Transcription Factor-Dependent Chromatin Remodeling of Il18r1 during Th1 and Th2 Differentiation This information is current as Qing Yu, Hua-Chen Chang, Ayele-Nati N. Ahyi and Mark of October 2, 2021. H. Kaplan J Immunol 2008; 181:3346-3352; ; doi: 10.4049/jimmunol.181.5.3346 http://www.jimmunol.org/content/181/5/3346 Downloaded from References This article cites 26 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/181/5/3346.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 • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Transcription Factor-Dependent Chromatin Remodeling of Il18r1 during Th1 and Th2 Differentiation1 Qing Yu, Hua-Chen Chang, Ayele-Nati N. Ahyi, and Mark H. Kaplan2 The IL-18R␣-chain is expressed on Th1 but not Th2 cells. We have recently shown that Stat4 is an important component of programming the Il18r1 locus (encoding IL-18R␣) for maximal expression in Th1 cells. Il18r1 is reciprocally repressed during Th2 development. In this report, we demonstrate the establishment of DH patterns that are distinct among undifferentiated CD4 T, Th1, and Th2 cells. Stat6 is required for the repression of Il18r1 expression and in Stat6-deficient Th2 cultures, mRNA levels, histone acetylation, and H3K4 methylation levels are intermediate between levels observed in Th1 and Th2 cells. Despite the repressive effects of IL-4 during Th2 differentiation, we observed only modest binding of Stat6 to the Il18r1 locus. In contrast, we observed robust GATA-3 binding to a central region of the locus where DNase hypersensitivity sites overlapped with conserved non-coding sequences in Il18r1 introns. Ectopic expression of GATA-3 in differentiated Th1 cells repressed Il18r1 mRNA and surface expression of IL-18R␣. These data provide further mechanistic insight into transcription factor-dependent establishment Downloaded from of Th subset-specific patterns of gene expression. The Journal of Immunology, 2008, 181: 3346–3352. he regulation of gene expression in differentiated Th cells acetylation and increased DNA methyltransferase associated with occurs through a network of genetic and epigenetic inter- the promoter corresponding to increased DNA methylation (7). actions (1, 2). During Th1 development, the expression This suggests that during Th2 differentiation, the Il18r1 locus is T http://www.jimmunol.org/ and function of instructive transcription factors such as Stat4 and actively repressed. However, a mechanism for the establishment of T-bet mediate epigenetic changes at relevant target genes (3–7). these distinct transcriptional states has not been described. The most detailed studies have been of the Ifng locus where Th1- In this report, we have compared chromatin remodeling of the promoting transcription factors stimulate increased transcription Il18r1 locus in Th1 and Th2 cells. We identified DNase hypersen- and histone acetylation and decreased DNA methylation, while sitivity (DH) sites that are independent of Stat4 and distinguish the Th2 inducing factors inhibit Ifng expression and promote repres- induced state in Th1 cells from the repressed state in Th2 cells. sive events in Ifng programming (3–6, 8–12). Whether this para- GATA-3 binds to conserved non-coding sequences (CNC)3 in the digm is consistent when other loci are examined is not clear. Il18r1 locus. Moreover, ectopic expression of GATA-3 results in The Il18r1 gene that encodes IL-18R␣ is expressed in Th1 but decreased IL-18R␣ expression. Thus, factors that promote Th2 not Th2 cells (13). Expression of IL-18R␣ is induced by an IL- differentiation repress Il18r1 expression via binding directly to the by guest on October 2, 2021 12/Stat4 pathway and inhibited by an IL-4/Stat6 pathway (7, 14– Il18r1 gene. 16), although the mechanism of this regulation is only beginning to be understood. We have recently described the regulation of this Materials and Methods locus during Th differentiation and observe three states of gene Mice expression: high level of expression in Th1 cells (an induced state), Ϫ Ϫ intermediate expression that is observed in Stat4-deficient Th1 The generation of C57BL/6 Stat4 / mice was previously described (17). Wild-type (WT) C57BL/6 and BALB/c mice were purchased from Harlan cells (an intermediate state) and very low transcription in Th2 cells Bioproducts for Science. BALB/c Stat6Ϫ/Ϫ mice (18) were purchased (a repressed state). We demonstrated that at least one distinction from The Jackson laboratory. All experiments used C57BL/6 mice, except between the basal state and the Stat4-dependent induced state was for BALB/c WT and Stat6Ϫ/Ϫ mice as noted in figure legends. Mice were transient hyperacetylation of the 5Ј end of the locus resulting in maintained in pathogen-free conditions in barrier facilities in the Labora- tory Animal Resource Center (Indiana University School of Medicine). All decreased association of DNA methyltransferases with the Il18r1 experiments were performed following approval of the Indiana University promoter and exon 1 (7). IL-4 has been previously shown to re- Animal Care and Use Committee. press IL-18R␣ expression during Th2 development. The repressed state of Il18r1 in Th2 cells was characterized by decreased histone Th differentiation and analysis CD4 cells were isolated from spleen and lymph nodes of mice using mag- netic beads (Miltenyi Biotec). For Th differentiation, CD4 cells (1 ϫ 106 ␮ ␮ Departments of Pediatrics, Herman B. Wells Center for Pediatric Research and De- cells/ml) were cultured with plate bound anti-CD3 (4 g/ml), 0.5 g/ml partment of Microbiology and Immunology, Indiana University School of Medicine, soluble anti-CD28, under Th1 (2 ng/ml IL-12 and 10 ␮g/ml anti-IL-4) or Indianapolis, IN 46202 Th2 (10 ng/ml IL-4 and 10 ␮g/ml anti-IFN-␥) skewing conditions and Received for publication March 26, 2008. Accepted for publication July 3, 2008. expanded after 3 days. After 5–7 days of culture, cells were harvested for FACS analysis with anti-IL-18R␣ (R&D Systems), RNA isolation and The costs of publication of this article were defrayed in part by the payment of page quantitative PCR (performed as described) (19), or assays described below. charges. This article must therefore be hereby marked advertisement in accordance Transduction with GATA-3-hCD4 expressing bicistronic retroviruses was with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by U.S. Public Health Service Award AI45515 (to M.H.K.) from the National Institutes of Health. 3 Abbreviations used in this paper: CNC, conserved non-coding sequence; WT, wild 2 Address correspondence and reprint requests to Dr. Mark H. Kaplan, Department of type; TSS, transcriptional start site; DH, DNase hypersensitivity; ChIP, chromatin Pediatrics, and Microbiology and Immunology, Herman B. Wells Center for Pediatric immunoprecipitation. Research, Indiana University School of Medicine, 702 Barnhill Drive, RI 2600, In- dianapolis, IN 46202. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 3347 Downloaded from FIGURE 1. IL-18R expression in Th1 and Th2 cells. A, Schematic of http://www.jimmunol.org/ Il1r family genes. Arrows indicate transcriptional orientation. B, Flow cy- tometric analysis of IL-18R␣ expression in C57BL/6 or BALB/c purified CD4 T cell cultures activated with anti-CD3, anti-CD28 and polarized to Th1 (IL-12 plus anti-IL-4) or Th2 (IL-4 plus anti-IFN-␥) phenotypes. Cells were isolated each day during differentiation and stained with anti- IL-18R␣. Shaded histograms indicate control Ab staining; open histograms represent staining with anti-IL-18R␣. C, mRNA analysis of Il18r1 and Il18rap expression in differentiating C57BL/6 Th1 and Th2 cultures. RNA was isolated from cells during each day of differentiation and mRNA levels were determined using quantitative PCR. Results shown are relative to by guest on October 2, 2021 levels in undifferentiated CD4 T cells. performed as previously described (20), except that cells had been dif- FIGURE 2. DH of the Il18r1 locus. A, Schematic of the Il18r1 gene ferentiated toward the Th1 phenotype. Five days after transduction, indicating exons (open, non-coding exons; closed, coding exons) and tran- hCD4ϩ cells were analyzed for expression of IL-18R␣ or CXCR3. In par- scriptional initiation site. The restriction enzymes used for DH analysis are ϩ allel experiments, hCD4 cells were purified from control and GATA-3 indicated below a map of the fragments used for analysis. Bars indicate transduced cells, and RNA was isolated for analysis. probes for the adjacent fragment and are noted as p1–3. Arrowheads with roman numerals indicate DH sites described below. RI, EcoRI; RV, DH analysis EcoRV. B, Nuclei were isolated from WT or Stat4Ϫ/Ϫ CD4 T cells di- Isolation and DNase I digestion of nuclei was performed as described (21). rectly ex vivo or cultured for 1 or 2 wk under Th1 or Th2 conditions as ϩ In brief, CD4 T cells were washed in cold PBS and resuspended in 0.3 M indicated.