Twist1 Regulates Ifng Expression in Th1 Cells by Interfering with Runx3 Function Duy Pham, Joshua W. Vincentz, Anthony B. Firulli and Mark H. Kaplan This information is current as of October 3, 2021. J Immunol 2012; 189:832-840; Prepublished online 8 June 2012; doi: 10.4049/jimmunol.1200854 http://www.jimmunol.org/content/189/2/832 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Twist1 Regulates Ifng Expression in Th1 Cells by Interfering with Runx3 Function

Duy Pham,*,† Joshua W. Vincentz,* Anthony B. Firulli,* and Mark H. Kaplan*,†

A network that includes STAT4, T-bet, and Runx3 promotes the differentiation of Th1 cells and inflammatory immune responses. How additional transcription factors regulate the function of Th1 cells has not been defined. In this study we show that the negative regulatory factor Twist1 decreases expression of T-bet, Runx3, and IL-12Rb2 as it inhibits IFN-g production. Ectopic expression of Runx3, but not T-bet or IL-12Rb2, compensates for the effects of Twist1 on IFN-g production, and Twist1 regulation of Ifng depends on complex formation with Runx3. Twist1 decreases Runx3 and T-bet binding at the Ifng locus, and it decreases chromatin looping within the Ifng locus. These data define an IL-12/STAT4–induced negative regulatory loop that impacts multiple components of the Th1 transcriptional network and provide further insight into regulation of Th1 differentiation. The Journal of Immunology, 2012, 189: 832–840. Downloaded from network of transcription factors regulates the develop- the transcriptional activity of NF-kB (10, 11). In Th1 cells, NF- ment of Th1 cells, cells that play a crucial role in an- kB, NFAT, and IL-12/STAT4 signaling can induce Twist1 ex- A tibacterial and inflammatory immunity. IL-12 and IFN-g pression, and Twist1 limits inflammation by suppressing IFN-g promote Th1 differentiation by the respective activation of STAT4 and TNF-a production (12). However, a detailed mechanism of and STAT1, leading to the induction of additional transcription how Twist1 regulates cytokine production in Th1 cells has not

factors, including T-bet and Runx3. Th1 differentiation requires been described. http://www.jimmunol.org/ both T-bet and STAT4 for complete development of the Th1 ge- In this study, we define the role of Twist1 in the Th1 cell netic program (1) and is mediated by the interlinked and se- transcriptional network. Using retroviral transduction studies, we quential effects of TCR/IFN-g/STAT1/T-bet and IL-12/STAT4/ demonstrate that Twist1 negatively regulates Th1 expression T-bet signaling pathways (2). Runx3 cooperates with T-bet to by decreasing expression and function of transcription factors, activate Ifng and silence Il4 in Th1 cells, but it can regulate Ifng including T-bet, STAT4, and Runx3. Twist1 functions through expression independently of T-bet and STAT4 (3, 4). Although several mechanisms, including physical interaction with Runx3 some factors that impact this network, including GATA3 (5–7), and interfering with the association of T-bet and Runx3 at the Ifng have been described, regulation of these pathways is still not locus. The results suggest that Twist1 acts as a modulator that clearly defined. regulates multiple transcription factors in Th1 cells to limit po- by guest on October 3, 2021 Twist1 is a transcriptional repressor and a member of the basic tentially harmful inflammatory responses. helix-loop-helix family of that plays a positive role in dorsoventral patterning in Drosophila embryos (8). Although the Materials and Methods role of Twist1 in developmental and cancer biology has been Mice explored (9), the function of Twist1 in the immune response C57BL/6 mice were purchased from Harlan Sprague Dawley (Indianapolis). is only beginning to be understood. Twist1 regulates cytokine 2 2 Stat4 / mice were previously described (13). Twist1fl/fl mice (14) were production in macrophages through a negative feedback loop crossed with CD4-Cre transgene mice to generate Twist1fl/fl CD4-Cre+ by targeting NF-kB activation, resulting in decreased TNF-a mice with Cre2 littermates as wild-type (WT) mice. Twist1cc/wt mice (15) and IL-1b (10, 11). The repressive mechanism may involve were crossed with Twist1fl/fl CD4-Cre+ mice to generate Twist1fl/cc CD4- + fl/wt + binding of Twist1 to E-boxes in cytokine promoters to inhibit Cre and Twist1 CD4-Cre as WT control. Mice were maintained under specific pathogen-free conditions. All experiments were performed with the approval of the Indiana University Institutional Animal Care and Use *Department of Pediatrics, Herman B. Wells Center for Pediatric Research, Indiana Committee. University School of Medicine, Indianapolis, IN 46202; and †Department of Micro- biology and Immunology, Indiana University School of Medicine, Indianapolis, IN In vitro T cell differentiation 46202 Naive CD4+CD62L+ T cells were isolated from spleen and lymph nodes Received for publication March 19, 2012. Accepted for publication May 16, 2012. using a MACS isolation system (Miltenyi Biotec). CD4+ T cells were This work was supported by Public Health Service Grants AI045515 (to M.H.K.) and activated with plate-bound anti-CD3 (2 mg/ml; 145-2C11) and soluble AR061392 and HL061677 (to A.B.F.). D.P. was supported by National Institutes of anti-CD28 (0.5 mg/ml; BD Pharmingen for Th0, Th1, Th2, and Th17 Health Grant T32 HL007910. cells or 1 mg/ml for Th9 and regulatory T cells [Tregs]) with additional Address correspondence and reprint requests to Dr. Mark H. Kaplan, Herman B. cytokines (all from PeproTech) and Abs to generate Th1 (5 ng/ml IL-12 Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 and 10 mg/ml anti–IL-4 11B11), Th2 (10 ng/ml IL-4 and 10 mg/ml anti– West Walnut Street, Room 202, Indianapolis, IN 46202. E-mail address: mkaplan2@ IFN-g XMG), Th9 (20 ng/ml IL-4, 2 ng/ml TGF-b, and 10 mg/ml anti– iupui.edu IFN-g XMG), or Th17 (100 ng/ml IL-6, 10 ng/ml IL-23, 10 ng/ml The online version of this article contains supplemental material. IL-1b, 2 ng/ml TGF-b,10mg/ml anti–IL-4 11B11, and 10 mg/ml anti– IFN-g XMG) or Treg (2ng/ml TGF-b,and10mg/ml anti–IL-4, 11B11) Abbreviations used in this article: 3C assay, conformation capture as- say; ChIP, chromatin immunoprecipitation; CNS, conserved noncoding sequence; culture conditions. Cells were expanded after 3 d without additional EGFP, enhanced GFP; RT-qPCR, real-time quantitative PCR; shRNA, short hairpin cytokines (Th0, Th1, and Th2), with 50 U/ml human IL-2 (Tregs), or RNA; qPCR, quantitative PCR; Treg, regulatory T cell; WT, wild-type. with full concentration (Th9) or half concentration of IL-6 (Th17) of the original cytokines in fresh medium. Cells were harvested on day 5 Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 for analysis. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200854 The Journal of Immunology 833

Retroviral expression vectors and retroviral transduction were incubated with either rabbit polyclonal T-bet (4B10), PEBP2b (FL- 182) (Santa Cruz Biotechnology), or normal rabbit IgG (12-370; Milli- Bicistronic retrovirus expressing enhanced GFP (EGFP) only (MIEG) and pore) overnight at 4˚C. The immunocomplexes were precipitated with T-bet and EGFP (T-bet) were previously described (16, 17). pBMN-IRES- A-agarose beads at 4˚C for 2 h, washed, eluted, and reverse GFP-IL-12Rb2c was a gift from Dr. Takashi Usui (Kyoto University). crosslinked at 65˚C overnight. DNA was purified, resuspended in H2O, Il12rb2c and Twist1 cDNAs (Open Biosystems) were digested and andanalyzedbyqPCRwithTaqManorSYBRprimersaspreviously subcloned into MIEG3-EGFP or MIEG3-hCD4. Flag-tagged Twist1 (18) described (19, 21, 22) and Flag-tagged Twist1cc pCDNA3.1 that was made using a QuikChange site-directed mutagenesis kit (Stratagene) with primer pair forward (59- Chromosome conformation capture assay TCAGCTACGCCTTCCCCGTCTGGAGGATG-39)andreverse(59- CATCCTCCAGACGGGGAAGGCGTAGCTGA-39) were digested and A chromosome conformation capture (3C) assay was performed as subcloned into MIEG3-EGFP. Runx3 cDNA (Open Biosystems) was am- described (23–25) with some modifications. Cells (107)werecross- plified, digested, and subcloned into MSCV-Thy1.1. Twist1-targeting short linked with 2% formaldehyde for 10 min at room temperature and hairpin RNA (shRNA) oligonucleotide was designed as described (12) quenched with 0.125 M glycine for 5 min. Cells were lysed with ice- and introduced into RNAi-Ready pSIREN-RetroQ-ZsGreen (Clontech) cold lysis buffer (10 mM Tris-HCl [pH 8], 10 mM NaCl, 0.2% Nonidet according to the manufacturer’s directions. Retroviral stocks were pre- P-40, protease inhibitor mixture) for 30 min. Nuclei were resuspended pared by calcium phosphate transfection of Phoenix GP cells. The medium in 0.5 ml restriction enzyme buffer (NEB3) containing 0.3% SDS and was replaced after 12 h, and viral supernatants were collected 24 and 48 h shaken for 1 h at 37˚C. Triton X-100 (final concentration, 1.8%) was later. Purified CD4+ T cells were cultured under Th1 cell differentiation added and shaken for 1 h at 37˚C to sequester the SDS. Crosslinked conditions. On day 2, cells were transduced with retrovirus-expressing DNA was digested overnight with 400–800 U BglII containing 1 mM vector control or gene of interest by centrifugation at 2000 rpm at 25˚C ATP that has been shown to enhance digestion efficiency (26). Enzyme for 1 h in the presence of 8 mg/ml polybrene. Viral supernatant was was inactivated by addition of SDS (final concentration, 1.6%), and replaced with the former culture supernatant supplemented with 50 U/ml samples were shaken for 20 min at 65˚C. The reaction was diluted with human IL-2. After spin infection, cells were expanded on day 3 and an- 7 ml ligation buffer (50 mM Tris-HCl [pH 8], 10 mM MgCl2,10mM Downloaded from alyzed on day 5. DTT, 1 mM ATP, 1 mg/ml BSA), and Triton X-100 (final concentration, 1%) was added and shaken for 1 h at 37˚C. DNA fragments were ligated Cell sorting, analysis of , and flow cytometry with4000UT4ligase(NEB)for4hat16˚Cfollowedby40minat room temperature. Crosslinks were reversed by incubation with 300 mg Transduced cells were collected on day 5, stained with anti-human CD4 proteinase K overnight at 65˚C. The samples were further incubated Alexa Fluor 647 (BD Biosciences) and anti-rat CD90/mouse CD90.1 FITC with 300 mg RNase for 30–45 min at 37˚C, and the DNAs were purified Abs (BioLegend), and sorted using a FACSVantage SE cell sorter (BD by phenol-chloroform extraction and ethanol precipitation. Ligation http://www.jimmunol.org/ Biosciences). Sorted cells were rested or restimulated with 2 mg/ml anti- products were quantified by quantitative TaqMan real-time PCR using CD3 for real-time quantitative PCR (RT-qPCR; 6 h) and ELISA (24 h). primers as described (23, 25). Additional primers and TaqMan probes Quantitative RT-PCR and ELISA were performed as previously described (with the location in kb from the Ifng transcription start site in paren- (17). For surface staining, resting T cells were stained with anti–IL- thesis) are as follows: (+35) 59-GGAGACACAGAAGTTCGAAG- 12Rb2-PE (R&D Systems) or anti–IL-18Ra-Alexa Fluor 647 (BioLegend) TTAGAA-39, (+46) 59-AAAAACCAACCTGTGTTATTTTG-39,(235) and fixed with 2% paraformaldehyde for 10 min before analysis. For 6FAM-CCTCTGCAAGCCTCACAGAGCA-TAMRA, and (+51) 6FAM- phospho-STAT4 and T-bet analyses, cells were fixed, permeabilized using CCATCTACTGCAAAAAGAAGCT-TAMRA. To generate control tem- 100% ice-cold methanol, and stained for anti–phospho-Stat4-PE (BD plates for the positive controls and to correct for differences in ligation and Pharmingen) and anti–T-bet Alexa Fluor 647 (BioLegend) before analysis. PCR efficiency between different templates, BAC clones were used to generate control template containing all possible ligation products. Equi- Immunoblot, immunoprecipitation, and DNA affinity molar amounts of three BAC clones spanning the mouse Ifng locus (RP23- by guest on October 3, 2021 precipitation assay 353P23, RP23-138P22, and RP23-55O21) and BAC spanning the mouse Gapdh Whole-cell protein lysates were extracted from sorted T cells and immu- locus (RP23-410F11) from CHORI were mixed, digested, phenol- noblotted with Runx3 (6821C3a), Stat4 (C-40), Twist1 (Twist2C1a), T-bet chloroform extracted, ethanol precipitated, and ligated at a DNA concen- m (4B10), or b-actin (C4) (Santa Cruz Biotechnology) as a control. For tration of 300 ng/ l. This sample was used as the DNA reference standard. immunoprecipitation, whole-cell protein lysates were generated from Th1 Relative crosslinking frequencies between the analyzed pairs were calcu- cells or from 293T cells transfected with constructs expressing Twist1, lated as described (23) and were normalized to control interaction fre- Gapdh Runx3, T-bet, Flag-tagged Twist1, or Flag-tagged Twist1cc using calcium quencies using primer pairs within the locus. phosphate. Whole-cell protein lysates were precleared with protein A- agarose (Thermo Scientific) before immunoprecipitation of equal amounts of protein with Runx3, T-bet (Santa Cruz Biotechnology) or Flag (M2; Results Sigma-Aldrich) Abs overnight at 4˚C. Immunocomplexes were captured Twist1 is induced by Th1 cell activation by protein A-agarose and eluted at 100˚C for 5 min in Laemmli sample Twist1 regulates Th1 cytokine production through an unidentified buffer. Immunoprecipitates were separated by 10% SDS-PAGE and immunoblotted with Twist1, Runx3, and T-bet Abs. A DNA affinity pre- mechanism (12). To begin to define that mechanism, we deter- cipitation assay was performed as described (19). Oligonucleotides con- mined the expression of Twist1 in T cell subsets. Twist1 mRNA taining a Twist1 binding site were described previously (20). Additional expression was highest in resting and activated Th1 cells, com- oligonucleotides (biotinylated at the 59 end) containing WT or mutated pared with other T cell subsets (Fig. 1A). Previous reports iden- (bold) Runx3 and T-bet binding sites (underlined) are: WT Runx3 and tified Twist1 as a STAT4 target gene (1, 12). Although Twist1 T-bet, 59-ACCTATGTGGTCTGCCTTTTCTTCTTTCTGGGCACGTTGA- 39; mutant Runx3, 59-ACCTAAGAGGACTGCCTTTTCTTCTTTCTGG- mRNA expression was detected during differentiation, expression 2/2 GCACGTTGA-39;mutantT-bet,59-ACCTATGTGGTCTGCCTTTTCT- was lower in Stat4 than in WT Th1 cells (Fig. 1B). Stimulation TCTTTCTGGGCTCGTTGA-39; and mutant Runx3-T-bet, 59-ACCTA of Th1 cells with anti-CD3 resulted in the induction of Twist1 AGAGGACTGCCTTTTCTTCTTTCTGGGCTCGTTGA-39. Biotinylated mRNA and protein (Fig. 1C, 1D). Thus, Twist1 is expressed in the oligonucleotides were incubated with streptavidin-agarose beads for 30 min at 4˚C. The complex was washed with pull-down buffer (25 mM HEPES, 15 greatest amounts in Th1 cells compared with other T cell subsets mM NaCl, 0.5 mM DTT, 0.5% Nonidet P-40, 0.1 mM EDTA [pH 7.5], 10% and is induced by TCR stimulation. glycerol). Nuclear extracts were added and incubated at 4˚C for 2 h. The complex was washed with pull-down buffer, eluted, and separated with 10% Twist1 negatively regulates the Th1 transcription factor SDS-PAGE. network Chromatin immunoprecipitation To define how Twist1 regulates Th1 cell function, Twist1 was ectopically expressed or targeted by shRNA in Th1 cells using Chromatin immunoprecipitation (ChIP) assay was performed as described (1). In brief, resting Th1 cells were crosslinked for 10 min with 1% retroviral transduction (Fig. 1E, 1F). In agreement with a previous formaldehyde and lysed by sonication. After preclearing with salmon report (12), ectopic Twist1 expression in Th1 cells reduced IFN-g sperm DNA, BSA, and protein A-agarose bead slurry (50%), cell extracts and TNF-a mRNA and protein levels, and decreasing Twist1 ex- 834 Twist1-DEPENDENT REGULATION OF Th1 CELLS Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 1. Twist1 negatively regulates Th1 gene expression and cytokine production. (A) Naive WT lymphocytes were differentiated under neutral conditions (Th0) or Th1 (IL-12 plus anti–IL-4), Th2 (IL-4 plus anti–IFN-g), Th9 (IL-4 plus TGF-b), Th17 (IL-6 plus TGF-b), or Treg (TGF-b)-polarizing conditions in vitro. Twist1 mRNA expression in resting or anti-CD3–activated Th1 cells was determined by RT-qPCR. Data represent means 6 SD of three mice and are representative of two independent experiments. (B) Kinetics of Twist1 mRNA expression during Th1 differentiation in WT and Stat42/2 cells. Average of replicate samples 6 SD. (C and D) Kinetics of Twist1 mRNA expression (C) and Twist1 protein (D) in Th1 cells after stimulation with 2 mg/ml anti-CD3 for 2, 4, 8, 16, and 24 h. Results are representative of two to three independent experiments with similar results. (E–J) Naive WT T cells were stimulated under Th1-polarizing condition. On day 2, cells were transduced with control EGFP (MIEG) or retroviral vector expressing Twist1 and EGFP (Twist1) (E, G, I), or control or short hairpin Twist1 (shTwist1) (F, H, J). On day 5, GFP+ cells were sorted for analysis. (E and F) Altered Twist1 protein expression in cells transduced with Twist1 (E) and altered Twist1 expression in shRNA-transduced Th1 cells (F). (G and H) Th1 gene expression in ectopic Twist1- (G) or short hairpin (H)-transduced cells was assessed by RT-qPCR before (Tbx21 and Runx3) or after (Ifng and Tnfa) 6 h restimulation with 2 mg/ ml anti-CD3. (I and J) Sorted GFP+ cells were stimulated with 2 mg/ml anti-CD3 for 24 h, and supernatants were collected and analyzed for IFN-g and TNF-a by ELISA. Data are (I) means 6 SD of three independent experiments or (J) means of replicate samples 6 SD and are representative of three independent experiments with similar results. *p , 0.05. pression resulted in increased IFN-g and TNF-a production (Fig. cells transduced with Twist1-expressing retrovirus compared with 1G–J). The difference in IFN-g and TNF-a production in Th1 vector control, following restimulation with increasing doses of cells was not due to altered expression of other cytokines, in- IL-12 (Fig. 2A). Decreased induction of pSTAT4 was correlated cluding Il4 and Il17a (data not shown). Coincident with decreased with reduction in IL-18ra expression, a STAT4-target gene (data cytokine production, the expression of Th1-related transcription not shown) (19, 27). Consistent with this result, reduced Twist1 factors such as T-bet, Hlx1, and Runx3 were decreased upon ec- expression results in increased IL-12–induced STAT4 phosphor- topic Twist1 expression and increased upon Twist1 shRNA ylation (Fig. 2B). We next examined the expression of that transduction (Fig. 1G, 1H and data not shown). contribute to STAT4 activation. STAT4 mRNA and protein ex- Because STAT4 is required for the expression of many Th1- pression, as well as mRNA of suppressors of cytokine signaling specific genes (1), we examined whether Twist1 was a compo- and protein tyrosine phosphatase-basophil like that negatively nent in a feedback mechanism to control STAT4 activation. regulate STAT4 activation (28, 29), were not altered. However, Phospho-STAT4, assessed by flow cytometry, was lower in Th1 Il12rb2 mRNA expression was decreased ∼50% by ectopic Twist1 The Journal of Immunology 835

Runx3 rescues the inhibitory effect of Twist1 independent of T-bet and STAT4 Because Twist1 regulates multiple genes in Th1 cells, we wanted to determine whether a single factor could rescue the inhibitory effect of Twist1. Initially, we asked whether ectopic expression of IL- 12Rb2 could compensate for the repressive effect of Twist1 by cotransducing Th1 cells using transduction of both IL-12Rb2 and Twist1. IL-12Rb2 surface expression was decreased in cells with ectopic Twist1 expression but was higher in double-transduced cells, compared with vector control (Fig. 3A). Recovery of IL- 12Rb2 expression resulted in increased IL-12–induced phospho- STAT4 (Fig. 3B). Despite recovery of T-bet expression and phospho-STAT4 (Fig. 3B and data not shown), ectopic IL-12Rb2 expression was unable to rescue IFN-g production, although there was recovery of TNF-a production (Fig. 3C). Coexpression of T- bet and Twist1 resulted in higher T-bet expression compared with vector control but failed to recover IFN-g and TNF-a production (Fig. 3D, 3E). Thus, although recovery of IL-12 signaling was

able to induce TNF-a in the presence of Twist1, neither recovery Downloaded from FIGURE 2. Twist1 regulates Il12rb2 expression and STAT4 activation. of IL-12 signaling nor T-bet expression was able to compensate (A) Naive WT T cells were stimulated and transduced as described in Fig. for the effects of Twist1 on IFN-g production. + 1. Transduced Th1 cells were stimulated with IL-12 for 4 h, and GFP cells Because Runx3 regulates IFN-g independently of T-bet and B were analyzed for phospho-STAT4 (pSTAT4). ( ) Phospho-STAT4 was STAT4 (4), we hypothesized that regulation of Runx3 by Twist1 determined in control or shRNA-transduced Th1 cells following sorting for might be a critical target. Ectopic Twist1 expression resulted in GFP expression. (C and D) Total cell extracts were immunoblotted for decreased Runx3, and retroviral expression of Runx3 resulted in STAT4 and b-actin as a control (C), and densitometry analysis of STAT4 http://www.jimmunol.org/ protein is indicated (D). (E and F) Stat4 and Il12rb2 gene expression in higher Runx3 levels in double-transduced cells compared with Twist1 (E) or shTwist1 (F) transduced Th1 cells sorted for GFP expression. control cells (Fig. 3F). IFN-g and TNF-a production in Runx3/ Data are means of three to four independent experiments 6 SD (A, B)or Twist1-transduced cells was comparable to that of control- the average of replicate samples 6 SD and are representative of two to transduced cells (Fig. 3G). The recovery of IFN-g and TNF-a three independent experiments with similar results (C–F). *p , 0.05. production by Runx3 in Th1 cells was independent of T-bet and STAT4 since there was no recovery of T-bet expression or phos- expression compared with vector control (Fig. 2C–E and data pho-STAT4+ cells (data not shown). Thus, although Twist1 can not shown). In parallel, transduction of Th1 cells with retroviral negatively regulate many Th1 genes, the recovery of T-bet and IL-

Twist1 shRNA resulted in increased Il12rb2 expression (Fig. 2F). 12Rb2 expression (and, as a consequence, STAT4 phosphoryla- by guest on October 3, 2021

FIGURE 3. Ectopic Runx3 expression compensates for the repressive activity of Twist1 in Th1 cells. Naive WT T cells were stimulated under Th1- polarizing conditions. On day 2, cells were transduced with control retrovirus vectors or (A–C) retroviral vector-expressing Twist1-GFP and IL-12Rb2- hCD4, (D, E) Twist1-hCD4 and T-bet-GFP, or (F, G) Twist1-GFP and Runx3-Thy1.1. Th1 cells were stained for IL-12Rb2 by surface staining (A)or phospho-STAT4 with or without IL-12 stimulation by intracellular cytokine staining (B), or T-bet by intracellular cytokine staining (D). Analysis was performed by gating on doubly transduced cells. (E and G) Double-positive cells were sorted and restimulated with 2 mg/ml anti-CD3 for 24 h. Supernatants were collected before IFN-g and TNF-a production was measured by ELISA. (F) Whole-cell lysates were extracted from sorted double-positive Th1 cells and were immunoblotted for Runx3 and b-actin as a control. Data are means of three to four independent experiments 6 SD (A, B) or averages of replicate samples 6 SD and representative of two to three independent experiments with similar results (C–G). *p , 0.05. 836 Twist1-DEPENDENT REGULATION OF Th1 CELLS tion) did not compensate for the effects of Twist1. These results (Twist1fl/fl CD4-Cre+). We confirmed the absence of Twist1 ex- suggested that Runx3, or a Runx3-induced gene, is at least one of pression in Twist1-deficient Th1 cells (Supplemental Fig. 1). the important Twist1 targets in the regulation of IFN-g. Twist1 deficiency did not alter normal lymphocyte development in thymus, spleen, and lymph nodes (Supplemental Fig. 1). Twist1- Twist1 exists in a complex with Runx3 or T-bet deficient Th1 cells produced significantly greater amounts of Twist1 inhibits osteoblast differentiation by interacting with the IFN-g on a per cell basis and in frequency of IFN-g+ cells com- Runt domain of Runx2, a highly conserved domain shared with pared with WT cells (Fig. 4B–E). We also confirmed the increase related proteins, including Runx3 (15). We hypothesized that in gene expression of Tbx21, Runx3, and Il12rb2 in Twist1-defi- Twist1 physically interacts with Runx3 and inhibits its regulatory cient Th1 cells, compared with WT Th1 cells. There was no sig- function. Using immunoprecipitation of extracts from resting and nificant difference in Eomes expression between WT and Twist1- early activated Th1 cells, we observed that Twist1 copurified with deficient Th1 cells (data not shown). Runx3, and we confirmed the association between Runx3 and T- We next tested the effect of Twist1 on Runx3 and T-bet DNA bet in Th1 cells (3). We also found an association between T-bet binding by DNA affinity precipitation assay using extracts from and Twist1 following precipitation with T-bet Ab (Fig. 4A). Twist1-deficient mice and littermate control (WT)-activated Th1 To examine the function of Runx3 and T-bet in the absence cells. Extracts from WT and Twist1-deficient Th1 cells were in- of Twist1, Twist1fl/fl mice (14) were mated with mice carrying a cubated with biotinylated oligonucleotides containing a Twist1- CD4-Cre transgene to generate mice with Twist1-deficient T cells binding sequence. Although T-bet and Runx3 were precipitated Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 4. Twist1 physically interacts with Runx3 and T-bet. (A) Whole-cell lysates were extracted from activated WT Th1 cells. Immunoblots were performed for the indicated proteins following immunoprecipitation with Runx3 or T-bet or control Abs as indicated. Input for immunoprecipitation is indicated on the right. (B–E) Naive CD4+CD62Lhi WT or Twist1fl/fl CD4-Cre+ T cells were cultured under Th1-polarizing conditions. IFN-g production was measured by ELISA (B) after reactivating with 2 mg/ml anti-CD3 for 24 h or intracellular cytokine staining (C–E) after reactivating with PMA/ionomycin for 6 h. (D and E) Graphs indicate the mean fluorescence intensity (D) or average percentage IFN-g+ cells (E) of Th1 cells from mice of the indicated genotype. (F–H) Nuclear extracts from activated (F) or resting (G) WT and Twist1fl/fl CD4-Cre+ Th1 cells were incubated with biotinylated oligonucleotides containing Twist1-specific binding site (F) or Runx3/T-bet–specific binding sites (WT or mutant as indicated) (G). (H) Immunoblots of precipitated proteins, with densitometry measurements for (G). (I) Mixture of whole-cell lysates collected from 293T cells transfected with constructs expressing Runx3, T-bet, or Twist1 were incubated at 4˚C overnight. Immunoblots indicate proteins immunoprecipitated with Runx3. Results are average 6 SEM (B)or6SD (D, E, H) of three independent experiments (*p , 0.05, ***p , 0.001) or representative of three or more independent experiments with similar results (A, C, F, I). The Journal of Immunology 837 with DNA-bound Twist1 in WT cell extracts, T-bet and Runx3 pression but also to diminished association and DNA binding were absent from precipitates of Twist1-deficient extracts, further activity of Runx3 and T-bet. supporting the interaction of these transcription factors and sug- gesting that interactions can occur when Twist1 is bound to DNA Twist1 interferes with the binding of Runx3 and T-bet to the (Fig. 4F). We observed that Runx3 and Tbx21 mRNA expression Ifng locus increased in Twist1-deficient Th1 cells compared with WT cells, Because Twist1 interferes with T-bet and Runx3 DNA binding, although there was a modest increase at the protein level, sug- and we observed only modest binding of Twist1 to the Tbx21, gesting the importance of protein complex formation in the Ifng, or other Th1 gene promoters (data not shown), we hy- function of Twist1. Because there is an association between pothesized that the interaction of Twist1 with Th1 transcription Twist1, Runx3, and T-bet, we tested whether, in the absence of factors decreased their binding to target genes, including Ifng. Twist1, there would be greater binding of Runx3 and T-bet to the T-bet and Runx3 regulate Ifng gene expression by binding to oligonucleotides containing Runx3- and T-bet–specific binding several conserved noncoding sequences (CNS) (4). To examine sites. In the absence of Twist1, we detected greater binding of T-bet and Runx3 binding in the absence of Twist1, we performed Runx3 and T-bet to the respective oligonucleotides compared with a ChIP assay using WT and Twist1-deficient Th1 cells and ex- WT samples (Fig. 4G, 4H, lane 2 versus 1). When the binding amined the binding at previously documented regions, including sites of Runx3, T-bet, or both were mutated, the binding of Runx3 CNS234, CNS26, Ifng promoter, and CNS+46 of the Ifng gene and T-bet was diminished, regardless of Twist1 expression (Fig. (Fig. 5A) (4, 22), using ChIP for T-bet or CBF-b, the binding 4G). We then tested whether Twist1 mediates its inhibitory effect partner of Runx3 (4). Our data showed that more T-bet was in Th1 cells by interfering with the T-bet/Runx3 interaction. Using bound in Twist1-deficient Th1 cells than in WT cells at the Ifng Runx3 immunoprecipitation, we detected a Runx3 and T-bet in- promoter and CNS+46 whereas there were no differences at Downloaded from teraction in the absence of Twist1 (first lane), although this in- CNS234 and CNS26 (Fig. 5B). In contrast, more CBF-b was teraction was diminished in the presence of Twist1 (Fig. 4I). These bound at CNS234 and CNS26inTwist1-deficient Th1 cells results suggested that the decrease in IFN-g production in Th1 compared with littermate controls (Fig. 5B). This result sug- cells might not only be due to the reduction in Runx3 gene ex- gested that Twist1 interferes with T-bet and CBF-b/Runx3 http://www.jimmunol.org/

FIGURE 5. Twist1 interferes with transcription factor binding and chromatin conformation at the Ifng locus in Th1 cells. (A) Representation of Ifng locus indicating CNS used for analysis. (B–G) Naive CD4+CD62hi fl/fl T cells from WT and Twist1 CD4- by guest on October 3, 2021 Cre+ were stimulated under Th1-po- larizing conditions. (B) ChIP assays were performed on Th1 cells using T- bet and CBFb Abs. (C) Th1 cells were fixed and digested with BglII enzyme. Undigested and digested sam- ples were subjected for qPCR using primer pairs spanning the restriction sites. Percentage digestion was calcu- lated using the formula: 100 – 100/ 2[(CtR 2 CtC)D 2 (CtR 2 CtC)UND].(D) Th1 cells as described above were fixed (crosslinked) and/or ligated as indicated. qPCR was performed us- ing primer pair 13F-9R. PCR prod- ucts were run on 2% agarose gel. BAC clones were titrated for qPCR and used as control. (E–G) 3C assay showing the relative crosslinking frequencies between the Ifng pro- moter (E), CNS234 (F), or CNS+46 (G) as the fixed anchor fragments and other BglII fragments containing the indicated CNS regions. Results are the average 6 SD of replicated sam- ples and are representative of four independent experiments with similar results. *p , 0.05. C, Internal con- trol; D, digested; R, restriction site; UND, undigested. 838 Twist1-DEPENDENT REGULATION OF Th1 CELLS complex binding at specific regulatory regions of the Ifng gene. Ifng promoter and CNS26 is too short to provide consistent Because T-bet and Runx3 bind to many CNS regions of the Ifng results in this assay. Crosslinking of the CNS region at 271 was locus, and the association between T-bet and Runx3 is required for not altered. Taken together, these results suggest that Twist1 optimal Ifng expression (3, 4), it is likely that Runx3 binding to regulates Ifng expression by altering the binding of the Th1 the Ifng locus enhances T-bet binding to regulatory elements. This transcription factors T-bet and Runx3, and altering the confor- is consistent with the recovery of IFN-g production in the Runx3 mation of the Ifng locus. transduction experiment (Fig. 3G). The effect of Twist1 on T-bet and CBF-b/Runx3 binding at Twist1/Runx3 interactions are required to regulate Ifng distinct elements suggested that interfering with T-bet/Runx3 A mutation in Twist1 at aa 192 from serine to proline (Twist1cc) interactions might also alter chromatin looping at the Ifng locus results in diminished interaction between Twist1 and Runx2 (15). (24, 25). To determine whether Twist1 interferes with chromatin Thus we wanted to examine whether Twist1 S192P (Twist1cc) looping at the Ifng locus, a 3C assay was performed with WT and could interact with Runx3. A DNA affinity precipitation assay Twist1-deficient Th1 cells that examined the interactions among using extracts from cells transfected with Runx3, WT Twist1 Ifng CNS regions using an established assay (23, 25) (Fig. 5C, (Twist1), and Twist1cc expressing vectors that were incubated 5D). Using three different anchor points, our results showed in- with biotinylated oligonucleotides containing a Twist1-binding creased crosslinking frequency between CNS234, CNS+46, and sequence demonstrated decreased interaction between Runx3 the Ifng promoter, and increased crosslinking of CNS26 with and Twist1cc (Fig. 6A, 6B). The mutation in Twist1cc did not CNS234 and CNS+46 (Fig. 5E–G). The distance between the affect the association of Twist1 with T-bet (data not shown). Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 6. The Twist1/Runx3 interaction is required for regulation of Ifng.(A and B) Nuclear extracts from 293T cells cotransfected with either Flag- tagged Twist1 or Flag-tagged Twist1cc with or without Runx3 were incubated with biotinylated oligonucleotides containing Twist1-specifc sequence. Immunoblot demonstrates protein expression of Twist1 and Runx3 (A) with densitometry measurements (B). (C–H) Naive CD4+CD62hi T cells from WT were cultured under Th1-polarizing conditions and were infected with retrovirus expressing Twist1 or Twist1cc. (C) Twist1 protein was assessed in transduced Th1 cells using immunoblot, with b-actin as a control. (D–G) IFN-g production was assessed by intracellular staining and gating on GFP+ populations following 6 h activation with anti-CD3 (D–F) or by ELISA following 24 h stimulation with anti-CD3 of sorted GFP+ populations (G). (E and F) Graphs indicate the mean fluorescence intensity (E) or average percentage IFN-g+ cells (F) of each transduced population. (H) Th1 gene expression in ectopic Twist1 or Twist1cc expression was assessed by RT-qPCR before (Il12rb2, Runx3, and Tbx21) or after (Ifng) 6 h restimulation with anti-CD3. (I–K) Naive CD4+CD62hi T cells from mice of the indicated genotypes were cultured under Th1-polarizing conditions. Th1 cells were stimulated with anti-CD3 for 6 or 24 h, respectively, for testing Ifng expression by RT-qPCR (I) or IFN-g production by ELISA with the ratio of production from Twist1-deficient or Twist1 mutant Th1 cells to the respective controls (J, K). Data are means of three to four independent experiments 6 SD (A–F) or are mean of replicate samples 6 SD and are representative of two independent experiments with similar results (G–K). *p , 0.05. The Journal of Immunology 839

The mutant Twist1 provided a tool to mechanistically distinguish sensitivity and Ag-induced arthritis (12). In this study, we have the effects of Twist1 that rely on interactions with Runx3 at the identified a mechanism by which Twist1 modulates inflammatory protein level, versus effects independent of Runx3 binding. To test cytokine production in Th1 cells. We showed that Twist1 nega- the effects of mutant Twist1, a retrovirus expressing Twist1cc was tively regulates transcription factors, including T-bet, STAT4, and introduced into Th1 cells, and cytokine production and gene ex- Runx3, leading to decreased IFN-g production in Th1 cells. The pression were analyzed. Introduction of Twist1cc did not repress formation of the Twist1/Runx3 complex is required for Twist1 to IFN-g production in Th1 cells compared with vector control and decrease IFN-g production. In the absence of Twist1, there is WT Twist1-transduced cells (Fig. 6D–H). Importantly, Twist1cc increased transcription factor binding and increased looping at the was able to repress expression of several Th1 genes, including Ifng locus, resulting in increased IFN-g production. Thus, Twist1 Il12rb2, Runx3, and Tbx21, as effectively as WT Twist1 (Fig. 6H). impairs the activity of the Th1 transcription factor network. To further define the function of Twist1cc in Th1 cells, we used Our data suggest that Twist1 regulates Th1 gene expression a mouse mutant strain termed Charlie Chaplin (Twist1cc/wt) that through several mechanisms. First, it negatively regulates the encodes Twist1 S192P and results in hindlimb polydactyly (15, expression of Th1 genes. In macrophages, Twist1 has been shown 30). We mated Twist1cc/wt with Twist1fl/fl CD4-Cre+ mice gener- to regulate TNF-a and IL-1b by binding to E-boxes in gene ating Twist1fl/wt CD4-Cre+ and Twist1fl/cc CD4-Cre+ (Twist1fl/cc) promoters (15). However, we did not observe Twist1 binding to mice that have T cells expressing one WT or one mutated allele of the promoters of Ifng, Tnfa,orIl12rb2, and we found only min- Twist1, respectively. IFN-g production in Twist1fl/cc Th1 cells was imal binding to the promoter of Tbx21. It is still possible that increased compared with control cells (Fig. 6I–K). These results Twist1 binds directly to Th1 genes in regions other than the demonstrate that although Twist1cc retains some repressive promoter that we tested. The second mechanism of Twist1- function in Th1 cells independent of Runx3, Twist1 control of dependent gene regulation is through complex formation with Downloaded from IFN-g production is primarily through association with Runx3 Runx3. This mechanism appears to be the primary mechanism for (Fig. 7). regulating Ifng. However, the Twist1 S192P mutant that reduced interactions with Runx3 was still able to transcriptionally regulate Discussion other Th1 genes, suggesting that Twist1 has gene regulatory ac- Twist1 is expressed in Th1 effector memory cells and limits Th1- tivity independent of Runx3. The direct regulation of genes other mediated inflammation in mouse models of delayed-type hyper- than Ifng might further impact the Th1 phenotype, and further http://www.jimmunol.org/ analysis will be required to further refine this regulatory network. In this study, we showed that Twist1 is expressed highest in resting Th1 cells compared with other T cell subsets, although its expression increased in all T cell subsets upon TCR stimulation. During differentiation, Twist1 expression peaked at day 2 in both Th1 and Th2 cells and gradually decreased, although Twist1 ex- pression was lower in Th2 cells than Th1 cells after day 3. Thus, Twist1 might be a negative regulator of Th1 cytokine production by guest on October 3, 2021 in other T cell subsets. Although Twist1 is a STAT4 target in Th1 cells, it might be targeted by other STATs in other Th lineages. In breast cancer cell lines, STAT3binds directly to the Twist1 promoter, and amounts of phospho-STAT3 correlate with expression of Twist1 (31). Be- cause STAT3 has a critical role in Th2 and Th17 differentiation, and STAT3-deficient Th2 cultures show increased IFN-g produc- tion compared with WT cultures, Twist1 might play a role in regulating cytokine production in these lineages as well (32–34). Twist2 promotes IL-10 production by activating the transcription factor c-Maf in myeloid cells, and because c-Maf is an important transcription factor for Th2 and Th17 cells, Twist1 might play an analogous role in these cells (35, 36). The function of Twist1 in other Th subsets will be the focus of further study. Twist1 requires dimerization to bind DNA (37). In Drosophila, dimerization partners determine the activity of Twist1 (38). Twist1 homodimers have opposing effects on mesoderm gene expression as heterodimers of Twist1 and the Drosophila E protein homolog, Daughterless (38). The Daughterless gene has 79% identity to E12 and E47, two alternative splice products of the E2a gene (39, FIGURE 7. Transcriptional regulatory network in Th1 cells. IL-12/IL- 40). Th1 cells differentiated from E2a-deficient mice showed a 12Rb2/STAT4 induces T-bet expression that positively regulates IL-12Rb2 decrease in IFN-g production compared with WT cells (41). It is expression. Both STAT4 and T-bet are required for the induction of Runx3, possible that E12/E47 sequesters Twist1 in heterodimer complexes which contributes to the optimal IFN-g production. Twist1, a transcription to inhibit its repressive function in Th1 cells. In the absence of repressor that is induced by IL-12/STAT4, suppresses IFN-g production by E2A gene products, there is more “free” Twist1 to form homo- two mechanisms: the repression of Il12rb2, Tbx21, and Runx3 expression potentially by directly binding to the conserved motif E-box (CANNTG) in dimers and thus inhibit cytokine production. E47 and the HLH gene regulatory regions, and the interference with the function of Runx3 inhibitor Id3 regulate the balance between Treg and Th17 cell through physical interaction. The latter mechanism is critical for regulation differentiation (42). The balance among Twist1, Id, and E proteins of Ifng since complementation of Il12rb2 or Tbx21 expression did not is likely important in defining Twist1 activity, as Id proteins rescue IFN-g production. interacting and titrating E proteins would favor the formation of 840 Twist1-DEPENDENT REGULATION OF Th1 CELLS

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