Differentiation During Th1 and Th2 Il18r1 Remodeling of Transcription

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

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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 © 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. Nuclei were subjected to DNase I (DNI) digestion and isolated sucrose. Membranes were disrupted with 0.2% Nonidet P-40 and cells genomic DNA was digested with EcoRV before Southern blotting with probe were incubated on ice for 10 min before Dounce homogenization. The cell p1. C, DNA from cells prepared as in B were Southern blotted with probe suspension was layered onto 1.7 M sucrose cushion, and nuclei were pu- p2 for the central region of the Il18r1 gene. D, DNA from cells prepared rified by centrifugation at 13,000 rpm for 15 min. The pellet was resus- as in B was digested with EcoRI before Southern blotting with probe p3. pended in cold buffer (0.3M sucrose, 5% glycerol). Aliquots of purified E, Nuclei were isolated from WT or Stat4Ϫ/Ϫ CD4 T cells activated for nuclei were digested with DNase I (Worthington) in 5 mM CaCl2 and 1 3 days and cultured for 24 h in the presence or absence of IL-12. Southern mM MgCl2 for 3 min at room temperature. Reactions were terminated with 5% SDS, 100 mM EDTA, 20 mM Tris (pH 7.4), and 2 mg/ml Proteinase analysis was performed using probe p2. K. DNA was purified using phenol/chloroform and digested to completion with EcoRI or EcoRV as indicated. DNA was separated through 1% agarose and fragments were detected using Southern analysis. Sequences of H3K4me2, H3K4me3, Stat4, Stat6, T-bet, GATA-3, or control IgG as in- primers used to generate probes for Southern blotting are available upon dicated. Abs were purchased from Millipore/Upstate Biotechnology or request. Santa Cruz Biotechnology. Complexes were precipitated with protein A- agarose beads. Supernatants from the control precipitation were used as Chromatin immunoprecipitation (ChIP) input material. Precipitates were washed with low- and high-salt buffers, and DNA was eluted from the precipitate. DNA crosslinks were reversed ChIP was performed as described (7). In brief, cells were fixed in form- at 65°C and DNA was purified using phenol-chloroform. Quantitative PCR aldehyde and nuclei were isolated after cell lysis and centrifugation. Sim- was performed with site-specific primer sets using ABI PRISM7500. Re- ilar numbers of cells for each condition were used in an experiment to sults are expressed as percent input (PCR from specific ChIP – PCR from facilitate comparisons among samples. Following nuclear lysis, chromatin control IgG)/PCR of input DNA Ϯ SD of replicates from representative was fragmented by sonication. Extracts were pre-cleared with salmon data of two to three experiments. Statistics were performed using a two- sperm DNA, BSA, and protein A-agarose slurry. The supernatant was in- tailed student’s t test. The sequences of the primer pairs are available upon cubated overnight with Abs to acetyl-H3, aceytl-H4, acetyl-H3K9, request. 3348 REGULATION OF Il18r1 IN Th SUBSETS Downloaded from

FIGURE 3. Histone modification of the Il18r1 5-prime end. Undiffer- entiatied CD4 T cells or cells differentiated under Th1 and Th2 conditions as described in Fig. 1B were used for ChIP analysis. ChIP was performed for acetyl-H3 (AcH3), acetyl-H4 (AcH4), di-methyl-H3K4 (2mH3K4), or tri-methyl-H3K4 (3mH3K4) and fragments specific for DHI or the TSS were amplified and detected using quantitative PCR. Results are expressed http://www.jimmunol.org/ as percent input Ϯ SD.

Results Chromatin remodeling at the Il18r1 gene The IL-18R locus on mouse chromosome 1 contains the closely FIGURE 4. Identification and histone modification of Il18r1 CNCs. A, linked Il18r1 and Il18rap genes encoding, respectively, the IL- VISTA plot of CNCs between human and mouse Il18r1 gene sequences. 18R␣ and IL-18R␤ receptor components, and the Il1rl1 gene, en- DH sites are indicated by arrowheads with roman numerals. CNC are in- by guest on October 2, 2021 coding the T1/ST2/IL-33R protein, within a 100 kb chromosome dicated by letters A-E. B, Chromatin from undifferentiated CD4 T cells or cells differentiated under Th1 and Th2 conditions as described in Fig. 1B segment that is ϳ70 kb telomeric from other IL-1R family genes was isolated for ChIP analysis of AcH3, AcH4, 2mH3K4, and 3mH3K4 (Fig. 1A). The IL-18R genes are expressed in undifferentiated ϩ levels at the indicated CNC sites. C, CD4 T cells were activated with CD4 T cells and expression is dynamically modulated during anti-CD3 and anti-CD28 for three days. Cells were washed and either left Th1 or Th2 differentiation (Fig. 1, B and C). Surface expression of unstimulated or stimulated with IL-12 or IL-4 for 24 h. ChIP was per- IL-18R␣ is decreased 24 and 48 h after activation in both Th1 and formed as described in A. Results are expressed as percent input Ϯ SD. Th2 cultures, but while it continues to be expressed on only a small portion of Th2 cells, it is induced during Th1 differentiation and is expressed on the majority of differentiated Th1 cells (Fig. 1B). tant to DNaseI treatment following 2 wk of culture in Th2 condi- Similar patterns of expression during differentiation are observed tions (Fig. 2B). As we have previously shown that Il18r1 is a Stat4 in BALB/c and C57BL/6 cultures (Fig. 1B). Message levels as- target gene (7), we also tested the appearance of DHI in Stat4Ϫ/Ϫ sessed using quantitative PCR demonstrates a similar pattern of cultures. Despite the ability of Stat4 to mediate increased locus expression for both Il18r1 and Il18rap being repressed in Th2 and acetylation, the sensitivity of DHI in Stat4-deficient Th1 cultures induced in Th1 C57BL/6 cultures, with similar expression patterns was indistinguishable from WT Th1 cells (Fig. 2B). These results in BALB/c cells (Fig. 1C and data not shown). suggest that chromatin remodeling of the 5Ј region of the Il18r1 To further explore the regulation of the Il18r1 gene, we per- gene progresses independently of IL-12 signaling. formed DH analysis on regions near the transcriptional start site To define alterations in chromatin structure in the central portion (TSS) (3). A genomic DNA probe (p1) was used to define DH sites of the Il18r1 locus, we performed similar experiments to examine of an EcoRV fragment that spanned the promoter, exon 1, and the DH in a second EcoRV fragment that spanned introns 1–7 (probe 5Ј end of intron 1 of Il18r1 (Fig. 2A). Th1 or Th2 cultures were p2). We observed a number of DH sites in this fragment that we differentiated for 1 wk and isolated nuclei were treated with in- termed DHII-DHV (Fig. 2C). DHII was observed most promi- creasing doses of DNaseI. One DH site (identified as DHI) was nently in Th1 cultures after 2 wk of culture but was detected in identified in the 5Ј end of Il18r1 that was present in undifferenti- undifferentiated T cells and 1-wk Th1 and Th2 cells (Fig. 2C). ated CD4 T cells. DHI sensitivity was increased following differ- DHIII was most apparent in Th2 cultures, particularly after 2 wk entiation and showed more sensitivity to DNaseI in Th1 than in of culture. DHIV was prominent in undifferentiated T cells, and Th2 cells, comparing the ratio of the EcoRV band to the DHI band was similar in Th1 and Th2 cells, though after 2 wk of culture it (Fig. 2B). To test whether chromatin was progressively remodeled was less sensitive to DNaseI in Th2 cultures. DHV was not a with increasing time in culture, we differentiated Th1 or Th2 cells strong DH site and was equally seen in Th1 and Th2 cells (Fig. for 2 wk and repeated the analysis. DHI appeared to show in- 2C). As was observed in the 5Ј end of the locus, the establishment creased sensitivity in Th1 cells, whereas it was increasingly resis- of the Th1 chromatin configuration was independent of Stat4. The Journal of Immunology 3349

FIGURE 5. Stat4 and Stat6 regulate Il18r1 expression. A, Flow cyto- metric analysis of IL-18R␣ expression on C57BL6 WT Th1, BALB/c WT Th2, C57BL/6 Stat4Ϫ/Ϫ Th1 and BALB/c Stat6Ϫ/Ϫ Th2 cells differentiated for 5 days as described in Fig. 1A. Shaded histograms indicate control Ab staining; open histograms represent staining with anti-IL-18R␣. Mean ﬂuorescence intensity for cells in each population is shown in the bar graph on the right. B, RNA was iso-

lated from cells of the indicated ge- Downloaded from notypes cultured under Th1 or Th2 conditions as shown and was used for quantitative PCR to determine Il18r1 mRNA levels relative to levels observed in Th2 cells. C, ChIP analysis was performed for AcH3, AcH4, and

dimethyl- or tri-methyl H3K4 with http://www.jimmunol.org/ cells cultured as in A. Quantitative PCR was performed for CNC regions, and results are expressed as percent input Ϯ SD. by guest on October 2, 2021

We examined DH sites in the 3Ј end of the Il18r1 using an (Fig. 4A). DHI or DHII did not correspond to CNC, and CNC D EcoRI fragment that spanned exon 8 to the 3Ј end of the locus but and E did not correspond to observed DH sites. To determine did not observe any DH sites in that region (Fig. 2D). We further whether the CNC had functional impact on chromatin structure, we examined whether acute stimulation with IL-12 would affect the examined histone acetylation in these regions using ChIP from DH pattern. WT and Stat4 Ϫ/Ϫ cells were activated for 3 days and undifferentiated Th, Th1, and Th2 cells. As with the DHI region incubated in the presence or absence of IL-12 for 24 h. However, (Fig. 3), there were significant increases in histone acetylation in IL-12 stimulation did not significantly alter the DH pattern (Fig. the CNC A-D regions (including DHIII-V) in Th1 cells compared 2E). The results of all DH analyses are summarized in Fig. 2A. with undifferentiated cells (Fig. 4B). Similar increases in H3K4 Il18r1 histone modifications during Th differentiation methylation were also observed (Fig. 4B). Modifications of CNC E were present at a lower level than those at other CNC regions. Since histone acetylation of the Il18r1 locus is Stat4-dependent, Following Th2 differentiation, there were generally lower levels of but DH patterns appeared to be independent of Stat4 activity, we histone acetylation and H3K4 methylation compared with undif- next wanted to determine whether the differences in chromatin ferentiated cells, though patterns differed slightly among the CNC structure among undifferentiated Th, Th1, and Th2 cells were also regions, with acetyl-H4 differing less in the CNC C and D regions, reflected by altered histone acetylation in that region. We used and tri-methyl H3K4 differing less in the CNC B and C regions ChIP followed by quantitative PCR with primers spanning the TSS B and DHI to assess histone acetylation and methylation of the H3K4 (Fig. 4 ). We then assessed the ability of cytokine stimulation to directly residue, modifications associated with active gene expression (22). ϩ While the levels of all modifications were significantly increased alter histone acetylation across the locus. CD4 T cells were ac- following Th1 development, changes between undifferentiated tivated with anti-CD3 and anti-CD28 in the absence of any skew- cells and Th2 cells were more modest, showing only slight de- ing cytokines for 3 days. Cells were then washed and incubated for creases in acetylated H3 levels and H3K4 methylation (Fig. 3). 24 h with IL-12, IL-4, or in the absence of any cytokine before To determine whether the DH pattern corresponded to CNCs, ChIP. While IL-12 only had minor effects at the TSS and DHI we overlayed the identified DH sites with a VISTA plot of Il18r1 sites, IL-12 induced acetylation of H3 and H4 at all CNC sites highlighting CNC between mouse and human sequences. DHIII, except CNC E (Fig. 4C). In contrast, IL-4 did not decrease acet- DHIV, and DHV corresponded to CNC A, B. and C, respectively ylation of H3 or H4 at any of the CNC regions (Fig. 4C). 3350 REGULATION OF Il18r1 IN Th SUBSETS

FIGURE 6. GATA-3 represses IL- 18R␣. A, ChIP analysis of Stat4 and GATA-3 binding to the promoter, DHI, or CNC A-E regions of Il18r1 in differentiated Th1 and Th2 cells. Quanti- tative PCR was performed and results are expressed as fold increase over control IgG ChIP. B, CD4 T cells were activated with anti-CD3 and anti-CD28 for 3 days. Cells were washed and either left unstimulated or stimulated with IL-12 or IL-4 for 24 h. ChIP was performed as described in A. Results are expressed as fold enrichment over control IgG ChIP. C, CD4 T cells were differentiated under Th1 conditions for 1 wk. Cells were then transduced with a control or GATA-3-express- Downloaded from ing bicistronic retroviral vector that also expresses hCD4. Four days after transduction, IL-18R␣ and CXCR3 expression was examined on hCD4- positive cells by ﬂow cytometry. D, Cells prepared as in C were sorted for hCD4 expression and RNA was iso- http://www.jimmunol.org/ lated for analysis of Il18r1 expression.

STAT protein regulation of Il18r1 expression and histone Transcription factor binding in the Il18r1 locus modiﬁcation Il18r1

In previous work, we identified that Stat4 binds to the pro- by guest on October 2, 2021 While the expression of Il18r1 is vastly different in Th1 and Th2 moter (7). To test whether factors that activate, or repress Il18r1 cells, Stat4 is required for the induced expression in Th1 cells and expression bind to specific regions of the locus, we performed Stat6 is required for the repression observed in Th2 cells (Fig. 5, ChIP for Stat4, Stat6, and GATA-3, and performed quantitative A and B). The intermediate level of inactivated Il18r1 expression PCR for the promoter and CNC regions. Stat4 was observed bind- in Stat4Ϫ/Ϫ Th1 and Stat6Ϫ/Ϫ Th2 is observed at the level of ing to the promoter region of Th1 cells but had lower levels of ␣ mRNA (Fig. 5B) and surface expression of IL-18R where these binding at DHI and the CNC regions and minimal levels detected populations have an intermediate staining intensity (Fig. 5A). in Th2 cells (Fig. 6A). Conversely, GATA-3 binding was detected To determine whether STAT proteins have a role in establishing primarily in Th2 cells with maximal levels in the CNC A region chromatin modifications in the CNC regions, we differentiated (Fig. 6A). Ϫ Ϫ C57BL/6 WT and Stat4 / Th1 cultures and BALB/c WT and To test whether these factors bound in response to cytokines Stat6Ϫ Ϫ / Th2 cultures and examined the level of histone acety- during the differentiation process, CD4 T cells were activated for lation at each of the CNC regions. As expected, levels of acetyl-H3 3 days and left unstimulated or stimulated with IL-4 or IL-12 for and acetyl-H4 were decreased in Stat4-deficient Th1 cultures com- 24 h. Stat4 binding to the promoter following IL-12 stimulation pared with WT Th1 cultures at CNC regions A-D (Fig. 5C). was increased 150-fold (Fig. 6B). Stat4 was also detected at the Acetyl-H3 and acetyl-H4 levels were very low in WT Th2 cells but CNC A and CNC B sites where binding was increased 80- to were increased in the absence of Stat6 at CNC regions A-C (Fig. 200-fold with only modest binding at the DH1 site (Fig. 6B). In 5C). There was no significant difference between levels of histone acetylation in WT Th2 cells derived from C57BL/6 or BALB/c contrast to Stat4 binding which was 10-fold over control binding in mice (data not shown). unstimulated cells, Stat6 binding was increased less than 5-fold We then tested whether there were differences in the levels of over control at any of the regions in unstimulated or IL-4 stimu- H3K4me2 and H3K4me3 at the Il18r1 CNC regions. Di-methyl- lated cells (data not shown). GATA-3 binding was not detected in H3K4 and tri-methyl-H3K4 were detected at CNC regions A-D in the promoter but was significantly enriched at the CNC A and WT Th1 cultures (Fig. 5D). In contrast to the patterns of acetylated CNC B regions although IL-4 had only modest effects on GATA-3 histones, H3K4 methylation was not decreased in the absence of binding (Fig. 6B). Stat4, correlating with the diminished but detectable Il18r1 mRNA These results suggested that IL-4 signaling and the induction of in Stat4Ϫ/Ϫ Th1 cells. Methyl-H3K4 levels were lower in Th2 GATA-3 would repress Il18r1 expression in Th1 cells. Transduc- cells but were increased in Stat6Ϫ/Ϫ Th2 cultures to levels ob- tion of GATA-3 into differentiating cells would divert them to a served in Stat4Ϫ/Ϫ Th1 cultures (Fig. 5D). Thus, H3K4 di- and Th2 phenotype where IL-18R␣ would be low and it would be tri-methylation is reduced in Th2 cultures, coincident with the re- difficult to discern direct effects of GATA-3 from indirect effects pression of Il18r1 mRNA. on differentiation of the cells. To avoid this problem and examine The Journal of Immunology 3351

Il18r1 regulation in absence of altered differentiation, we first dif- velopment, with DH sites I and IV becoming less sensitive, and the ferentiated cells under Th1 conditions for 1 wk before transduction appearance of DHIII (Fig. 2). These changes are most significant with control-hCD4 or GATA-3-hCD4 expressing bicistronic ret- after 2 wk of culture suggesting progressive changes correlating roviruses. After 5 days of culture, hCD4ϩ cells were analyzed for with increased commitment to the Th2 phenotype. While IL-4 did expression of IL-18R␣. Expression of GATA-3 reduced IL-18R␣ not have acute effects on altering chromatin modifications (Fig. 4), on Th1 cells and Il18r1 mRNA levels (Fig. 6, C and D). To con- Stat6 is required for repression of the locus during Th2 develop- firm that cells transduced with GATA-3 did not convert to a Th2 ment. In the absence of Stat6, there are increases in H3K4 meth- phenotype, we also analyzed expression of CXCR3, a chemokine ylation and histone acetylation, compared with undifferentiated receptor expressed on Th1 cells. GATA-3 did not have an effect on CD4 T cells or WT Th2 cells that results in Il18r1 expression CXCR3, demonstrating specificity in the effects on IL-18R␣ ex- increased to an intermediate level (Fig. 5). The precise function of pression (Fig. 6C). Stat6 in this process is unclear. We did not detect significant increases in Stat6 binding to the Il18r1 locus in the regions we ex- Discussion amined. It is still possible that Stat6 binds to another region of Gene expression is dynamically regulated during cellular differ- Il18r1 or to sites in adjacent genes. However, gene repression may entiation. This process has been studied in the development of Th be indirect, through the Stat6-mediated induction of GATA-3. subsets primarily by examining cytokine genes that are differen- GATA-3 was bound to the Il18r1 locus in Th2 cells, most signif- tially expressed in Th1 and Th2 subsets. In previous work, we icantly in the CNC A and B regions, where CNC A corresponds to examined the Il18r1 gene, a receptor protein that is expressed in DHIII a Th2 site, and CNC B is DHIV, which is less sensitive to and functionally linked to Th1 cells. We identified Stat4-dependent DNase in Th2 cells (Figs. 2, 4, and 6). GATA-3 is expressed in mechanisms for high IL-18R␣ expression in Th1 cells that in- naive CD4 T cells, but is induced 10- to 20-fold during Th2 de- Downloaded from volved Stat4-induced changes in histone modification and regula- velopment, and it is likely that the induced level is required to tion of the level of DNA methylation (7). In this report, we ex- mediate Il18r1 repression. While GATA-3 is predominantly con- amine chromatin structural changes by assessing DH across the sidered as an activator of the Th2 genetic program, it can repress locus. Several DH sites correspond to CNCs and show distinct gene expression during cellular differentiation (23–25). Impor- patterns of sensitivity in Th1 or Th2 cells. We further show that tantly, we demonstrate that ectopic expression of GATA-3 in dif- these DH/CNC regions are targets of histone modification follow- ferentiated Th1 cells decreases IL-18R␣ surface expression and http://www.jimmunol.org/ ing cytokine stimulation and during differentiation. Moreover, Il18r1 mRNA expression, without affecting CXCR3, another Th1- Stat4 and GATA-3 are associated with these regions during Th1 expressed receptor (Fig. 6). The fact that IL-18R␣ expression is and Th2 development, respectively, and ectopic expression of not reduced to a greater extent in retrovirus-transduced cells may GATA-3 represses IL-18R␣ expression, suggesting that both fac- be due to the presence of T-bet in Th1 cells, which is known to tors directly affect Il18r1 gene expression. have antagonistic activity upon GATA-3 (26), as well as differ- Unlike cytokine genes that have been the focus of previous work ences between the half-life of Il18r1 mRNA and IL-18R␣ protein. on chromatin differences involved in Th1 and Th2 programming, Together, these data define a mechanism for the repression of a Il18r1 is expressed in undifferentiated CD4 T cells (Fig. 1). During locus that is active in undifferentiated CD4 T cells and repressed by guest on October 2, 2021 the differentiation period, Il18r1 expression is induced in Th1 cells during Th2 differentiation. and repressed in Th2 cells at the level of mRNA and surface ex- Th cell differentiation provides a model for understanding pression (Fig. 1). The pattern of DH sites I, II, and IV is similar changes in gene architecture that culminate in gene expression or between undifferentiated T cells and Th1, suggesting that overall repression. Much of the analysis of gene expression in Th1 and chromatin structure does not change significantly during Th1 de- Th2 cells has been on cytokine genes. However, the changes that velopment, perhaps accounting for the lack of an effect of Stat4- occur in these genes might be unique as they are inducible genes deficiency on DH pattern (Fig. 2). In contrast, histone modifica- that are strictly controlled by stimulation from Ag or cytokine tions are impacted more dramatically during Th1 development, receptors. In a previous report and in this report, we have described with histone acetylation and methylation being decreased only the programming of the Il18r1 locus, a gene that is highly ex- modestly in Th2 cells, compared with undifferentiated CD4 T cells pressed in Th1 cells and is rapidly repressed during Th2 develop- (Figs. 3 and 4). However, there are also differentiation independent ment. We have shown that Stat4 is required for maximal expres- changes in H3K4 methylation, presumably resulting from T cell sion and that Stat6 is required for maximal repression, possibly activation and expansion, that are observed in Stat4Ϫ/Ϫ Th1 and through a mechanism involving GATA-3 binding to the locus and Stat6Ϫ/Ϫ Th2 cells at similar levels to those observed in Th1 cells repressing expression. This suggests that direct repression of genes (Fig. 5). Together, these data provide a sequence of remodeling during differentiation by a phenotype promoting transcription fac- events during programming in Th1 differentiation. Il18r1 exists in tor, in addition to interference with the expression or function of a poised state in undifferentiated CD4 T cells (Figs. 2–4). Follow- reciprocal program promoting factors, may be an important com- ing T cell activation, and in the absence of Stat6- and GATA-3- ponent of the ability of transcription factors to program lineage dependent repression, there is an increase in H3K4 methylation decisions. that correlates with the establishment of gene expression patterns intermediate between Th1 and Th2 cells (Figs. 3–5). IL-12 induces Acknowledgments increased histone acetylation as early as 24 h following stimulation We thank Dr. S. Goenka for review of this manuscript. (Fig. 4). Stat4 binds to Il18r1 following IL-12 stimulation and is required for the induction of histone acetylation, decreased DNA methylation, and establishment of the high level of gene expres- Disclosures sion observed in Th1 cells (Figs. 5 and 6) (7). Thus, gene induction The authors have no financial conflict of interest. during Th1 development is controlled by several distinct chromatin remodeling events. References Similarly, several processes are involved in gene repression dur- 1. Ansel, K. M., D. U. Lee, and A. Rao. 2003. An epigenetic view of helper T cell ing Th2 differentiation. The DH pattern changes during Th2 de- differentiation. Nat. Immunol. 4: 616–623. 3352 REGULATION OF Il18r1 IN Th SUBSETS

2. Lee, G. R., S. T. Kim, C. G. Spilianakis, P. E. Fields, and R. A. Flavell. 2006. T 14. Lawless, V. A., S. Zhang, O. N. Ozes, H. A. Bruns, I. Oldham, T. Hoey, helper cell differentiation: regulation by cis elements and epigenetics. Immunity M. J. Grusby, and M. H. Kaplan. 2000. Stat4 regulates multiple components of 24: 369–379. IFN-␥-inducing signaling pathways. J. Immunol. 165: 6803–6808. 3. Fields, P. E., S. T. Kim, and R. A. Flavell. 2002. Cutting edge: changes in histone 15. Nakahira, M., M. Tomura, M. Iwasaki, H.-J. Ahn, Y. Blan, T. Hamaoka, T. Ohta, acetylation at the IL-4 and IFN-␥ loci accompany Th1/Th2 differentiation. J. Im- M. Kurimoto, and H. Fujiwara. 2001. An absolute requirement for Stat4 and a munol. 169: 647–650. role for IFN-␥ as an amplifying factor in IL-12 induction of the functional IL-18 4. Mullen, A. C., A. S. Hutchins, F. A. High, H. W. Lee, K. J. Sykes, L. A. Chodosh, receptor complex. J. Immunol. 167: 1306–1312. and S. L. Reiner. 2002. Hlx is induced by and genetically interacts with T-bet to 16. Smeltz, R. B., J. Chen, J. Hu-Li, and E. M. Shevach. 2001. Regulation of inter- promote heritable T(H)1 gene induction. Nat. Immunol. 3: 652–658. leukin (IL)-18 receptor ␣ chain expression on CD4ϩ T cells during T helper 5. Shnyreva, M., W. M. Weaver, M. Blanchette, S. L. Taylor, M. Tompa, (Th)1/Th2 differentiation: critical downregulatory role of IL-4. J. Exp. Med. 194: D. R. Fitzpatrick, and C. B. Wilson. 2004. Evolutionarily conserved sequence 143–153. elements that positively regulate IFN-␥ expression in T cells. Proc. Natl. Acad. 17. Kaplan, M. H., Y.-L. Sun, T. Hoey, and M. J. Grusby. 1996. Impaired IL-12 Sci. USA 101: 12622–12627. responses and enhanced development of Th2 cells in Stat4-deficient mice. Nature 6. Lewis, M. D., S. A. Miller, M. M. Miazgowicz, K. M. Beima, and 382: 174–177. A. S. Weinmann. 2007. T-bet’s ability to regulate individual target genes requires 18. Kaplan, M. H., U. Schindler, S. T. Smiley, and M. J. Grusby. 1996. Stat6 is the conserved T-box domain to recruit histone methyltransferase activity and a required for mediating responses to IL-4 and for the development of Th2 cells. separate family member specific transactivation domain. Mol. Cell. Biol. 27: Immunity 4: 313–319. 8510–8521. 19. Mathur, A. N., H. C. Chang, D. G. Zisoulis, R. Kapur, M. L. Belladonna, 7. Yu, Q., V. T. Thieu, and M. H. Kaplan. 2007. Stat4 limits DNA methyltransferase G. S. Kansas, and M. H. Kaplan. 2006. T-bet is a critical determinant in the recruitment and DNA methylation of the IL-18R␣ gene during Th1 differentia- instability of the IL-17-secreting T-helper phenotype. Blood 108: 1595–1601. tion. EMBO J. 26: 2052–2060. 20. Chang, H. C., S. Zhang, V. T. Thieu, R. B. Slee, H. A. Bruns, R. N. Laribee, 8. Agarwal, S., and A. Rao. 1998. Modulation of chromatin structure regulates M. J. Klemsz, and M. H. Kaplan. 2005. PU.1 expression delineates heterogeneity cytokine gene expression during T cell differentiation. Immunity 9: 765–775. in primary Th2 cells. Immunity 22: 693–703. 9. Chang, S., and T. M. Aune. 2005. Histone hyperacetylated domains across the 21. Wu, C., P. M. Bingham, K. J. Livak, R. Holmgren, and S. C. Elgin. 1979. The Ifng gene region in natural killer cells and T cells. Proc. Natl. Acad. Sci. USA chromatin structure of specific genes. I. Evidence for higher order domains of 102: 17095–17100. defined DNA sequence. Cell 16: 797–806. 10. Zhang, F., and M. Boothby. 2006. T helper type 1-specific Brg1 recruitment and 22. Ruthenburg, A. J., C. D. Allis, and J. Wysocka. 2007. Methylation of lysine 4 on Downloaded from remodeling of nucleosomes positioned at the IFN-␥ promoter are Stat4 depen- histone H3: intricacy of writing and reading a single epigenetic mark. Mol. Cell. dent. J. Exp. Med. 203: 1493–1505. 25: 15–30. 11. Chang, S., and T. M. Aune. 2007. Dynamic changes in histone-methylation 23. Ouyang, W., M. Lohning, Z. Gao, M. Assenmacher, S. Ranganath, A. Radbruch, “marks” across the locus encoding interferon-␥ during the differentiation of T and K. M. Murphy. 2000. Stat6-independent GATA-3 autoactivation directs IL- helper type 2 cells. Nat. Immunol. 8: 723–731. 4-independent Th2 development and commitment. Immunity 12: 27–37. 12. Schoenborn, J. R., M. O. Dorschner, M. Sekimata, D. M. Santer, M. Shnyreva, 24. Ranganath, S., and K. M. Murphy. 2001. Structure and specificity of GATA D. R. Fitzpatrick, J. A. Stamatoyannopoulos, and C. B. Wilson. 2007. Compre- proteins in Th2 development. Mol. Cell. Biol. 21: 2716–2725.

hensive epigenetic profiling identifies multiple distal regulatory elements direct- 25. Obara, N., N. Suzuki, K. Kim, T. Nagasawa, S. Imagawa, and M. Yamamoto. http://www.jimmunol.org/ ing transcription of the gene encoding interferon-␥. Nat. Immunol. 8: 732–742. 2008. Repression via the GATA box is essential for tissue-specific erythropoietin 13. Xu, D., W. L. Chan, B. P. Leung, D. Hunter, K. Schulz, R. W. Carter, gene expression. Blood 111: 5223–8232. I. B. McInnes, J. H. Robinson, and F. Y. Liew. 1998. Selective expression and 26. Hwang, E. S., S. J. Szabo, P. L. Schwartzberg, and L. H. Glimcher. 2005. T functions of interleukin-18 receptor on T helper (Th) type 1 but not Th2 cells. helper cell fate specified by kinase-mediated interaction of T-bet with GATA-3. J. Exp. Med. 188: 1485–1492. Science 307: 430–433. by guest on October 2, 2021