, various 5′ and 3′ deletion mutants were generated by PCR amplification and cloned into pXP2. To generate deletion mutants of putative EGR1 binding elements, PCR-generated deletion mutants [E1 (− 583 to − 137/− 128 to +171); E2 (−583 to − 104/− 94 to +171); E3 (− 583 to +52/+ 62 to +171); E4 (−583 to +52/+ 62 to + 171)] were cloned into the pXP vector. An oligonucleotide encoding an shRNA against EGR1 (5′-GTTACTACCTCTTAATCCAT-3′) was synthesized and inserted into the pSuper-puro vector, which contains an H1 promoter and T5 terminal sequences (Oligoengine). The PCR-amplified EGR1 cDNA was subcloned into the Myc-tagged pcDNA3 vector to generate pcDNA3EGR1.

Cell culture and transfection The human T cell lines, Jurkat T (ATCC) and CCRF-CEM (Korean Cell Line Bank), and the human B cell lines, IM-9 (Korean Cell Line Bank) and Raji (ATCC), were cultured in RPMI1640 containing 10% FBS and 1% antibiotics, at 37 °C in a humidified incubator with 5% CO2. For transfection, the Tbet promoter constructs (5 μg) were cotransfected along with pCMV-β-galactosidase (1 μg) into Jurkat T cells, using a microporator (Digital Bio Technology) according to the manufacturer's recommendations.

Chromatin immunoprecipitation (ChIP) assay The ChIP assay was performed using a Chromatin Immunoprecipitation Kit (Upstate) according to the manufacturer's recommendations. Briefly, 5 × 106 Jurkat T cells were cultured in the presence or absence of PMA (10 ng/ml) and ionomycin (500 ng/ml) for 3 h, and then fixed with 1% formaldehyde at 37 °C for 10 min. The cells were subsequently harvested and sonicated in lysis buffer (Upstate). Pre-cleared chromatin was incubated with 2 μg of antibodies against EGR-1 (Santa Cruz), SP1 (Santa Cruz), rabbit-IgG (Santa Cruz), acetyl-histone H3 (Santa Cruz), methyl-histone H3 (at Lys 9, Upstate), or normal IgG (Santa Cruz) for 16 h at 4 °C. The input DNA was separated from the sheared DNA prior to immunoprecipitation, and the purified and eluted chromatin were reverse crosslinked, purified, and subjected to PCR amplification using primers designed based on the T-bet promoter sequence (forward, 5′-GGGAGCCGGAGAGCTTCATAA-3′, reverse, 5′-CGGCTCGGTGCCCGTCAGC-3′; amplifying a fragment spanning nt −128 to +222).

EGR1 transactivates T-bet expression

Immunoblotting For immunoblotting analysis, cells were harvested, washed twice in cold PBS, and lysed in extraction buffer [20 mM HEPES (pH 7.6), 20% glycerol, 250 mM NaCl, 1.5 mM MgCl2, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol (DTT), and protease inhibitor cocktail (Roche)]. Protein concentrations were determined using the Bio-Rad Protein Assay, with bovine serum albumin as the standard. Samples containing equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked, and probed with antibodies specific for EGR-1 (Santa Cruz), SP1 (Santa Cruz), T-bet (Santa Cruz), phospho-STAT1 (Chemicon), and actin (Sigma).

Quantitative real-time PCR The RNeasy RNA extraction kit (Qiagen) was used to extract total RNA from human T and B cells. First-strand cDNA was synthesized using the Improm II Reverse transcriptase kit (Promega). The quantitative PCR reactions (total volume, 15 μl) included the cDNA (0.2 μl for EGR1 and GAPDH; 0.6 μl for T-bet), specific primers (final concentration of 200 nM) and SYBR Green PCR Master Mix (Applied Biosystems). Each reaction was performed in triplicate, and the mRNA levels were normalized relative to that of GAPDH. The primer sequences were as follows: EGR1, 5′-AAAGTTTGCCAGGAGCGATG-3′ and 5′-CAGGGGATGGGTATGAGGTG-3′; T-bet, 5′-GCCTACCAGAATGCCGAGATTA-3′ and 5′-GGACTCAAAGTTCTCCCGGAAT-3′; GAPDH, 5′-TGCACCACCAACTGCTTAGC-3′ and 5′-GGCATGGACTGTGGTCATGAG-3′.

Polarization of mouse CD4+ T cells, and FACS OTII TCR-transgenic mice were purchased from Jackson Laboratory. Splenocytes from OTII TCR-transgenic mice were stimulated with 1 μM ovalbumin peptide (OVA) 323-339 (ISQAVHAAHAEINEAGR) for 7 days in the presence of αIL-4 (10 μg/ml, Biolegend) and αIFN-γ (10 μg/ml, Biolegend) for Th0 polarization, with rIL-12 (10 ng/ml, R&D) and αIL-4 for Th1 polarization, or rIL-4 (10 ng/ml, R&D) and αIFN-γ for Th2 polarization. Human rIL-2 (R&D) was added to the polarized cultures 72 h after stimulation. At day 5, cells were harvested from each culture and stimulated with PMA and ionomycin for 5 h, subjected to intracellular staining with anti-CD4-Cy3 (Biolegend), anti-IFN-γ-FITC (Biolegend) and anti-IL-4-PE (Ebioscience), and analyzed by flow cytometry. Plots were gated on CD4+ cells. After day 7, the CD4+ Tcells were purified by negative selection using magnetic bead separation (MACS) according to the manufacturer's instructions (Miltenyi Biotec). The Th0-, Th1- or Th2-polarized cells were then restimulated with PMA (10 ng/ml) and ionomycin (500 ng/ml) for 3 h prior to harvesting for flow cytometric and immunoblotting analyses.

Results EGR1 binds to the T-bet promoter and transactivates T-bet expression during T cell development To investigate the transcriptional mechanism responsible for T-bet expression, we determined the T-bet promoter

387 region. First, we identified the transcription start site (+ 1) using SMART RACE cDNA amplification (data not shown, Fig. 1A). We then amplified a ∼ 760-bp fragment containing the 5′ flanking region upstream of the translational initiation site (ATG). The luciferase activity of this promoter sequence was about 60-fold higher than that observed in cells transfected with the promoterless luciferase backbone vector (Figs. 1A and B), indicating that we had isolated a functional promoter. To define the minimal sequences required for the transcription of T-bet, we generated a series of luciferase reporter gene constructs containing 3′ and 5′ deletion mutants of the identified promoter sequence. Our results revealed that the region from − 247 to +171 was sufficient to confer the full transcriptional activity of the T-bet promoter. Next, we searched for potential cis-acting regulatory elements within the minimal T-bet promoter region, using the MatInspector version 2.2 and MATCH version 1.0 computer programs. We identified putative binding sites for multiple transcription factors, including SP1, EGR1, OCT1, ATF and CREB (Fig. 1A). Interestingly, the results of our deletion mapping (Fig. 1B) suggested that four cis-acting regions, designated E1, E2, E3 and E4, appeared to be important for T-bet transcriptional activity. These regions included the putative EGR1 and SP1 binding sites. Therefore, we generated five internal deletion mutants of the T-bet promoter, selectively targeting the E1, E2, E3 and E4 elements. We found that disruption of regions −143 to −129 (ΔE1), − 109 to −95 (ΔE2), and +70 to +88 (ΔE3) singly or in combination significantly reduced T-bet promoter activity (Fig. 1C). When all three elements (ΔE2/ΔE3/ΔE4) were internally deleted, the reporter construct completely lost its promoter activity. These findings suggest that EGR1 and/or SP1 may be critical for T-bet induction. To examine whether EGR1 and/or SP1 bind directly to the T-bet proximal promoter, we performed chromatin immunoprecipitation (ChIP) using primers encompassing the T-bet minimal promoter region (−247 to + 171). As shown in Figure 1D, ChIP using anti-EGR1 and -SP1 antibodies showed that both EGR1 and SP1 bound to the T-bet promoter. In contrast, no binding was seen with immunoprecipitation using control IgG. Interestingly, the binding of EGR1 to the T-bet promoter was significantly enhanced in Jurkat T cells stimulated with PMA and ionomycin, whereas this stimulation did not induce apparent changes in the binding of SP1 to the T-bet promoter. We observed that stimulation with PMA-ionomycin yielded a marked decrease of methylated-histone H3 (at Lys 9) binding but a significant increase of acetylatedhistone H3 binding to the T-bet promoter; these findings were consistent with those reported in a previous study, and were used as controls [32]. An electromobility shift assay of Jurkat T cell nuclear extracts confirmed that EGR1 physically interacted with the cis-acting elements in the Tbet promoter (Supplemental Fig. S1). Next, we compared the expression levels of T-bet, EGR1 and SP1 in Jurkat T cells activated by PMA-ionomycin. Stimulation with PMAionomycin was associated with increased expression of Tbet and EGR1, but no significant change was seen in the expression of SP1 (Fig. 1E). Together, these results led us to hypothesize that EGR1 plays a role in transactivating T-bet expression during T cell development.

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EGR1 is an upstream regulator of T-bet expression To determine whether EGR1 directly activates T-bet transcription, we generated expression plasmids encoding EGR1 or an EGR1-targeting shRNA, and transfected them separately into Jurkat T cells along with the T-bet promoter construct.

H.-J. Shin et al. The transfected Jurkat Tcells were cultured and treated with PMA-ionomycin, and we examined T-bet promoter activity (Fig. 2A). The luciferase reporter activity driven by the T-bet promoter was significantly increased by EGR1 overexpression, but sharply decreased by depletion of EGR1. Moreover, the transcriptional activation of T-bet mediated by EGR1

EGR1 transactivates T-bet expression expression was further enhanced under stimulation with PMA-ionomycin, indicating that EGR1 transactivates T-bet expression to a higher degree in activated T cells. As shown in Figures 1 and 2A, stimulation with PMAionomycin activated binding of EGR1 to the T-bet promoter element, and overexpression of EGR1 transactivated expression of the T-bet reporter gene. Based on these results, we generated various T-bet promoter mutant constructs with internal deletions of each EGR1-binding element, and cotransfected them into Jurkat T cells along with an EGR1 expression vector or control backbone (Fig. 2B). Although vectors lacking the various EGR1-binding elements showed luciferase activities similar to that driven by the wild type Tbet promoter in unstimulated cells, the deletions (particularly of E1, E2 or E3) significantly reduced the promoter activity in PMA-ionomycin-treated cells, compared to that driven by the wild type promoter. To further assess this effect, we transfected an EGR1 expression plasmid into Jurkat T cells, and cultured the cells in the absence of PMA-ionomycin. Notably, overexpression of EGR1 in the unstimulated Jurkat T cells significantly activated the transcription of both wild type and mutant T-bet promoters to the same degree as seen in chemically stimulated cells. These results indicate that EGR1 may be an upstream regulator of T-bet expression. As binding of EGR1 to the T-bet promoter and T-bet promoter activity are both increased following PMA-ionomycin stimulation, we next examined whether EGR1 contributed to the expression or induction of T-bet protein. Jurkat T cells were transfected with an expression plasmid encoding EGR1, an EGR1-targeting shRNA, or luciferase (negative control), and cultured in the absence or presence of PMA-ionomycin. As shown in Figure 2C, endogenous EGR1 was efficiently induced by PMA-ionomycin treatment, and this effect was depleted by transfection with the EGR1-targeting shRNA. Under these conditions, the levels of T-bet protein appeared to directly correlate with those of EGR1. Furthermore, overexpression of EGR1 led to a marked induction of T-bet in the PMAionomycin-stimulated cells. The amount of T-bet protein in EGR1-overexpressing cells stimulated with PMA-ionomycin was highly augmented compared to that in cells cultured in the absence of PMA-ionomycin. In contrast, there was no apparent induction of T-bet protein levels in Jurkat T cells overexpressing SP1 (data not shown). Together, these data suggest that the expression of T-bet in activated T cells

389 appears to be required for the EGR1-mediated transcriptional activation.

T-bet expression in human T and B cells appears to rely on EGR1-mediated signaling T-bet expression is known to be mediated by INF-γ/STAT1 and TCR signaling [4,6,10]. In addition, EGR1 expression is also rapidly elevated in response to TCR signaling [23,24]. To evaluate the correlation of EGR1 expression with these signaling pathways, we treated Jurkat T cells with INF-γ and/ or PMA-ionomycin and immunoblotted using antibodies against EGR1, phosphorylated-STAT1 (pSTAT1), T-bet or actin. As shown in Figure 3A, our results revealed that EGR1 increased following treatment with PMA-ionomycin but not INF-γ, whereas pSTAT1 was induced by both treatments. The expression level of T-bet protein was slightly increased in INF-γ-treated cells, but was much higher in PMA-ionomycin-treated cells. Interestingly, Tbet expression was synergistically activated in cells treated with both PMA-ionomycin and INF-γ, whereas co-treatment did not increase the levels of pSTAT1 above those seen in response to either treatment alone. These findings strongly suggest that EGR1-mediated signaling plays an important role in triggering Tbet expression in response to cytokine- and TCR-mediated signaling. To further confirm that T-bet expression is mediated via the EGR1 pathway following PMA-ionomycin stimulation, we transfected the EGR1 expression plasmid into Jurkat T cells, cultured the cells in the presence of PMA-ionomycin, and examined the correlation between EGR1 and T-bet expressions. As shown in Figure 3B, the overexpression of EGR1 significantly induced the expression of T-bet, whereas there was no apparent change in pSTAT1 expression. Several studies have found that T-bet performs diverse physiological functions in different cell types, including CD8+ T cells, NK cells and DCs [19–21]. To examine the effect of EGR expression on T-bet transcription in other cell types, expression plasmids encoding EGR1 or the EGR1-targeting shRNA were transfected into CCRF-CEM cells (T cells), IM-9 and Raji cells (B cells) and Jurkat T cells (control) (Figs. 3C and D). Transfected cells were stimulated, total RNA was purified, and the mRNA expression levels of T-Bet and EGR1 were measured by quantitative PCR. As shown in Figure 3C, T-bet expression was highly associated with the levels of EGR1 in IM-9, CCRF-

Figure 1 EGR1 binds to the T-bet promoter and activates T-bet transcription. (A) Sequence of the core T-bet promoter (−247 to + 171). The major transcription start site (+ 1) was determined by 5′-RACE. Putative transcription factor binding sites in the minimal Tbet promoter sequence are underlined. E1, E2, E3 and E4 indicate the putative EGR1 and SP1 binding elements. (B) To define the minimal promoter sequence, several mutants of the T-bet promoter were generated by progressive deletion of sequences from the 3′ and 5′ ends. The promoter activities of the mutant T-bet promoter were compared with the background activity from the promoterless luciferase backbone vector, pXP2. The relative activity of luciferase to β-galactosidase is presented. (C) Truncation of the putative EGR1 and SP1 binding elements in the T-bet promoter (ΔE1, lacking −143 to −129; ΔE2, lacking −109 to −95; ΔE3, lacking + 70 to +88; ΔE4, lacking +120 to + 138; ΔE1/ΔE2, lacking − 143 to −129 and −109 to − 95; ΔE3/E4, lacking + 70 to + 88 and +120 to + 138; ΔE2/E3/E4, lacking −109 to − 95, +70 to + 88 and + 120 to +138). The five constructs were transfected into Jurkat T cells along with a βgalactosidase expression plasmid. WT, full-length (−583 to + 171) T-bet promoter. (D) ChIP analyses of the association of EGR1 and SP1 with the T-bet promoter region in Jurkat T cells treated with or without PMA-ionomycin. Crosslinked cell lysates were prepared from Jurkat Tcells that had been cultured in the absence or presence of PMA-ionomycin (P/I). Proteins immunoprecipitated with antibodies against EGR1, SP1, acetylated-histone H3 (AcH3), methylated-histone H3 (MeH3) or control IgG were decrosslinked. DNA extracted from each immunoprecipitated chromatin sample was PCR-amplified using T-bet promoter-specific primers designed to amplify the region from − 128 to + 222. (E) Extracts from Jurkat Tcells cultured in the absence (−) or presence (+) of PMA-ionomycin (for 6 and 12 h) were immunoblotted with anti-EGR1, anti-SP1, anti-T-bet and anti-actin antibodies.

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Figure 2 T-bet induction is regulated by EGR1 expression in the activated T cells. (A) Jurkat T cells were cotransfected with vectors encoding EGR1 or an EGR1-targeting shRNA along with the T-bet promoter, and then cultured in the absence (−) or presence (+) of PMAionomycin (P/I). The relative luciferase to β-galactosidase activity was determined. The results were confirmed by three independent experiments. (B) Truncation mutants of each EGR1 binding element were generated from the full-length T-bet promoter (−583 to +171), as described in Figure 1. The resulting constructs were transfected into Jurkat T cells with either a control backbone vector (pMyc) or a plasmid encoding EGR1, and the cells were cultured in the absence or presence of PMA-ionomycin. The relative luciferase activity was measured as described in Figure 1B. (C) Jurkat T cells were transfected with plasmids encoding EGR1, the EGR1-targeting shRNA, or luciferase (control). After 24 h post-transfection, cells were further cultured in the absence (−) or presence (+) of PMAionomycin (P/I). The cells were harvested and analyzed by immunoblotting using anti-EGR1, anti-T-bet or anti-actin antibodies. (D) Jurkat T cells were transfected with plasmids encoding EGR1, STAT1 α/β, or SP1. The cells were cultured as described above, and analyzed by immunoblotting with anti-EGR1, anti-phospho-STAT1, anti-SP1, anti-T-bet or anti-actin antibodies.

CEM and Jurkat T cells. No such effect was seen in Raji cells, but these cells showed relatively low levels of EGR1 mRNA expression, possibly due to transfection resistance. We did not observe significant changes in IFN-γ production during or after EGR1-mediated T-bet induction. Furthermore, knockdown of EGR1 expression by shRNA transfection was associated with a significant reduction of the T-bet transcription in all cells tested (Fig. 3D). Together, these results indicate that T-bet expression appears to rely on EGR1-mediated signaling.

Expression of T-bet during T helper cell differentiation appears to be mediated by the status of EGR1 induction To examine the correlation between EGR1 and T-bet expression during T helper cell differentiation, we established an in vitro assay using a homogenous population of naïve CD4+ T cells. CD4+ T cells were isolated from OTII mice, labeled with

EGR1 transactivates T-bet expression

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Figure 3 T-bet expression is synergistically activated by EGR1 and TCR signaling. (A) Jurkat T cells were treated with recombinant IFN-γ (3000 U/ml) and/or PMA-ionomycin for 18 h and harvested for immunoblotting using anti-EGR1, anti-phospho-STAT1, anti-T-bet or anti-actin antibodies. (B) Jurkat Tcells were transfected with either a control backbone vector (pCDNA3) or an EGR1 expression plasmid (pDNA3-EGR1), and then cultured in the presence of PMA-ionomycin. The cells were harvested and analyzed by immunoblotting with anti-EGR1, and phospho-STAT1, anti-T-bet or anti-actin antibodies. (C) Human Tcell (Jurkat and CCRF-CEM) and B cell (IM-9, Raji) lines were transfected with a control backbone vector or an EGR1 expression vector, and then cultured in the presence of PMA-ionomycin. Total RNA was extracted from the transfected cells, and mRNA was amplified by quantitative real-time PCR (qRT-PCR), as described in the Materials and methods section. (D) IM-9, Raji, Jurkat and CCRF-CEM cells were transfected with plasmids expressing control luciferase shRNA (shLuc) or EGR1-targeting shRNA (shEGR1), and cultured in the presence of PMA-ionomycin. The relative mRNA levels of EGR1 and T-bet were measured by qRT-PCR.

carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with anti-CD3 and anti-CD28, and cultured under Th0 cell-, Th1 cell- and Th2 cell-skewing conditions (Figs. 4A and B). We analyzed the correlation between EGR1 and T-bet inductions by immunoblotting using antibodies against EGR1, T-bet or actin. Consistent with previous reports [5,8–10], T-bet protein was expressed under Th1 cell-skewing conditions but not under Th0 cell- or Th2 cell-skewing conditions in the absence of restimulation with PMA-ionomycin (Fig. 4C). Interestingly, we were also unable to detect EGR1 under the

latter two conditions. After restimulation, EGR1 was greatly induced under Th0 cell- and Th1 cell-polarizing conditions but not under Th2 cell-polarizing conditions. Notably, the levels of T-bet protein were markedly increased in accordance with those of EGR1 under the Th0 cell- and Th1 cell-polarizing conditions (Fig. 4C). In contrast, the levels of both EGR1 and Tbet were highly decreased under Th2 cell-skewing conditions. Together, these data indicate that the expression of T-bet during T helper cell differentiation appears to be mediated by the status of EGR1 induction. However, after restimulation,

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Figure 4 EGR1 and T-bet expression levels are directly correlated during T helper cell differentiation. (A and B) Naïve T cells were activated with OVA peptide-APCs under Th0 cell-, Th1 cell-, or Th2 cell-polarizing conditions. On day 5, CD4+ T cells were purified by two-color sorting. The sorted cells were expanded for another two days, collected on day 7, and restimulated with PMA-ionomycin (P/I). The CD4+ T cells were subjected to intracellular staining (ICS) with anti-CD4-Cy3, anti-IFN-γ-FITC and anti-IL-4-PE antibodies, and analyzed by flow cytometry. The plots were gated on CD4+ T cells, and the numbers represent the percentage of gated cells in each quadrant. (C) The Th0-, Th1- or Th2-polarized cells were restimulated with PMA-ionomycin on day 7 as described above, and then harvested and immunoblotted with anti-EGR1, anti-T-bet and anti-actin antibodies.

both EGR1 and T-bet proteins were significantly induced in the non-skewing Th0 cells, to approximately the same degree seen under the Th1 cell-skewing conditions. These unexpected observations raise the new possibility that T-bet induction may directly rely on the condition of EGR1-mediated signaling rather than the condition of T helper cell differentiation. Alternatively, it may be possible that the Th2 cell polarization is due, at least in part, to negative regulation via impairment of EGR1-mediated T-bet induction.

Discussion T-bet was initially proposed to be induced by IL-12 and STAT4 activation [33]. However, another study placed T-bet

expression upstream of IL-12 and STAT4 during Th1 cell development, showing that T-bet expression was dependent on IFN-γ and STAT1, but not STAT4 [5]. Still another study found that IL-12-STAT4 binds to the T-bet enhancer, and induces IFN-γ-STAT1-independent T-bet expression in CD8+ T cells [34]. Thus, the molecular mechanism by which IFN-γ and STAT1 direct T-bet expression remains unclear. Moreover, most prior studies have focused on the functional role of T-bet in T helper cells and its etiological role in disease. The molecular mechanisms responsible for transcriptional activation of T-bet in response to cell surface receptor-generated signals have not yet been elucidated. Here, we characterized the essential transcriptional regulatory elements in the promoter region of the human T-bet gene, and found that EGR1 binds to four cis-acting elements in the T-bet promoter,

EGR1 transactivates T-bet expression thereby activating T-bet transcription. The induction of T-bet in response to TCR and chemical activators in human T and B cells appears to rely on EGR1-mediated signaling. T-bet is a Th1-specific transcription factor that is directly linked to three important pathways for Th1 cell differentiation, namely TCR signaling, and the IFN-γ-STAT1 and IL-12STAT4 pathways [5,7,34,35]. However, recent studies have strongly indicated that T-bet not only acts a master switch for Th1 development, it also plays a vital role in innate immunity [18], which controls the spread of infection in the host. In this context, it is likely that T-bet performs diverse physiological functions in cells other than T lymphocytes (e.g. DCs, CD8+ T cells and NK cells), and may trigger a variety of intracellular signals. Interestingly, TCR-generated intracellular signals rapidly change the activity and expression of transcriptional regulators, including T-bet, which then elicit the signaling cascades that trigger the gene expression necessary for establishment of the mature T cell phenotype [23]. Together, these observations suggest that Tbet induction may be coupled to the pathogen-associated signal, and its time- and location-specific expression appears to be critical for the maintenance of immune homeostasis in response to a variety of extracellular stimuli. However, it is unlikely that T-bet is a rapid response mediator between cell surface receptor-generated signals and downstream changes in gene expression. Notably, EGR1 seems to be a good candidate as an immediate early gene responding to a number of extracellular stimuli, including TCR signaling [22]. However, the precise function of EGR1 still remains to be elucidated in most systems. In this study, consistent with previous studies [23,25,26], we showed that EGR1 is efficiently induced in human T and B cell lines and mouse T helper cells upon stimulation with PMA-ionomycin. We further showed for the first time, that EGR1 binds to cisacting elements in the T-bet promoter, thereby activating Tbet transcription. These new findings may explain how T-bet could be rapidly induced upon stimulation. Introduction of T-bet in developing Th2 cells has been shown to trigger the production of IFN-γ and the concomitant reduction of Th2 cytokines such as IL-4 and IL-5 [10], thereby impeding Th2 differentiation. Similarly, it has been suggested that the principle function of T-bet in developing Th1 cells may be to negatively regulate GATA-3, thereby inhibiting the production of Th2 cytokines [36]. However, one notable biochemical aspect of Th2 cells is the widespread suppression of T-bet expression. Although this lack could be potentially counteracted by IFN-γ in Th2 cells, a recent report showed that IFN-γ is not sufficient to maintain T-bet induction after initial stimulation [34]. Thus, our present results suggest that EGR1 could be an alternative pathway for maintaining T-bet induction. Indeed, EGR1 is induced both acutely and chronically by a variety of stimuli, including cytokines and inflammatory signals. Moreover, EGR1 expression is very low in resting T cells, but is upregulated following stimulation by TCR and/or chemical activators. Interestingly, we were unable to detect significant induction of EGR1 in Th2polarized cells following stimulation with PMA-ionomycin, and these cells also showed minimal induction of T-bet. Therefore, it is likely that the lack of EGR1 induction in Th2 cells is an additional important mechanism underlying the lack of T-bet expression in these cells. Future experiments will be required to address this important issue.

393 In summary, this study provides novel evidence that EGR1 is a critical transcriptional regulator of T-bet expression, and that T-bet induction during T cell development appears to be regulated by EGR1-mediated signaling.

Acknowledgments This study was supported by research grants from the Korea Science and Engineering Foundation through the Rheumatism Research Center (RII-2002-098-05003, 2008) and the Korea Health 21 R&D Project, Ministry of Health and Welfare (03-PJ10-PG13-GD01-0002).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.clim.2009.02.009.

References [1] O. Avni, D. Lee, F. Macian, S.J. Szabo, L.H. Glimcher, A. Rao, T(H) cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes, Nat. Immunol. 3 (2002) 643–651. [2] I.C. Ho, L.H. Glimcher, Transcription: tantalizing times for T cells, Cell 109 (2002) S109–S120. [3] J.D. Farrar, H. Asnagli, K.M. Murphy, T helper subset development: roles of instruction, selection, and transcription, J. Clin. Invest. 109 (2002) 431–435. [4] A.A. Lighvani, D.M. Frucht, D. Jankovic, H. Yamane, J. Aliberti, B.D. Hissong, B.V. Nquven, M. Gadina, A. Sher, W.E. Paul, J.J. O'Shea, T-bet is rapidly induced by interferon-gamma in lymphoid and myeloid cells, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 15137–15142. [5] M. Afkarian, J.R. Sedy, J. Yang, N.G. Jacobson, N. Cereb, S.Y. Yang, T.L. Murphy, K.M. Murphy, T-bet is a STAT1-induced regulator of IL-12R expression in naïve CD4+ T cells, Nat. Immunol. 3 (2002) 549–557. [6] K.M. Murphy, S.L. Reiner, The lineage decisions of helper Tcells, Nat. Rev., Immunol. 2 (2002) 933–944. [7] M. Shi, T.H. Lin, K.C. Appell, L.J. Berg, Janus-kinase-3-dependent signals induce chromatin remodeling at the Ifng locus during T helper 1 cell differentiation, Immunity 28 (2008) 763–773. [8] A.C. Mullen, F.A. High, A.S. Hutchins, H.-W. Lee, A.V. Villarino, D.M. Livingston, A.L. Kung, N. Cereb, T.P. Yao, S.Y. Yang, S.L. Reiner, Role of T-bet in commitment of TH1 cells before IL-12dependent selection, Science 292 (2001) 1907–1910. [9] I.M. Djuretic, D. Levanon, V. Negreanu, Y. Groner, A. Rao, K.M. Ansel, Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells, Nat. Immunol. 8 (2007) 145–153. [10] S.J. Szabo, S.T. Kim, G.L. Costa, X. Zhang, C.G. Fathman, L.H. Glimcher, A novel transcription factor, T-bet, directs Th1 lineage commitment, Cell 100 (2000) 655–669. [11] E.-S. Hwang, S.J. Szabo, P.L. Schwartzberg, L.H. Glimcher, T helper cell fate specified by kinase-mediated interaction of Tbet with GATA-3, Science 307 (2005) 430–433. [12] S.J. Szabo, B.M. Sullivan, C. Stemmann, A.R. Satoskar, B.P. Sleckman, L.H. Glimcher, Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells, Science 295 (2002) 338–342. [13] S. Finotto, M.F. Neurath, J.N. Glickman, S. Qin, H.A. Lehr, F.H. Green, K. Ackerman, K. Haley, P.R. Galle, S.J. Szabo, J.M. Drazen, G.T. De Sanctis, L.H. Glimcher, Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet, Science 295 (2002) 336–338.

394 [14] M.C. Munthe-Kaas, K.H. Carlsen, G. Håland, C.S. Devulapalli, K. Gervin, T. Egeland, K.L. Carlsen, D. Undlien, T cell-specific T-box transcription factor haplotype is associated with allergic asthma in children, J. Allergy Clin. Immunol. 121 (2008) 51–56. [15] K.G. Tantisira, E.-S. Hwang, B.A. Raby, E.S. Silverman, S.L. Lake, B.G. Richter, S.L. Peng, J.M. Drazen, L.H. Glimcher, S.T. Weiss, TBX21: a functional variant predicts improvement in asthma with the use of inhaled corticosteroids, Proc. Natl. Acad. Sci.U. S. A. 101 (2004) 18099–18104. [16] G. Lugo-Villarino, S. Ito, D.M. Klinman, L.H. Glimcher, The adjuvant activity of CpG DNA requires T-bet expression in dendritic cells, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 13248–13253. [17] B.M. Sullivan, O. Jobe, V. Lazarevic, K. Vasquez, R. Bronson, L.H. Glimcher, I. Kramnik, Increased susceptibility of mice lacking T-bet to infection with Mycobacterium tuberculosis correlates with increased IL-10 and decreased IFN-gamma production, J. Immunol. 175 (2005) 4593–4602. [18] W.S. Garrett, G.M. Lord, S. Punit, G. Lugo-Villarino, S.K. Mazmanian, S. Ito, J.N. Glickman, L.H. Glimcher, Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system, Cell 131 (2007) 33–45. [19] M.J. Townsend, A.S. Weinmann, J.L. Matsuda, R. Salomon, P.J. Farnham, C.A. Biron, L. Gapin, L.H. Glimcher, T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells, Immunity 20 (2004) 477–494. [20] J. Wang, J.W. Fathman, G. Lugo-Villarino, L. Scimone, U. von Andrian, D.M. Dorfman, L.H. Glimcher, Transcription factor Tbet regulates inflammatory arthritis through its function in dendritic cells, J. Clin. Invest. 116 (2006) 414–421. [21] N.S. Joshi, W. Cui, A. Chandele, H.-K. Lee, D.R. Urso, J. Hagman, L. Gapin, S.M. Kaech, Inflammation directs memory precursor and short-lived effector CD8(+) T cell fates via the graded expression of T-bet transcription factor, Immunity 27 (2007) 281–295. [22] A. Gashler, V.P. Sukhatme, Early growth response protein 1 (Egr1): prototype of a zinc-finger family of transcription factors, Prog. Nucleic Acid Res. Mol. Biol. 50 (1995) 191–224. [23] H. Shao, D.H. Kono, L.Y. Chen, E.M. Rubin, J. Kaye, Induction of the early growth response (Egr) family of transcription factors during thymic selection, J. Exp. Med. 185 (1997) 731–744. [24] M. Bettini, H. Xi, J. Milbrandt, G.J. Kersh, Thymocyte development in early growth response gene 1-deficient mice, J. Immunol. 169 (2002) 1713–1720.

H.-J. Shin et al. [25] V.L. Seyfert, S. McMahon, W. Glenn, X.M. Cao, V.P. Sukhatme, J.G. Monroe, Egr-1 expression in surface Ig-mediated B cell activation. Kinetics and association with protein kinase C activation, J. Immunol. 145 (1990) 3647–3653. [26] D. Makover, M. Cuddy, S. Yum, K. Bradley, J. Alpers, V. Sukhatme, J.C. Reed, Phorbol ester-mediated inhibition of growth and regulation of proto-oncogene expression in the human T cell leukemia line JURKAT, Oncogene 6 (1991) 455–460. [27] C. Skerka, E.L. Decker, P.F. Zipfel, A regulatory element in the human interleukin 2 gene promoter is a binding site for the zinc finger proteins Sp1 and EGR-1, J. Biol. Chem. 270 (1995) 22500–22506. [28] J.X. Lin, W.J. Leonard, The immediate-early gene product Egr1 regulates the human interleukin-2 receptor beta-chain promoter through noncanonical Egr and Sp1 binding sites, Mol. Cell. Biol. 17 (1997) 3714–3722. [29] E.L. Decker, N. Nehmann, E. Kampen, H. Eibel, P.F. Zipfel, C. Skerka, Early growth response proteins (EGR) and nuclear factors of activated T cells (NFAT) form heterodimers and regulate proinflammatory cytokine gene expression, Nucleic Acids Res. 31 (2003) 911–921. [30] R. Dzialo-Hatton, J. Milbrandt, R.D. Hockett Jr., C.T. Weaver, Differential expression of Fas ligand in Th1 and Th2 cells is regulated by early growth response gene and NF-AT family members, J. Immunol. 166 (2001) 4534–4542. [31] R.Q. Cron, R. Bandyopadhyay, A. Genin, M. Brunner, G.J. Kersh, J. Yin, T.H. Finkel, M.K. Crow, Early growth response-1 is required for CD154 transcription, J. Immunol. 176 (2006) 811–818. [32] A. Morinobu, Y. Kanno, J.J. O'Shea, Discrete roles for histone acetylation in human T helper 1 cell-specific gene expression, J. Biol. Chem. 279 (2004) 40640–40646. [33] J. Rengarajan, S.J. Szabo, L.H. Glimcher, Transcriptional regulation of Th1/Th2 polarization, Immunol. Today 21 (2000) 479–483. [34] Y. Yang, J.C. Ochando, J.S. Bromberg, Y. Ding, Identification of a distant T-bet enhancer responsive to IL-12/Stat4 and IFNγ/ Stat1 signals, Blood 110 (2007) 2494–2500. [35] L.H. Glimcher, Trawling for treasure: tales of T-bet, Nat. Immunol. 8 (2007) 448–450. [36] T. Usui, J.C. Preiss, Y. Kanno, Z.J. Yao, J.H. Bream, J.J. O'Shea, T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription, J. Exp. Med. 203 (2006) 755–766.