The Journal of Immunology

Phylogenetic and Functional Analysis Identifies Ets-1 as a Novel Regulator of the Th2 Cytokine

Jannine M. Strempel,* Roland Grenningloh,†,‡ I-Cheng Ho,†,‡ and Donata Vercelli*,x,{

The Th2 cytokine gene locus has emerged as a remarkable example of coordinated , the regulation of which seems to be rooted in an extensive array of cis-regulatory regions. Using a hypothesis-generating computational approach that integrated multispecies (n = 11) sequence comparisons with algorithm-based binding-site predictions, we sought to identify evolutionarily conserved noncoding regions (ECRs) and motifs shared among them, which may underlie coregulation. Twenty-two transcription factor families were predicted to have binding sites in at least two Th2 ECRs. The ranking of these shared motifs according to their distribution and relative frequency pointed to a regulatory hierarchy among the transcription factor families. GATA sites were the most prevalent and widely distributed, consistent with the known role of GATA3 as a Th2 master switch. Unexpectedly, sites for ETS-domain were also predicted within several Th2 ECRs and the majority of these sites were found to support Ets-1 binding in vitro and in vivo. Of note, the expression of all three Th2 cytokines (IL-5, -13, and -4) was significantly and selectively decreased in Th2 cells generated from Ets-1–deficient mice. Collectively, these data suggest that Ets-1 contributes to Th2 cytokine gene regulation by interacting with multiple cis-regulatory regions throughout the Th2 locus. The Journal of Immunology, 2010, 184: 1309–1316.

he spatio-temporal control of eukaryotic gene expression tensively in the Th2 lineage and was shown to provide a remarkable is a complex process, orchestrated to a large extent by example of coordinated gene expression (4, 5). Such coordination T mechanisms that control transcription. Regulation of in- seems to be highly adaptive, in that the distinct components of the dividual loci often relies on multiple cis-regulatory elements, lo- Th2 cytokine trio cooperate, ensuring the deployment of comple- cated proximal (promoters) and distal (enhancers, silencers, and mentary antiparasite defense pathways (6). In contrast, when dys- insulators) to the transcription start site. Clusters of functionally regulated by genetic and/or environmental influences, coordinated related may exhibit additional complexity and share mul- Th2 cytokine expression becomes critical for the pathogenesis of tiple cis-regulatory elements that transduce developmental and/or allergic inflammation (7, 8). environmental cues to specify sequential expression of individual An extensive array of highly conserved cis-regulatory elements genes, as in the b-globin gene cluster (1), or coordinated ex- essential for the coordinated expression of Th2 cytokine genes pression of several genes, as is the case for the Th2 cytokine gene has been identified by a combination of DNase I hypersensitive locus (2), which includes IL5, IL13, and IL4. site (HS) mapping and comparative sequence analysis (Fig. 2A). The Th2 cytokine genes are grouped within a 150-kb region of HSs 1 and 2 (9), located between Il13 and Il4, map to conserved human 5q31 and the syntenic region of mouse chro- noncoding sequence (CNS)-1, a potent enhancer of cytokine mosome 11. IL13 and IL4 are adjacent to one another, whereas IL5 is gene expression in T cells (10–12) (Fig. 2A). Two other regions, separated from these two by a large (85 kb) DNA repair gene, which map to the second intron of Il4 (HS II and III; Il4 intronic RAD50, a configuration that has been preserved for $300 million enhancer [IE]) and downstream of Il4 (HSV/Va; CNS-2), exhibit years (3). The regulation of this locus has been studied most ex- enhancer activity in Th2 cells and mast cells (13–15). Several HSs (RAD50 HS [RHS]5–7) clustered within the 39 end of the DNA repair gene RAD50 (16, 17) collectively function as a locus *Functional Genomics Laboratory, Arizona Respiratory Center, xArizona Center for { control region (LCR), conferring robust Th2-specific, position- the Biology of Complex Diseases, and Department of Cell Biology, College of Medicine, University of Arizona, Tucson, AZ 85719; †Division of Rheumatology, independent and copy number-dependent expression to the Il4 Immunology, and Allergy, Department of Medicine, Brigham and Women’s Hospital; ‡ and Il13 genes (16, 18). Finally, a single silencer located at the 39 and Harvard Medical School, Boston, MA 02115 end of Il4 (HSIV) is required to suppress Il4 expression in naive Received for publication December 12, 2008. Accepted for publication November CD4+ T and Th1 cells (14, 19). Of note, the major Th2 cis-reg- 24, 2009. ulatory elements typically map to extensive (300–600 bp) regions This work was supported by National Institutes of Health Grant 5RO1HL66391 (to D.V.) and a National Science Foundation Integrative Graduate Education and Re- that are highly conserved between mice and humans. search Traineeship in Genomics (to J.M.S.). Several lines of evidence point to a significant degree of in- Address correspondence and reprint requests to Dr. Donata Vercelli, 1657 East Helen terdependency among distant regulatory regions within the Th2 Street, 321 Thomas W. Keating Bioresearch Building, Tucson, AZ 85719. E-mail address: [email protected] locus. Indeed, analysis of transgenic models has shown that no The online version of this article contains supplemental material. single element is sufficient to fully recapitulate Th2 cytokine gene Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; CNS, con- expression at the levels driven by an intact Th2 locus (16, 18). served noncoding region; ECR, evolutionarily conserved region; HS, hypersensitive Conversely, deletion of any enhancer region is sufficient to site; IE, intronic enhancer; LCR, locus control region; MULAN, multiple sequence local alignment and visualization tool; P, promoter; PWM, position weight matrix; markedly inhibit the expression of more than one Th2 cytokine rhEts-1, recombinant human Ets-1; RHS, RAD50 hypersensitive site; TF, transcrip- gene (11, 15, 20). Moreover, characterization of long-range in- tion factor. trachromosomal interactions at the murine Th2 locus determined Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 that in T cells, the Il5, Il13, and Il4 promoters are physically

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804162 Downloaded by guest on October 1, 2021 1310 Ets-1 CONTRIBUTES TO Th2 CYTOKINE GENE COREGULATION

clustered with one another and with the distant regulatory ele- EMSA ments, forming a distinctive chromatin hub (21). Single-stranded complementary oligonucleotides were annealed and PAGE TheadvancedstructuralandfunctionalcharacterizationoftheTh2 purified. Annealed oligonucleotides were end-labeled with g-[32P] ATP using locus provides a unique opportunity to decipher molecular cues that T4 polynucleotide kinase. Binding reactions consisted of 50 ng human re- might dictate the function of individual regulatory regions, as well as combinant Ets-1 (Axxora, San Diego, CA) in binding buffer (25 mM Tris-Cl, 1 mM EDTA, 60 mM KCl, 6 mM MgCl, 10 mM DTT, 1 mgPoly(dI·dC), 10% specify coordinated gene expression. A simple model to orchestrate glycerol). Competition assays were performed by preincubation for 30 min at the coexpression of distinct genes predicts that their promoters will room temperaturewith unlabeled oligonucleotide competitors (30- and 90-fold contain a common set of transcription factor binding sites critical for molar excess) prior to addition of the probe. Samples were incubated with transducing relevant signals. Building on such a model, we took radiolabeled probes for 30 min at room temperature and run on a 5% (w/v) a comprehensive in silico approach, and complemented it with polyacrylamide gel (20 mAmp, 4 h, 4˚C), which was dried and subjected to autoradiography. Oligonucleotide sequences used as probes and/or com- functionalinvitroandinvivostudies,todeciphertheregulatorylogic petitors included a previously defined high-affinity Ets-1 motif (SC1_ETS1 underlying the coexpression of the Th2 cytokine gene cluster. Al- CGGCCAAGCCGGAAGTGAGTGCC; the core consensus is underlined) though computational predictions of transcription factor binding (27), a mutant of this motif (SC1_ETS1mut CGGCCAAGCCttAAGT- sites have been hindered by considerable false-positive rates, such GAGTGCC), and the conserved ETS sites predicted within the human Th2 cis- regulatory regions (IL5P: TGTCTTTGAGGAAATGAATAA, IL13P: GTT- predictions are substantially improved by integrating motif-finding CGGGGAGGAAGTGGGTAG, IL4P.1: GATTTCACAGGAACATTTTAC, algorithms with phylogenetic comparisons (22–24) and further IL4P.2: TTTTCTCCTGGAAGAGAGGTG, IL13P: GTTCGGGGAGGAA- strengthened by functional validation. In this study, we show that GTGGGTAG, RHS5: GGTAACACAGGAAGTCAGCAG, IL4IE: AT- our hypothesis-generating, multipronged approach succeeded in GAAAACAGGAACTGAAATG, HSIV: TCTGCCACAGGATATGGGTAG, identifying Ets-1 as a novel, important regulator of coordinated Th2 CNS2: TGGGTCACAGGAAGCCCAAGA). The intensity of the Ets-1 complexes was determined by densitometry. cytokine gene expression. Mice Materials and Methods Female 3–6-wk-old C57BL/6 mice were obtained from The Jackson Multispecies comparative analysis Laboratory (Bar Harbor, ME). Ets-1–deficient mice were described pre- viously (28). The animals were housed under specific pathogen-free con- Genomic sequences corresponding to the human Th2 cytokine gene cluster ditions and experiments were performed in accordance with the (20 kb downstream of IL5 through the 39 end of KIF3A) and the syntenic institutional guidelines for animal care at the University of Arizona and the regions in Pan troglodytes (chimpanzee), Papio anubis (baboon), Callithrix Dana-Farber Cancer Institute (Boston, MA) under approved protocols. jacchus (marmoset), Otolemurgarnetti (bush baby), Bos taurus (cow), Canis familiaris (dog), Rattus norvegicus (rat), Mus musculus (mouse), Mono- T cell isolation and in vitro differentiation of Th2 cells delphis domestica Gallus gallus + (opossum), and (chicken) were obtained Murine Th2 cells were generated essentially as described (29). Naive CD4 from NCBI. Some of the sequence used was generated by the National In- T cells were isolated from spleens by negative selection, followed by stitutes of Health Intramural Sequencing Center (www.nisc.nih.gov). enrichment using anti-CD62L–coated magnetic beads (Miltenyi Biotec, Generation of a multispecies alignment and identification of evolu- Auburn, CA). Cells (2–5 3 106) were cultured in the presence of anti- tionarily conserved regions (ECRs) were performed with the MUltiple CD3ε Ab (145.2C11, 1 mg/ml) and anti-CD28 (37.51, 1 mg/ml) (BD sequence Local AligNment and visualization tool (MULAN) program Pharmingen, San Diego, CA) in a flask coated with goat anti-hamster IgG (http://mulan.dcode.org/) (25). MULAN uses a local alignment strategy (0.2 mg/ml), under Th2 skewing conditions (1000 U/ml IL-4, 3 mg/ml anti- using the threaded blockset aligner program and uses the phylogenetic IL-12, and 5 mg/ml anti–IFN-g; complete medium) for 3 d. Then the cells relationships of the sequences provided to build the multispecies alignment were expanded in complete medium containing IL-2 (20 U/ml) for 7 d. (25). Repeat masking was performed on all sequences with the species- Cytokine expression at the single-cell level was examined by intracellular appropriate filters prior to alignment. A large gap due to incomplete se- staining using the following Abs: anti–IL-4 PE (11B11), anti–IL-13 Alexa quence data was detected at the 39 end of RAD50 in C. jacchus. Therefore, (eBio13A), and anti–IFN-g FITC (XMG1.2). For restimulation, cells were our analysis of this region (which spans RHS6.1, 6.2, and 7 within the Th2 incubated with increasing concentrations (0.3–3 mg/ml) of anti-CD3 mAb, LCR) did not include this species. Because of the very close relationship in the presence of constant amounts of anti-CD28 Ab (2 mg/ml), for 24 h. between human and marmoset, it is unlikely that exclusion of this species Cytokine secretion was quantified by ELISA (Quantikine Immunoassays, significantly altered the multispecies alignment or the transcription factor R&D Biosystems, Minneapolis, MN). binding-site profiles generated for this region. Chromatin immunoprecipitation Identification of conserved transcription factor binding sites Murine Th2 cells were treated with 1% formaldehyde for 10 min at room Genomic regions (excluding exons) identified by the MULAN program as temperature followed by the addition of glycine(125 mM finalconcentration) multispecies ECRs ($70% identity over $100 bp in all species examined) to halt cross-linking. Cells were harvested, washed twice with 13 PBS, and were examined for putative transcription factor binding sites. Ungapped resuspended in cell lysis buffer (5 mM PIPES [pH 8], 85 mM KCl, 0.5% sequences from each species were analyzed individually with the Mat- Nonidet P-40) supplemented with protease inhibitors (10 mg/ml Aprotinin, Inspector program, using the matrix library version 6.0, which contains 431 13 EDTA-free complete protease inhibitor mixture [Roche, Basel Swit- position weight matrices (PWMs) for vertebrate transcription factors, rep- zerland], and 1 mM PMSF). Nuclei were collected by centrifugation and resenting 148 families, (Genomatix, Munich, Germany, http://www.ge- lysed in nuclear lysis buffer (50 mM Tris-HCl [pH 8.1], 10 mM EDTA, 1% nomatix.de/). PWMs describe transcription factor binding sites in terms of SDS) supplemented with protease inhibitors as above. Chromatin was the complete nucleotide distribution for each single position, allowing for sheared by sonication to yield the majority of fragments in the 200–600-bp quantification of similarity between the weight matrix and the putative size range. An aliquot of chromatin (1 3 107 whole cell equivalents) was binding motif (26). Detection criteria were set to only report matches that precleared with a 50% A Sepharose slurry (150 ml) for 20 min at 4˚C. demonstrated a core similarity score $0.85 and a matrix similarity score that Precleared chromatin was then diluted 10-fold in IP dilution buffer (0.01% met or exceeded the optimization threshold defined for each PWM. The SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.1], 167 optimization threshold is a parameter designed to significantly reduce the mM NaCl) containing protease inhibitors. Immunoprecipitation reactions rate of false positives by limiting detection to no more than three hits within were performed with 5 3 106 whole-cell equivalents overnight at 4˚C with an 10 kb of nonregulatory sequence (26). We identified those transcription anti–Ets-1 Ab (C-20, Santa Cruz Biotechnology, Santa Cruz, CA) or normal factor binding sites predicted independently in 100% of the species exam- rabbit IgG (10 mg). Immunocomplexes were collected with Protein A aga- ined, including matches to different matrices within a family. Matrix families rose beads (Millipore, Bedford, MA), washed once each with low-salt wash, represent functionally related transcription factors that also have PWMs that high-salt wash, and LiCl wash, and washed twice with TE. Im- are essentially indistinguishable (26). Of these sites, those that mapped to the munocomplexes were eluted with IP elution buffer (1% SDS, 50 mM same location within the multispecies alignment were included in the profile. NaHCO3), and cross-links were reversed with an overnight incubation at 65˚ For those cases in which multiple matrix families met the conservation cri- C. DNA was purified by phenol/chloroform extraction and ethanol pre- teria and mapped to the same location in the alignment, all families were cipitation using glycogen as a carrier. Real-time PCR was performed with the included in the profile. Quantitect SYBR Green PCR kit (Qiagen, Valencia, CA) on an ABI Prism Downloaded by guest on October 1, 2021 The Journal of Immunology 1311

7900 Sequence Detection System (Applied Biosystems, Foster City, CA). human–mouse alignments (Fig. 2A) and validated in functional The following primer pairs were used: Il5 promoter: 59-ACCCTGAGTTT- studies (10, 11, 15, 17–20) were highlighted as ECRs in the seven CAGGACTCG-39,59-TCAGAACACTCAAGTGCAGAAG-39; Rad50H- S5: 59-GTTTGTCGTCTCAGAGTAAAGC-39,59-GTCACATCCTGAGC placental mammalian species examined (Fig. 2B). In addition, this ATGTTCC-39; Il13 promoter: 59-GTGAGAAG-ATGGAATCTGGC-39,59- analysis identified a novel noncoding ECR, located ∼6 kb up- CCCTACTCTTTCCTCATACTG-39; Il4 promoter: 59-CCT-ACGCTGA- stream of the IL5 gene (IL-5/-6). Of note, only a single noncoding TAAGATTAGTCTG-39,59-CACCAGATTGTCAGTTATTCTG-39; Il4 IE: element (110 bp within RHS5) was conserved in the syntenic 59-GATGTGACAGGCTGATAGTG-39,59 GAGACCACTGGTAAG-TC- AAGAC-39; HSIV: 59-GATTGAAACTCATTTGCATGTC-39,59-GTGT- region in chicken. In contrast, the majority of the noncoding ECRs TCTGACC-ATCCAAGTGTAG-39; CNS-2: 59-GAAGCAGTGTGGGAGA were highly conserved in the orthologous opossum locus (Fig. GCGT-39,59-GGGAAA-GTGATCGTGAGGAG-39; and Cd14 promoter: 2B), with the exception of the functionally undefined IL5/-6, the 59-GAATGCCCTAATGCCACTCTG-39 and 59-GCTTGTTCGACCAAT distal region of the IL13 promoter (which encompasses a con- TTGGC-39. PCR reactions were performed under the following cycling conditions: 15 min at 95˚C, followed by 40 cycles of 15 s at 95˚C, 30 s at 57˚ served GATA-3–responsive element) (36), and HSIV, which is C, and 30 s at 72˚C. Dissociation curve analysis and agarose gel electro- critical for Il4 silencing in murine Th1 cells (19). phoresis confirmed amplification of a single product for each primer set. In Collectively, these data indicate that the majority of conserved addition, PCR efficiency was optimized by adjusting the primer concentra- regulatory regions within the Th2 cytokine locus predate the di- tion and running serial dilutions of input DNA to generate a standard curve. ∼ Chromatin immunoprecipitation (ChIP) data are represented as the PCR vergence of marsupials and placental mammals 180 million years signal generated for the sample immunoprecipitated with anti–Ets-1 Ab ago (35, 37). The phylogenetic distance between chicken and relative to normal rabbit IgG (specific enrichment). mammals might preclude alignment-based detection of additional regulatory regions. Real-time PCR Phylogenetic profiling of Th2-responsive elements Total RNA was extracted using TRIzol reagent and reverse-transcribed using oligo(dT) priming. Negative control reactions without reverse tran- The Th2 cytokine locus ECRs identified by comparative analysis scriptase were always included. Semiquantitative RT-PCR was performed in were then examined to search for shared transcription factor duplicate on an ABI Prism 7900 Sequence Detection System using SYBR binding sites that might contribute to coordinated regulation of the green dye detection. mRNA levels were measured using predesigned primer Th2 locus. Because some of the Th2 ECRs were undetectable in the sets for murine Il4, Il5, Il13, Il10, Gata3, and Gapdh (Quantitect Primer Assays, Qiagen). Products were verified by melting curve analysis. Th2 opossum genome, we profiled transcription factor binding sites cytokine and Gata3 mRNA levels are expressed relative to Gapdh mRNA across nine placental mammalian species, representing four major abundance within the same sample. clades (primates, carnivores, artiodactyls, and rodents). Multi- species comparisons of ECRs increase the overall sequence di- Results vergence, which serves to refine alignments and reveal invariant positions more likely to reflect true functional constraints asso- Most Th2 cis-regulatory elements predate the divergence ciated with transcription factor binding sites (22–24, 38). Indeed, between placental and nonplacental mammals the sequence diversity within the noncoding Th2 ECRs captured ToinvestigatetheevolutionaryhistoryoftheTh2regulatoryelements by the multiple species alignment was ∼20% greater than that and establishan appropriate scopeofspecies for subsequent binding- obtained by pairwise comparison between human and mouse se- site analyses, we first assessed to what extent the extensive array of quences (data not shown). Th2 ECRs from placental mammals Th2 regulatory regions characterized in humans and mice is con- were examined for putative transcription factor binding sites using served in additional placental mammals and evolutionarily more the MatInspector program (26). Searches were conducted on un- distant species. Beyond placental mammals, orthologs for IL5, IL13, gapped sequence from each species using a vertebrate PWM li- and IL4 were recently identified for a marsupial, the laboratory brary and stringent match criteria. Sites predicted independently in opossum (Monodelphis domestica) (30), and the chicken (G. gallus) all species and residing in the same position within the multiple (3). Although a draft sequence of the platypus (Ornithorhynchus species alignment were included in the profile. anatinus) genome was recently released (31), the genomic region Within the Th2 ECRs, which collectively span ∼4.8 kb, we expected to contain orthologs of the Th2 cytokine genes is in- identified 103 conserved motifs representing targets for 52 distinct completely covered and could not be included in our analysis. transcription factor families (data not shown). Fig. 3 shows that 22 Furthermore, because comparative analysis of syntenic regions and transcription factor families were predicted to have binding sites mining of expressed sequence tag libraries failed to identify these within at least two Th2 regulatory regions. When multiple sites for genes in earlier vertebrate species (teleosts) (32), we did not extend a particular factor were found, they typically resided in different our analysis beyond the avian order.

Fig. 1 shows the phylogenetic relationships and estimated di- Homo sapiens vergence times (33–35) for the 11 species included in our study. A Pan troglodytes multiple DNA sequence alignment for the entire Th2 cytokine Papio anubis gene cluster (Fig. 2A) was generated with the MULAN program Callithrix jacchus (25), which relies on a local alignment strategy and integrates the Otolemur garnettii phylogenetic relationships among the sequences. Fig. 2B shows Mus musculus the MULAN-generated alignment for selected regions within the Rattus norvegicus Canis familiaris Th2 locus in a subset of species representative of the clades we Bos taurus analyzed. The ability to detect the exons for IL5, IL13, and IL4 by Monodelphis domestica multiple species alignment decreased with increasing phyloge- Gallus gallus netic distance, consistent with previous reports that the opossum (30) and chicken (3, 32) Th2 cytokines exhibit relatively low 300 200 100 0 homology to the human proteins. In contrast, all exons for the Million Years DNA repair gene RAD50 were highly conserved and readily FIGURE 1. Phylogenetic relationships and estimated divergence times identified in all species (Fig. 2B and data not shown). As expected, for species used in comparative analysis. Divergence time estimates were all of the Th2 regulatory regions previously identified in pairwise provided by the literature (33–35). Downloaded by guest on October 1, 2021 1312 Ets-1 CONTRIBUTES TO Th2 CYTOKINE GENE COREGULATION

A LCR

HS 1,2 (IL13P) HSS HSS3 1-2 (CNS-1) HS I (IL4P) HS IVHS VaHS V (CNS-2) RHS 1 (IL5P) RHS RHS 5 RHS 6 7 HS II (IL4 IE)

5LI RAD50 31LI IL4 KIF3A

B IL5 Th2 LCR (Rad50 3’ End) * FIGURE 2. Th2 cis-regulatory ele- P IL5/-6 RHS5 RHS6.1/6.2 RHS7 ments predate the divergence of pla- 100 100 Gallus cental and nonplacental mammals. A, 50 50 Map of the Th2 cytokine gene cluster 100 100 with the locations of murine DNase I Mono 50 50 hypersensitive sites (vertical arrows). B, 100 100 MULAN-generated graphical represen- Mus 50 50 tation of DNA sequence alignments be- 100 100 Canis

tween the indicated species and the Percent Identity 50 50 reference human sequence. Selected re- 100 100 gions of the Th2 cytokine locus are Oto 50 50 shown, with gene annotations and fea- 100 100 tures noted above the plots. Shaded re- Papio 50 gions indicate multispecies ECRs with 50 gene features specified by color (exon: blue; untranslated region: yellow; intron: IL13 IL4 IEP pink; intergenic: red). Dashed boxes P HSS3CNS-1 HSIV CNS-2 denote regions that are conserved among 100 100 the placental mammals but not in opos- Gallus 50 50 sum or chicken. P, promoter. 100 100 Mono 50 50 100 100 Mus 50 50 100 100 Canis Percent Identity 50 50 100 100 Oto 50 50 100 100 Papio 50 50

regions, rather than clustering within one or few regulatory ele- family member that is predominant in T cells (43). To functionally ments. In fact, the number of conserved sites and the number of validate the ETS motifs predicted within the Th2 ECRs, we used distinct regulatory regions they are predicted to target were pos- EMSA and compared and contrasted the ability of oligonucleo- itively correlated (R2 = +0.81). The number of motifs common to tides corresponding to individual putative ETS sites to bind re- distinct regions did not correlate with GC content nor linear combinant human Ets-1 (rhEts-1). Fig. 4A shows the results of an proximity (data not shown). Therefore, these data are unlikely to experiment representative of this approach. Incubation of rhEts-1 merely reflect genomic bias. Rather, they may highlight an un- with a high-affinity Ets-1 consensus probe (27) led to the forma- derlying regulatory network within the Th2 locus. tion of a complex (lane 1) that was competed almost completely upon incubation with a 30-fold molar excess of unlabeled Ets-1 Identification and functional validation of Ets-1 binding sites in consensus oligonucleotide (lane 2) and was specifically super- the Th2 locus shifted by an anti–Ets-1 Ab (data not shown). Oligonucleotides The ranking of transcription factor binding sites shared across the spanning the putative ETS sites in HSIV (lanes 4 and 5) and the Th2 locus, according to their distribution and relative frequency IL13 promoter (lanes 6 and 7) also competed Ets-1 binding, albeit (Fig. 3, Supplemental Fig. 1), pointed to a regulatory hierarchy less effectively. In contrast, an oligonucleotide encompassing one among transcription factor families. Interestingly, the most prev- of the two putative ETS sites within the IL4 promoter (IL4P.1, alent and widely distributed motifs were those for GATA proteins, lanes 12 and 13) failed to compete Ets-1 binding. Similar ex- consistent with the notion that GATA-3 acts as a master switch for periments were performed to test each predicted ETS motif, and Th2 cell differentiation and Th2 cytokine expression (36, 39–42). the intensity of the relevant bands was measured by densitometry This finding validated our approach and by extension raised the and compared with the intensity of the uncompeted Ets-1 com- possibility that members of the ETS and SORY/HMG transcrip- plex. The Ets-1 consensus and an Ets-1 oligonucleotide containing tion factor families, which like GATA proteins were predicted to two mutations in the ETS core were used as positive and negative target six or more Th2 cis-elements, might also play a role in the controls throughout this study. coordinated regulation of the Th2 cytokine locus. Fig. 4B shows the competition curves for the eight putative ETS Our subsequent analysis focused on the ETS family, which had sites identified within the Th2 locus. The motifs within RHS5 and not been previously implicated in orchestrating the coordinated the IL13 promoter competed Ets-1 binding almost as effectively as expression of the Th2 cytokines, and particularly on Ets-1, the the Ets-1 consensus oligonucleotide. The sites within HSIV, CNS2, Downloaded by guest on October 1, 2021 The Journal of Immunology 1313

LCR *

Description TF Family IL5P IL5 (-6) RHS5 RHS6.1 RHS6.2 RHS7 IL13P HSS3 CNS-1 IL4P IL4IE HSIV CNS-2 GATA binding factors GATA 1 11 3 111 3 Human and murine ETS1 factors ETSF 1 1 1 1112 6+ SOX/SRY and related HMG box factors SORY/HMG 1 1 111 1

Factors with moderate activity to homeo domain consensus sequence HOXF 11 1 1 Signal transducer and activator of transcription STAT 1 2 12 4

MAF and AP1 related factors AP1R 1 1 1 Nuclear factor of activated T-cells NFAT 1 1 1 CG/1PS srotcaf xob CG xob srotcaf CG/1PS F1PS 1113 nietorp gnidnib ATAT gnidnib nietorp FPBT 1 1 1

cAMP responsive element binding proteins CREB 12 EGR /nerve growth factor induced protein C & related factors EGRF 11 Ectopic viral integration site EVI 1 1 rotcaf niamod daehkroF niamod rotcaf DHKF 1 1 Growth factor independence-transcriptional repressor GFI1 21 sexelpmoc XBP - XOH - XBP sexelpmoc CXOH 1 1 2 Interferon regulatory factors IRFF 1 1 associated zinc fingers MAZF 11 Nuclear factor kappa B/c- NFKB 1 1 RBP-Jkappa/CBF1; Notch pathway RBPF 1 1 Human acute myelogenous leukemia factors (RUNX) RUNX 1 1 TALE homeodomain class recognizing TG motifs TALE 12 Zinc binding protein factors ZBPF 11 FIGURE 3. Common putative transcription factor binding motifs among the Th2 cis-regulatory regions. Shown are those transcription factor (TF) families that exhibit conserved target sites in more than one Th2 element. Noted within each box is the number of individual conserved sites predicted for that region. Transcription factors are ranked according to the number of Th2 cis-elements that contain conserved putative binding motifs. The DNA se- quence of each putative GATA and ETS motif for each species analyzed is provided as supplemental material (Supplemental Fig. 1).

and IL4IE exhibited intermediate binding ability. Competition by at IL4IE, but it barely exceeded the one generated for the Cd14 the IL5 promoter site was more modest but still appreciable. In promoter. Of note, our ChIP experiments detected Ets-1 and no contrast, individual ETS motifs within the IL4 promoter (IL4P.1 and other members of the large ETS family, because specific enrich- IL4P.2) failed to compete Ets-1 binding. ment was completely abrogated at all of the Th2 ECRs in Ets-1– Fig. 4C shows the core motif defined specifically for Ets-1 (27) deficient Th2 cells (data not shown). Overall, these data show that and the sequences of the Th2 Ets-1 binding sites, ranked by rel- Ets-1 binds to multiple Th2 regulatory regions in vivo, supporting ative Ets-1 binding ability. Relative to the Ets-1 core, all se- the possibility that Ets-1 participates in the coordinated regulation quences containing no or one mismatch supported Ets-1 binding, of the Th2 cytokine gene cluster. albeit to different extents. In contrast, two mismatches seemed to Ets-1 is required for optimal expression of all three Th2 compromise binding, as shown by the poor interactions detected cytokine genes for IL5 promoter, IL4P.1, and IL4P.2. Collectively, these data show that the majority of the ETS sites To test the role of Ets-1 in Th2 cytokine gene expression, we com- predicted within the Th2 ECR behave as bona fide Ets-1–binding pared the levels of Il4, Il13, and Il5 mRNA and protein produced by +/2 2/2 + motifs in vitro, suggesting that this protein (and/or possibly other Th2 cells from Ets-1 or Ets-1 mice. Naive CD4 T cells members of the ETS family) might bind at multiple locations isolated from these mice were cultured under Th2-skewing con- throughout the locus, thereby participating in the concerted reg- ditions for 7 d and stimulated for 24 h with increasing concentrations ulation of Th2 cytokine gene expression. of plate-bound anti-CD3 mAb, in the presence of a constant amount of anti-CD28 Ab. mRNA levels were assessed by real-time RT-PCR. Ets-1 binds several regulatory regions in the murine Th2 locus Fig. 6A shows that Ets-12/2 Th2 cells exhibited a marked decrease in vivo in Il4, Il5, and Il13 mRNA relative to Ets-1+/2 Th2 cells. In contrast, To examine whether Ets-1 binds to the endogenous Th2 ECRs, transcript levels for Il10, a cytokine gene expressed by Th2 cells and 2 ChIP experiments were performed on chromatin isolated from located on mouse chromosome 1, were comparable in Ets-1+/ and 2 2 naive CD4+ T cells cultured under Th2-skewing conditions for 7 d. Ets-1 / Th2 cells. Impaired Th2 cytokine transcription in cells Immunoprecipitation relied upon an Ab specific for Ets-1 or lacking Ets-1 was unlikely to reflect a defect in Th2 cell differen- normal rabbit IgG as a negative control. Real-time PCR was tiation per se, because Gata3 mRNA levels in these cells were un- performed using primers that target the individual Th2 ECRs affected (Fig. 6B). previously tested for Ets-1 binding in vitro. The promoter of Cd14, In parallel, we examined the impact of Ets-1 deficiency on Th2 a gene that is not expressed in murine Th2 cells (data not shown), cytokine secretion. Fig. 6C shows that, consistent with the de- 2 2 was amplified for comparison. crease observed in Th2 cytokine transcripts, Ets-1 / Th2 cells Fig. 5 shows that, relative to the Cd14 promoter, Ets-1–con- were also impaired in their ability to release IL-4, -13, and -5, but taining complexes were specifically enriched at all three Th2 cy- not IL-10. Collectively, these data demonstrate that Ets-1 acts as tokine gene promoters. The strongest occupancy was detected at a global positive regulator of the expression of the Th2 cytokine the Il4 promoter, a region that contains two ETS-binding motifs. gene cluster, thereby providing functional validation for our Notably, the two ETS motifs in this region failed to bind Ets-1 phylogenetic analysis. when tested individually in vitro, suggesting that the strong signal detected in vivo reflects cooperative binding. Ets-1 was also Discussion consistently found to bind distal Th2 regulatory regions, including The Th2 cytokine gene locus typically acts as a unit, the distinct RHS5 of the Th2 LCR, the Il4 silencer region (HSIV), and a Th2 components of which (IL5, IL13, and IL4) are coregulated and cytokine enhancer (CNS2). An Ets-1–specific signal was detected coexpressed in response to T cell activation and differentiation. Downloaded by guest on October 1, 2021 1314 Ets-1 CONTRIBUTES TO Th2 CYTOKINE GENE COREGULATION

Ets-1 Ets-1 A cons. HSIV IL13P cons. IL4P.1

Nil Nil Nil

30X 90X 30X 90X 30X 90X 30X 90X 30X 90X

FIGURE 4. Ets-1 binds to the majority of ETS motifs predicted in the Th2 ECR. A, Ets-1 pro- tein/DNA interactions were characterized using a g2[32P]-labeled Ets-1 consensus probe. Puri- Ets-1 fied rhEts-1 (50 ng) was preincubated with an increasing molar excess of the indicated com- petitors before addition of a high-affinity Ets-1 consensus probe. B, EMSA blots were analyzed by densitometry, and the results are expressed as 12345 6 7 8 9 10 11 12 13 the percentage inhibition of the Ets-1 band - C Ets-1 Core tive to the uncompeted Ets-1 complex. C, Se- B C A T quences of the Ets-1 binding site core, the high- CAGGA TGC affinity Ets-1 consensus probe, and the eight 100 Ets -1 consensus GGCCAAGCCGGAAGTGAGTGC conserved ETS motifs predicted within the Th2 Rad HS5 GGTAACACAGGAAGTCAGCAG 80 ECRs. Bold type indicates nucleotide sites within IL13P GTTCGGGgAGGAAGTGGGTAG the Ets-1 core; nucleotides that differ from the HSIV TCTGCCACAGGATaTGGGTAG defined core sequence are marked by lower case 60 CNS2 TGGGTCACAGGAAGCCCAAGA letters. 40 IL4IE ATGAAAACAGGAAcTGAAATG IL5P TGTCTTTgAGGAAaTGAATAA

Percent Inhibition 20 IL4P.1 GATTTCACAGGAAcaTTTTAC IL4P.2 TTTTCTCCtGGAAGaGAGGTG 0 Ets-1 Mut GGCCAAGCCttAAGTGAGTGC 03090 Molar Excess Competitor

This expression pattern is likely to be critical for effective immune The main exception seems to be a single region conserved in responses, but its molecular underpinnings remain only partially chicken that corresponds to one of several murine DNase I HSs understood. To investigate the molecular basis for coordinated within the Th2 LCR (RHS5). It is tempting to speculate that this regulation of Th2 cytokine genes, we combined comparative and region might participate in transcriptional regulation of the functional analyses, and we focused on transcription factor bind- chicken orthologs of mammalian type-2 cytokines. ing sites that are highly conserved across placental mammals and Despite remarkable conservation of the majority of Th2 regu- common to more than one Th2 regulatory region. Our comparison latory regions, a few elements were not identifiable in the opossum of the Th2 cytokine gene locus in evolutionarily distant vertebrate locus. These included a GATA-3–responsive element 59 of IL13 species (human to chicken) revealed that although gene content, and the Th1-specific IL4 silencer, HSIV. The emergence of a Th1- order, and orientation have been preserved for .300 million years, selective IL4 silencer suggests that a fine-tuning of the Th1/Th2 nearly all of the distal cis-regulatory elements known to underlie dichotomy may have occurred during mammalian evolution. Re- Th2 coregulation arose after the split between birds and mammals. sistance to certain mammalian pathogens (e.g., Leishmania spp.) requires extremely polarized Th cell responses (44, 45). Thus, regulatory mechanisms that place increasingly tighter restrictions 45 40 on alternative cytokine responses might be adaptive to the host. 35 Perhaps most importantly, our identification of highly conserved 30 Ets-1 binding sites in multiple cis-regulatory regions in the Th2 25 locus, and the finding that the expression of Il5, Il13, and Il4 20 15 transcripts and protein were substantially and selectively impaired Specific Enrichment Specific 10 in Ets-1–deficient murine Th2 cells, for the first time define Ets-1 5 as a global regulator of coordinated Th2 cytokine gene expression. 0 IL5P IL13P IL4IE CNS2 Previously, GATA-3, the Th2 master regulator, was the only RHS5 IL4P HSIV CD14P transcription factor known to have a broad influence on Th2 locus FIGURE 5. Ets-1 binds several regulatory regions in the endogenous regulation, directly controlling chromatin structure and transcrip- murine Th2 locus. Spleens from C57BL/6 mice were harvested, and naive tional activity (46). The phenotype of Ets-1 deficiency (global + CD4 T lymphocytes were isolated and cultured under Th2-skewing reduction of Th2 cytokine expression) is less severe than the conditions for 7 d. Chromatin was immunoprecipitated with an anti–Ets-1 phenotype of GATA-3 deficiency (complete inhibition of Th2 cell Ab or normal rabbit IgG as a negative control. The precipitated DNA was development) (47) but is similar in scope. Of note, GATA-3 and amplified by quantitative real-time PCR using primers specific for the Th2 ECRs or the Cd14 promoter as a comparison. All PCR reactions were Ets-1 were shown to regulate the Il5 promoter cooperatively by carried out in duplicate. Results are expressed as specific enrichment for binding to adjacent sites (48–50). This finding raises the possi- Ets-1 relative to the IgG control. Shown are mean 6 SE values for two bility that the Th2 cytokine defect detected in Ets-1 knockout mice independent experiments, one of which was conducted on chromatin iso- results from an impairment of the functional synergism between lated from cells pooled from four mice. GATA-3 and Ets-1. However, although GATA and ETS sites Downloaded by guest on October 1, 2021 The Journal of Immunology 1315

Il4 Il13 binding at HSIV may play a role in the abnormal regulation of Il4 A 0.05 0.4 +/- expression detected in the absence of Ets-1. Overall, our study 0.04 -/- 0.3 highlighted specific functional elements that Ets proteins may 0.03 0.2 target to orchestrate Th2 cytokine expression. Interestingly, our 0.02 analysis also revealed a remarkable heterogeneity in the sequence 0.01 0.1 0.00 0.0 of Ets-1–binding motifs, particularly their flanking regions. This 0 0.03 0.3 3 0 0.03 0.3 3 heterogeneity might result in a functional hierarchy among Ets-1–

0.15 Il5 0.2 Il10 binding sites, particularly under conditions of limited Ets avail- ability, as well as in differential interaction between Ets-1 and its

Relative Expression Relative 0.10 protein partners. 0.1 Synergism between homotypic factors that occupy distant DNA 0.05 elements is a regulatory arrangement that has been appreciated since

0.00 0.0 early studies conducted with phage l (52). The highly conserved, 0 0.03 0.3 3 0 0.03 0.3 3 common motifs we identified across the Th2 locus may provide µ anti-CD3 [ g/ml] a molecular basis for functional cooperation between distant Th2

Gata3 regulatory regions. Indeed, data describing the three-dimensional B 0.06 +/- architecture of the murine Th2 locus highlight the potential for di- -/- + 0.04 rect communication among distant Th2 cis-elements. In naive CD4 T cells, the Il5, Il13, and Il4 promoters are physically clustered with 0.02 one another and with distant Th2 cis-regulatory regions (21). Analogous to a single regulatory element containing tandem arrays Relative Expression Relative 0.00 0 0.03 0.3 3 of identical binding sites, three-dimensional clustering of homo- anti-CD3 [µg/ml] typic sites may serve as a strategy for the Th2 cytokine genes to effectively vie for limited amounts of trans-acting factors and, in 100 IL-4 300 IL-13 C turn, foster expression of the locus as a whole. 80 +/- 250 -/- 200 Very little is known about how long-range chromatin contacts are 60 150 initiated and/or maintained, yet the earliest evidence supporting 40 100 a DNA looping model pointed toward the homo-oligomerization of 20 50 transcriptional activators and/or repressors bound to distant target 0 0 0 0.03 0.3 3 0 0.03 0.3 3 sites (52–54). These early studies were conducted on artificial ng/ml 100 IL-5 100 IL-10 DNA templates, but more recent experiments in a native chro- 80 80 mosomal context also link sequence-specific DNA-binding pro- 60 60 teins to chromosomal looping and implicate distant, matching binding sites in this process. For example, the erythroid-specific 40 40 Kru¨ppel-like factor directly contributes to the formation of an 20 20 active chromatin hub in the b-globin locus by interacting with 0 0 0 0.03 0.3 3 0 0.03 0.3 3 multiple cis-regulatory elements distributed across the locus (55). anti-CD3 [µg/ml] Similarly, androgen -mediated regulation of the prostate- FIGURE 6. Ets-1 is required for optimal expression of all three Th2 cy- specific Ag gene proceeds via independent recruitment of andro- tokine genes. Naive CD4+ T cells were isolated from Ets-1+/2 or Ets-12/2 gen receptors to a distant upstream enhancer and the proximal mice and cultured under Th2-skewing conditions in vitro. After 7 d, cells promoter, which subsequently form a stable chromosomal loop were stimulated with the indicated concentrations of plate-bound anti-CD3 (56). The distribution of the highly conserved GATA motifs across mAb, in the presence of a constant amount of anti-CD28 mAb (2 mg/ml), for the Th2 locus seems to reflect the role that GATA-3 plays in 24 h. A and B, mRNA levels were assessed by quantitative real-time RT-PCR higher-order chromatin architecture, because ectopic expression of using SYBR green dye detection. All PCR reactions were carried out in GATA-3 in fibroblasts induces chromatin contacts between the duplicate. mRNA levels are expressed relative to Gapdh mRNA abundance. Th2 LCR and the Il4 and Il13 promoters (21). In contrast, these C, Th2 cell supernatants were collected and cytokine secretion was measured associations were relatively weak, suggesting that additional T by ELISA. All samples were measured in duplicate. The figure shows one representative experiment of three. cell lineage-restricted factors may be required to reinforce and/or maintain these long-range interactions (21). Our finding that functional binding sites for Ets-1 are distributed across the Th2 coexist in all but two of the Ets-1–binding ECRs, these sites are in locus raises the intriguing possibility that the contribution of Ets-1 close proximity only in the Il5 promoter and in CNS2, the en- to the coordinated regulation of Th2 cytokine genes may extend to hancer located 39 of Il4. This arrangement suggests that altered the higher-order chromatin architecture of the locus. cooperativity between Ets-1 and GATA-3 may contribute to, but not be the only mechanism for, the locus-wide defect in Th2 cy- Disclosures 2/2 tokine expression observed in Ets-1 mice. The authors have no financial conflicts of interest. The consequences of Ets-1 deficiency are not limited to Th2 cells. 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