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 Supplementary http://www.jimmunol.org/content/suppl/2012/06/08/jimmunol.120085 Material 4.DC1 References This article cites 44 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/189/2/832.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 3, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 transcription factor 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 gene 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 proteins 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, chromosome 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- protein 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.