Upstream Stimulating Factors Regulate the Expression of ROR γT in Human Lymphocytes

This information is current as Marcin Ratajewski, Aurelia Walczak-Drzewiecka, Anna of October 1, 2021. Salkowska and Jaroslaw Dastych J Immunol published online 13 August 2012 http://www.jimmunol.org/content/early/2012/08/12/jimmun ol.1200519 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 13, 2012, doi:10.4049/jimmunol.1200519 The Journal of Immunology

Upstream Stimulating Factors Regulate the Expression of RORgT in Human Lymphocytes

Marcin Ratajewski,* Aurelia Walczak-Drzewiecka,† Anna Sałkowska,† and Jarosław Dastych†

Retinoic acid-related orphan gT(RORgT) is the orphan that regulates the development of Th17 cells and the expression of IL-17. The differentiation of Th17 cells is associated with the upregulation of RORgT mRNA, and the mechanisms regulating that process in human cells are not well understood. We investigated the transcriptional regulation of RORgT in a human lymphocytic cell line and Th17 differentiated from naive CD4+ cells from human peripheral blood. A series of experiments, including 59 deletion and in situ mutagenesis analysis of the human RORgT promoter, chromatin immunoprecip- itation, and overexpression of selected transcription factors, revealed that the transcription factors upstream stimulatory factor 1 (USF-1) and USF-2 are indispensable for the transcription of RORgT in human lymphocytes. There was also upregulation of USF- Downloaded from 1 and USF-2 during the differentiation of Th17 cells from naive CD4+ cells. In this article, we report the first analysis, to our knowledge, of the human RORgT promoter and demonstrate the role of the USF-1 and USF-2 transcription factors in regulating the expression of RORgT in human lymphocytes. Thus, USFs are important for the molecular mechanisms of Th17 differen- tiation, and possible changes in the expression of USFs might be of interest for inflammatory conditions with a Th17 compo- nent. Furthermore, these observations suggest a possible link between metabolic disorders in which the role of glucose-induced USF expression has already been established and autoimmune diseases in which the upregulation of RORgT is frequently http://www.jimmunol.org/ detected. The Journal of Immunology, 2012, 189: 000–000.

he retinoic acid-related orphan receptors g (RORg) and gT IL-17RA, which is ubiquitously expressed in multiple cell types (RORgT) are products of the RORC transcribed (14). IL-17RA engagement triggers a signaling cascade leading to T from different transcription start sites under the control of the activation of the transcription factors NF-kB and AP1, which independent promoters (1–4). These two nuclear hormone receptors are major regulators that mediate changes in the expression of the differ in their amino acid sequences by 13 aa residues in the N- responsible for initiating and maintaining an inflammatory terminal region. The basic RORC variant (RORg) is expressed

state (14). The genes upregulated by IL-17 include cytokines, such by guest on October 1, 2021 ubiquitously and plays an important function in the regulation of as IL-6, and chemokines, such as IL-8, Rantes, and GRO (15–17). development and metabolism (5). Recently, the 7-oxygenated IL-17 expression is a major phenotypic marker of Th17 cells that sterol was identified as an endogenous ligand modulating the are capable of initiating an immune response that is distinct from function of this (6). The expression of RORgT those driven by Th1 and Th2 (18, 19). The Th17-driven immune (also termed RORC2) has a very restricted pattern, and its product response provides effective protection against certain extracellular is predominantly found in immune organs, such as thymus, and in pathogens (20, 21) and is also responsible for the development of particular lineages of immune cells, such as immature double- inflammatory processes associated with several human diseases, positive thymocytes and Th17 lymphocytes (2, 7), where this including autoimmune and allergic conditions (22, 23), athero- transcription factor plays important functions in the maturation of sclerosis (24), liver dysfunctions (25), and tumors (26). Differ- a/b T cells and the differentiation of Th17 cells, respectively (2, entiation of naive CD4+ lymphocytes into Th17 cells is supported 8). Th17 cells are a subset of Th cells capable of expressing the by the coordinated action of several cytokines, including IL-6, proinflammatory cytokine IL-17 (9–11). This cytokine, which is IL-23, and TGF-b (23). RORgT mRNA levels increase upon also expressed by several other immune cells such as gdT cells Th17 expansion in vitro, and increased RORgT mRNA levels in (12) and mast cells (13), exercises its function by triggering the tissues positively correlate with Th17 activity observed in differ- ent inflammatory diseases (27–29). Mechanisms regulating the *Laboratory of Transcriptional Regulation, Institute of Medical Biology, Polish † g Academy of Sciences, 93-232 Lodz, Poland; and Laboratory of Cellular Immunol- tissue-specific expression of ROR T are not well understood. ogy, Institute of Medical Biology, Polish Academy of Sciences, 93-232 Lodz, Poland Studies with knockout mice revealed the involvement of the Received for publication February 9, 2012. Accepted for publication July 11, 2012. transcription factors STAT3, IFN regulatory factor 4, and v- This work was supported by National Science Centre Grant N N301 507638. reticuloendotheliosis viral oncogene homolog (c-Rel) in the up- Address correspondence and reprint requests to Prof. Jaroslaw Dastych, Laboratory regulation of RORgT (4, 30–32). The mechanism by which of Cellular Immunology, Institute of Medical Biology, Polish Academy of Sciences, RORgT transcripts are transcriptionally regulated in human Lodowa 106, 93-232 Lodz, Poland. E-mail address: [email protected] cells is largely unknown. Our investigation of the transcrip- Abbreviations used in this article: GDAP1, ganglioside-induced differentiation- tional regulation of RORgT in human lymphocytes revealed associated ; HMBS, hydroxymethylbilane synthase; HPRT1, hypoxanthine phosphoribosyltransferase 1; ROR, retinoic acid-related orphan receptor; RORg, that the transcription factors upstream stimulatory factor 1 (USF-1) ROR g; RORC,RORC;RORgT, RORgT; RPLI3A, ribosomal protein LI3a; and USF-2 are indispensable for the transcription of RORgT,and siRNA, small interfering RNA; USF, upstream stimulatory factor. that the upregulation of USFs occurs in differentiating Th17 cells Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 in parallel with the upregulation of RORgT and IL-17.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200519 2 USFs REGULATE RORgT EXPRESSION

Materials and Methods EZ-ChIP kit (Millipore) according to the manufacturer’s protocol. PCR Cell culture amplification was performed using 2 ml DNA with primers specific to the RORgT promoter (forward: 59-AGGCTGCACCACACTGG-39, position Jurkat (human T cell lymphoblast-like), HeLa (cervix carcinoma), and 2599; reverse: 59-TTCTACTCCTCCTACCCCCG-39, position: 2429) for HepG2 (hepatocellular carcinoma) cell lines were obtained from American 36 cycles, and the ganglioside-induced differentiation-associated protein Type Culture Collection (Manassas, VA). They were maintained under (GDAP1) promoter (36) was used as a negative control for 32 cycles. The standard conditions in RPMI 1640 or DMEM containing 10% FBS at 37˚C amplification of soluble chromatin before immunoprecipitation was used in a 5% CO2 atmosphere. as an input control. PCR products were resolved on a 1.5% agarose gel and stained with ethidium bromide. Real-time RT-PCR EMSAs RNA was isolated from cells using TRI Reagent (Molecular Research Center, Cincinnati, OH) and reverse-transcribed with the Maxima First Nuclear extracts were prepared as described elsewhere (37) and subse- Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific/Fermentas, quently used in EMSA. Infrared dye-labeled annealed oligonucleotides Vilnius, Lithuania). Real-time RT-PCR amplification was carried out on were incubated with 1 mg nuclear extract in binding buffer containing 10 a LightCycler 480 (Roche, Basel, Switzerland) using SYBRGreen I Master fmol infrared dye-labeled probes, 5 mM Tris-HCl (pH 8.0), 50 mM NaCl, Mix (Roche) as follows: 5 min at 95˚C and then 45 cycles each at 95˚C for 5 mM DTT, 5 mM MgCl2, 0.5% Igepal, 25% glycerol, 100 mM ZnCl2, and 10 s, 60˚C for 10 s, and 72˚C for 10 s. Sequence of GATA-3 primers were 0.2 mg salmon testis DNA in a 20-ml reaction volume. For the competition published by Hoene et al. (33). Other intron-spanning primers used to assay, 10- and 50-fold molar excesses of unlabeled oligonucleotides (wt, m4, detect cDNA sequences were designed using Primer3 software: RORgT m3, m2, or m1) were added to the reaction mixture. The reactions were forward, 59-CTGCTGAGAAGGACAGGGAG-39;RORg forward, 59-CA- incubated on ice for 60 min before probe addition, followed by an addi- CAGAGACAGCACCGAGC-39;RORgT/RORg reverse (same for both tional 30-min incubation. The binding reactions were resolved by electro- isoforms), 59-AGTTCTGCTGACGGGTGC-39; USF-1, 59-AATTGCCC- phoresis on a 5% polyacrylamide gels in 0.5% Tris-acetate EDTA buffer.

CTAGGACTCACC-39 (forward) and 59-ACAAGCGGTGGTTACTCTGC- TheproductswerethenimagedonanOdyssey(LiCor,Lincoln,NE) Downloaded from 39 (reverse); USF-2, 59-TGATCCAAAATCCCTTCAGC-39 (forward) and infrared fluorescence scanner. Anti–USF-1 (H-86; Santa Cruz) and anti– 59-TCTTCTCCTCTCATCTCGGG-39 (reverse); and IL-17A, 59-AAAC- USF-2 (H-100; Santa Cruz) Abs were used for supershift experiments. AACGATGACTCCTGGG-39 (forward) and 59-CTTGTCCTCAGAATT- + TGGGC-39 (reverse). D cycle threshold values were transformed into relative Naive CD4 T cell isolation and differentiation copy number values (the number of copies of cognate mRNA per house- keeping gene index, calculated as the averaged cycle threshold of the The protocol used in this study was described in detail by Wilson and colleagues (23): PBMCs were isolated from buffy coats obtained from housekeeping genes hypoxanthine phosphoribosyltransferase 1 [HPRT1], + healthy donors and centrifuged through Ficoll, and the naive CD4 fraction http://www.jimmunol.org/ hydroxymethylbilane synthase [HMBS], and ribosomal protein LI3a + [RPLI3A]) as described in our previous study (34). was then isolated using the CD4 T Cell Isolation Kit II (Miltenyi Biotec GmbH Bergisch, Gladbach, Germany). Naive CD4+ T cells were main- RORgT promoter constructs and transfection tained in Yssel’s media containing human AB serum for 5 d in Th17 polarizing conditions with beads coated with anti-CD2, anti-CD3, and anti- Fragments of RORgT spanning its putative promoter region were prepared CD28 (T Cell Activation/Expansion Kit; Miltenyi Biotec), and with a cy- by PCR amplification of human genomic DNA and cloned into a pUC18 tokine mixture containing human IL-1b (50 ng/ml), human IL-6 (30 ng/ vector using HincII, a blunt restriction endonuclease. The following pri- ml), human IL-23 (10 ng/ml), and human TGF-b (10 ng/ml). The cyto- mers were used for amplification: reverse primer, 59-CTGTTAAGCTTA- kines were purchased from R&D Systems (Minneapolis, MN). CATCCTCCTTTCCAGAGGG-39 (position +78; all nucleotide locations are reported relative to the A in the ATG translation initiation codon) and Analysis of IL-17 production by guest on October 1, 2021 forward primers, 59-AGGATTGGTACCTCCACATAAAACATCGAGAA- + + CC-39 (position 2869), 59-CAGGCGGTACCACACTGGGTATGGCTGT- For the analysis of IL-17 production, CD4 T cells and CD4 cells stim- TGG-39 (position 2589), 59-ACCGCGGGTACCTGTCGCATGTGGTTT- ulated with the T Cell Activation kit, in the presence or absence of the TGG-39 (position 2341), and 59-GCCACCGGTACCGTGCAGAGCTT- cytokine mixture, were cultured for 5 d. The cells and supernatants were AAACCCCC-39 (position 2180). The sequences of the cloned inserts were collected for RNA isolation and ELISA assays, respectively. ELISA was verified by automated sequencing and then recloned into pGL3-Basic vector conducted using the Quantikine Human IL-17 Immunoassay kit (R&D (Promega, Madison, WI) using Acc65I/HindIII restriction endonucleases. Systems) according to the manufacturer’s protocol. Measurements were Exgene500 from Fermentas was used for the transfection of HeLa and performed at 450 nm using a Sunrise microplate reader (Tecan). HepG2 cells. Jurkat cells were transfected with Fugene HD (Roche). Lu- ciferase measurements were performed on an Infinite 200 PRO (Tecan, Small interfering RNA and nucleofection Ma¨nnedorf, Switzerland) and standardized as described previously (34). Duplexes of Stealth small interfering RNA (siRNA) against the USF-1 Expression vectors coding various transcription factors were purchased (USF1HSS144431) and USF-2 (USF2HSS144434) transcripts were pur- from OriGene Technologies (Rockville, MD) with the exception of USF-1 chased from Invitrogen (Carlsbad, USA). Negative control siRNA-A with (Open Biosystems, Lafayette, CO). a scrambled sequence was purchased from Santa Cruz. Jurkat cells were Site-directed mutagenesis nucleofected with 100 pmol of each target siRNA duplex using Amaxa SE Cell Line for 4D-Nucleofector X Kit L (Lonza, Basel, Switzerland) Mutagenesis was performed directly on plasmids (pUC18) containing according to the manufacturer’s protocol. After nucleofection, the cells sequences 2589/+78 and 2341/+78 of RORgT promoter using the PCR- were cultured for 96 h and then collected for RNA extraction. Th17 cells + based method followed by removal of the template by DpnI digestion. differentiated in vitro from naive CD4 were nucleofected with the same Introduced mutation of the E-BOX sequence was based on a previous work siRNA duplexes as described earlier using Amaxa P3 Primary Cell 4D- of Ciccone et al. (35). For mutagenesis, the following primer pairs were Nucleofector X Kit (Lonza) according to the manufacturer’s instruction. used: 59-TCCACAGGGTGTACGGTAGCACATGCCACCACC-39 (m4f) After nucleofection, the Th17 cells were cultured in the Yssel’s medium and 59-GGTGGTGGCATGTGCTACCGTACACCCTGTGGA-39 (m4r); 59- without cytokines for 16 h. This was followed by addition of cytokine GTCCCTGAAGTACCCTAGCAAAGAGGGTCAGGC-39 (m3f) and 59- mixture containing IL-1b (50 ng/ml), IL-6 (30 ng/ml), IL-23 (10 ng/ml), GCCTGACCCTCTTTGCTAGGGTACTTCAGGGAC-39 (m3r); 59-CCC- and TGF-b (10 ng/ml), and beads coated with anti-CD2, anti-CD3, and AGATTGGCACCGTAGGGCGCCTGTCATCCT-39 (m2f) and 59-AGGA- anti-CD28 and culture for an additional 5 d. TGACAGGCGCCCTACGGTGCCAATCTGGG-39 (m2r); and 59-GGTG- GGGGGTTGTACCTTAGCCACCTGTGTGGT-39 (m1f) and 59-ACCAC- Computational analysis and statistics 9 ACAGGTGGCTAAGGTACAACCCCCCACC-3 (m1r). The mutated Putative transcription factor elements within the RORgT gene promoter sequences were verified by sequencing and then recloned into pGL3-Basic were identified using MatInspector software (38). Alignment of the RORgT vector. promoters from various mammalian species was performed using the Chromatin immunoprecipitation assay CLUSTALW program (39). Genomic sequences were downloaded from the University of California Santa Cruz genome browser (40). Statistical Chromatin immunoprecipitation with normal mouse IgG (Millipore, Billerica, analysis was performed using paired t test or one-way ANOVA, followed MA), anti–USF-1 (H-86; Santa Cruz, Santa Cruz, CA), anti–USF-2 (H-100, by Dunn’s post hoc test. A p value #0.05 was considered statistically Santa Cruz), and anti–c- (9E11; Santa Cruz) was performed using the significant. The Journal of Immunology 3

FIGURE 1. (A) Jurkat, HeLa, and HepG2 cells were cultured under standard conditions and harvested by centrifugation. Cell pellets were lysed and RNA was analyzed for expression of RORg and RORgT using real-time RT-PCR. The relative level of gene expression is shown as the copy number of cognate mRNAs per housekeeping gene index calculated as described in Materials and Methods. Data represent mean 6 SEM from three independent experiments (n = 3). (B) Jurkat, HeLa, and HepG2 cells were transfected with indicated 59-deletion constructs of the human RORgT gene promoter constructs as described in Materials and Methods. Promoter activity of each construct was determined by luminometric assay of luciferase activity. Results are standardized against Downloaded from control reporter activity (pCMVSEAP, which contains a secreted alkaline phosphatase coding sequence un- der the control of a CMV promoter) and expressed as the fold induction over the activity of a promoterless pGL3-basic vector. Data represent mean 6 SEM (n = 5). The nucleotide positions are relative to the ATG translation initiation codon. http://www.jimmunol.org/

Results stream sequences of the human RORgT gene revealed four bind- by guest on October 1, 2021 Tissue-specific expression of RORC gene variants ing sites for USF-1 and USF-2 (E-BOX 1–4) that are preserved among mammalian species (Fig. 3). The expression of two known variants of the RORC gene was first analyzed in selected cell lines. HepG2 hepatocytes and cervix cell Effect of USF-1 and USF-2 overexpression on activity of carcinoma HeLa cells almost exclusively expressed RORg,whereas human RORgT promoter Jurkat lymphocytes predominantly expressed RORgT (Fig. 1A). Next, the effect of USF-1 and USF-2 overexpression on the activities of different human RORgT promoter constructs was assessed. Analysis of the human RORgT promoter activity Next, a series of 59-deletion mutants of the human RORgT promoter cloned into a luciferase reporter construct was used to analyze promoter activity in Jurkat lymphocytes and identify the responsible sequences. The transfection of Jurkat lymphocytes with RORgT promoter constructs resulted in significant promoter activity, with the highest activity observed for constructs containing the sequen- ces spanning from 2340 to +78 (Fig. 1B). The shortest analyzed sequence (2180/+78) and constructs representing sequences fur- ther 59 upstream of the RORgT translation start site also exhibited significant activity compared with the promoterless pGL3-Basic vector. The promoter activities in nonlymphatic cells (HepG2 hepatocytes and HeLa cells) were several fold lower than that observed in Jurkat lymphocytes. The longest promoter construct (2869/+78) had the highest activity. FIGURE 2. Identification of USF-1 and USF-2 as potent acti- vators of the human RORgT gene promoter. HeLa cells were cotransfected Screening for transcription factors interacting with human with expression vector encoding indicated transcription factors or with RORgT promoter empty CMV-XL5 plasmid (control) and with the phRORgT(2869/+78) Luc construct as described in Materials and Methods. Cells were next Several transcription factors were then overexpressed in HeLa cells cultured for 48 h under standard conditions, lysed, and analyzed for lu- cotransfected with the longest reporter construct. Overexpression ciferase activity using luminometric assay. Results are normalized as the of USF-1 and USF-2, but not the other tested transcription factors, fold induction over activity observed in cells transfected with pGL3-basic resulted in a significant (2.1- and 2.9-fold, respectively) increase in and shown as mean 6 SEM (n = 4). Asterisk indicates statistically sig- human RORgT promoter activity (Fig. 2). Analysis of the 59-up- nificant difference at p , 0.01. 4 USFs REGULATE RORgT EXPRESSION

FIGURE 3. Sequence conservation of the E-BOXes and their extended flanking regions identified in the RORgT gene promoters of var- ious mammalian species. Genomic sequences of the upstream regions of RORC in indicated spe- cies were aligned using CLUSTALW program. Squares represent putative E-BOX sequences identified with MatInspector software. The dif- ferences in the aligned sequences are underlined. The nucleotide positions are relative to the ATG translation initiation codon.

Overexpression of USF-1 and USF-2 did not change the activity of phRORgT(2341/+78)Luc. To gain further insights into the role of the shortest promoter construct (2180/+78), which does not USF in the regulation of RORgT, we used additional promoter contain a USF binding motif (Fig. 4A). In contrast, the activity of constructs (2589/+78) with mutations in E-BOX 3 alone, E-BOX the promoter construct containing sequences 2340 to +78, which 4 alone, or both E-BOX 3 and E-BOX 4 in a series of transfection overlap the E-BOX 1 and 2 sites, was significantly upregulated. experiments in HeLa cells. Mutation of E-BOX 4, the most distal The activities of the two longer promoter constructs (2869/+78 site, enhanced the responses elicited in HeLa cells by overexpressed and 2589/+78), which overlap all four USFs binding sites (E- USF-2 but did not change the response caused by overexpressed Downloaded from BOX 1–4), were upregulated slightly higher than the activity of USF-1 (Fig. 4B). In contrast, mutation of E-BOX 3 resulted in http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. Results of reporter gene assay to identify promoter sites mediating USF-1 and USF-2 induction of the human RORgT. (A–C) HeLa cells were cotransfected with empty CMV-XL5 plasmid or expression vector encoding USF-1 or USF-2 as indicated and with indicated RORgT reporter construct as described in Materials and Methods. Cells were next cultured for 48 h under standard conditions, lysed, and analyzed for luciferase activity using luminometric assay. The results are normalized as the fold induction over activity observed in cells transfected with pGL3-basic and are presented asmean6 SEM (n = 5). Asterisk indicates (A, B) statistically significant difference at p , 0.01 as compared with empty CMV-XL5 plasmid or (C)ascompared with the wild-type phRORgT(2341/+78)Luc; (C) caret (^) indicates statistically significant difference at p , 0.01 as compared with the mutated phRORgTm1(2341/+78)Luc; (C) dollar sign indicates statistically significant difference at p , 0.01 as compared with the mutated phRORgTm2(2341/+78) Luc. (D) Jurkat cells were transfected with indicated RORgT reporter construct as described in Materials and Methods. Cells were next cultured for 48 h under standard conditions, lysed, and analyzed for luciferase activity using luminometric assay. Results are normalized as the fold induction over activity observed in cells transfected with pGL3-basic and are presented as mean 6 SEM (n = 5). Asterisk indicates statistically significant difference at p , 0.01 as compared with the wild-type phRORgT(2589/+78)Luc; caret (^) indicates statistically significant difference at p , 0.01 as compared with the wild-type phRORgT(2341/+78)Luc. The Journal of Immunology 5 a small but significant decrease in response to USF-1 over- than that observed in the presence of either alone. The promoter expression and did not alter the level of upregulation caused by activities of mutated and wild type phRORgT(2341/+78)Luc and USF-2 overexpression. The construct containing both mutations phRORgT(2589/+78)Luc promoter constructs were compared in enhanced the response to USF-2 overexpression and caused a Jurkat lymphocytes. In the longer construct (2589/+78), mutation small decrease in response to USF-1 overexpression. When of E-BOX 3 alone or E-BOX 3 and E-BOX 4 (but not E-BOX a similar analysis was performed using the construct phRORgT 4 alone) resulted in a small but detectable decrease in pro- (2341/+78)Luc containing E-BOX 1 and E-BOX 2 and additional moter activity in the Jurkat lymphocytes (Fig. 4D). Mutations constructs with mutations in E-BOX 1 alone, E-BOX 2 alone, or in phRORgT(2341/+78)Luc, which depleted this construct of the both, the data showed a different pattern of effects on RORgT more distally located E-BOX 2 USF binding site, strongly (.2- promoter activity following the overexpression of USF-1 and fold) decreased promoter activity. Mutation of E-BOX 1 resulted USF-2 (Fig. 4C). Thus, mutation of each of these two binding sites in a significant but small (10%) decrease in promoter activity, and resulted in a significant decrease in promoter activity following double mutation of E-BOX 1 and E-BOX 2 resulted in promoter the overexpression of USF-1 and USF-2. Mutation of both E- activity comparable with those observed with the single E-BOX 2- BOXes resulted in promoter activity that was significantly lower deficient mutant. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. Binding of USF-1 and USF-2 to the human RORgT gene promoter. (A–D) Nuclear extracts were prepared from resting Jurkat cells as described in Materials and Methods. EMSAs were performed using infrared (IR) fluorescently labeled oligonucleotide probes and unlabeled “cold” oli- gonucleotides of indicated sequences. For the competition assay, the “cold” competitor oligonucleotides with wild-type (wt) or mutated (m4, m3, m1,or m2) sequences were added at indicated molar fold excess over the amount of the labeled probe. For supershift assay, anti–USF-1 and anti–USF-2 Abs were added to the reaction mixture as indicated. (E) Results of chromatin immunoprecipitation performed on living Jurkat cells. Chromatin was isolated from resting Jurkat cells fragmented and immunoprecipitated with normal mouse IgG, anti–USF-1, anti–USF-2, and anti–c-Myc Abs. Immunoprecipitated DNA sequences were amplified by PCR with primers specific to the RORgT promoter and GDAP1 promoter (negative control), and visualized on agarose gel stained with ethidium bromide. NS, Nonspecific; S, shift; SS, supershift. 6 USFs REGULATE RORgT EXPRESSION

Analysis of interaction of USFs with human RORgT promoter than the USF-1 siRNA, with the combination of both siRNAs sequences having the strongest inhibitory effect (65% decrease). To confirm that USFs regulate RORgT promoter activity in Effect of inhibition of USFs on RORgT expression in human Jurkat lymphocytes, EMSA assays were performed using Jurkat Th17 cells lymphocyte nuclear extracts and nucleotide probes matching the To gain evidence of USF involvement in the regulation of RORgT sequences of E-BOX 1–4. Incubation of the Jurkat-derived nuclear expression in Th17, the standard experimental protocol for in vitro extract with all four nucleotide probes resulted in the detection of differentiation of Th17 cells from peripheral blood naive CD4+ specific bands that were competed out with an excess of cold lymphocytes was used. The culture of naive CD4+ lymphocytes in nucleotide of matching but not with mutated sequences (Fig. 5). the presence of a standard cytokine mixture combined with acti- The addition of Abs against USF-1 and USF-2 resulted in the vation by cross-linking of CD2, CD3, and CD28 resulted in the appearance of additional bands representing supershifts. For the accumulation of a significant amount of IL-17 in the supernatant E-BOX 4 nucleotide (Fig. 5A), the weak but detectable super- (2.1 6 0.9 pg/ml for CD4+ and 403.1 6 103.8 pg/ml for Th17, shift band appeared following the addition of anti–USF-2, but statistically significant difference at p , 0.05) in parallel with an not anti–USF-1 Ab. For the E-BOX 3 nucleotide (Fig. 5B), in increase in IL-17 mRNA in the cell pellet (Fig. 7A). As expected, contrast, a supershift band appeared after the addition of anti– the differentiation of Th17 from CD4+ was also associated with USF-1, but not anti–USF-2 Ab. Supershift experiments with a significant upregulation of RORgT mRNA (Fig. 7B), but not nucleotide probes matching the sequences of E-BOX 2 and E- RORg mRNA (Fig. 7C) and GATA-3 (Fig. 7D) in these cells. BOX 1 (Fig. 5C, 5D) generated similar extra bands with both Interestingly, this in vitro differentiation process was associated anti–USF-1 and anti–USF-2 Ab. Thus, the USF-1 and USF-2 Downloaded from with significant upregulation of USF-1 and USF-2 mRNA (Fig. 7E, proteins in the nuclear extract of Jurkat lymphocytes interacted 7F). To further confirm the role of USFs in RORgT expression, with all four tested USF-binding motifs in vitro. The interaction Th17 cells obtained in vitro from CD4+ lymphocytes were nucleo- of USF-1 and USF-2 with the RORgT promoter was then in- fected with siRNAs targeting USF-1 and USF-2, and cultured for vestigated in intact Jurkat lymphocytes. Chromatin immuno- additional 5 d under optimal conditions. As shown in Table I, this precipitation experiments revealed the binding of both USF-1 treatment inhibited USF-1 and USF-2 expression and resulted in a and USF-2 but not v-myc myelocytomatosis viral oncogene significant decrease in the level of RORgT mRNA. The expression http://www.jimmunol.org/ homolog (c-Myc; used as negative control) proteins to the RORgT of RORg, GATA-3, and IL-17 were not significantly inhibited. promoter (Fig. 5E). In addition, USF-1 and USF-2 did not bind to the control GDAP1 promoter, which does not contain USF binding Discussion motifs. The transcriptional regulation of RORgT, one of the two RORC Effect of inhibition of USFs on RORgT expression in Jurkat variants, is an important element of the regulation of Th17 dif- cells ferentiation. Most data regarding the tissue-specific expression To confirm the functionality of USFs binding to the RORgT pro- of RORgT and its transcriptional regulation come from murine moter, we used siRNAs targeting USF-1 and USF-2. The trans- studies. Similar to those of the mouse, the two variants of the by guest on October 1, 2021 fection of Jurkat cells with siRNAs targeting USF-1 and USF-2 human RORC gene originate from different transcription start sites resulted in a significant and targeted gene-specific decrease in the and are regulated by different promoters. There was a cell line- respective mRNA levels (Fig. 6A, 6B). Jurkat cells treated with (Fig. 1A) and cell lineage-specific (data not shown) pattern of siRNAs targeting USF-1 alone, USF-2 alone, and both transcripts expression of RORg and RORgT. Also like the mouse, human showed RORg mRNA (data not shown) and GATA-3 mRNA (Fig. RORgT is preferentially expressed in selected lymphocytic line- 6C) levels similar to those seen in control cells. In contrast, Jurkat ages but is hardly detectable in nonlymphatic cells (Fig. 1A, Fig. cells treated with siRNAs targeting USF-1 alone, USF-2 alone, or 7B, data not shown). This preferential expression of RORgTin both transcripts showed significantly lower RORgT mRNA levels human Jurkat lymphocytes and peripheral blood-derived CD4+ (Fig. 6D). The USF-2 siRNA inhibited RORgT expression more Th17 cells was associated with a very low expression of RORg

FIGURE 6. Endogenous RORgT is a target gene for USF-1 and USF-2. Jurkat cells were nucleofected with siRNA duplexes targeting USF-1, USF-2, or control siRNA-A with scrambled sequence as in- dicated. After nucleofection, cells were cultured for 96 h and then collected for RNA extraction. Expression of USF-1 (A), USF-2 (B), GATA-3 (C), and RORgT(D) were determined by real-time RT-PCR. Results were normalized to the house- keeping gene index (HPRT1, HMBS,andRPLI3A) and are presented as mean 6 SEM from four in- dependent experiments (n = 4). Asterisk indicates statistically significant difference at p , 0.01 as compared with cells nucleofected with control siRNA-A. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 7. Expression of selected genes upon Th17 differentiation from CD4+ lymphocytes. Naive CD4+ fraction was isolated from PBMCs obtained from healthy donors using immunomagnetic beads and cultured for 5 d under Th17 polarizing conditions as described in Materials and Methods.RNA obtained from naive CD4+ and differentiated Th17 lymphocytes was analyzed for expression of IL-17 (A), RORgT(B), RORg (C), GATA-3 (D), USF-1 (E), and USF-2 (F) by real-time RT-PCR. Results were normalized to the housekeeping gene index (HPRT1, HMBS, and RPLI3A) and are presented as mean 6 SEM from six independent experiments performed using cultures originated from six different donors (n = 6). Asterisk indicates statistically significant difference at p , 0.05. that is similar to the previously reported pattern of RORg/RORgT mediated by sequences further upstream, up to 2340 bp relative to expression in murine thymocytes (2) and different from the pattern the RORgT translation start site (Fig. 1B). To our knowledge, this by guest on October 1, 2021 observed in murine Th17 cells, in which high expression of represents the first analysis of human RORgT promoter activity. RORgT was associated with comparable levels of RORg (4). This Our observations are consistent with the hypothesis that prefer- tissue-specific pattern of expression is consistent with the results ential transcription of the RORgT promoter in T cells participates of promoter activity analysis, which showed that RORgT promoter in the tissue-specific expression of RORgT in human T cells. constructs had significantly higher activity in Jurkat T lymphocytes Previously, a similar upstream sequence (2400 to +151) of murine than in nonlymphatic cells (Fig. 1B). Although significant pro- RORgT showed significant promoter activity in murine EL-4 moter activity in Jurkat lymphocytes was observed with sequences lymphocytes (4). Screening for transcription factors that up- spanning 2180 bp 59 upstream of RORgT, the highest activity was regulate the human RORgT promoter revealed possible roles for USF-1 and USF-2 in the regulation of human RORgT expression Table I. Effect of downregulation of the USF-1 and USF-2 mRNA on (Fig. 2). This regulation could be mediated by four USF binding the expression of selected genes in Th17 cells sites, E-BOX 1–4, in the promoter region that are preserved in several mammalian species (Fig. 3). In situ mutation analysis of Gene siRNA-A siUSF-1 + 2 promoter constructs (Fig. 4) and gel shift experiments (Fig. 5A–D) USF-1 209.3 6 71.6 129.3 6 50.0a confirmed that these binding sites can bind USF-1 and USF-2 and USF-2 150.4 6 44.1 54.8 6 23.9a are necessary for mediating the effects of USF-1 and USF-2 on the RORg 0.6 6 0.1 0.5 6 0.2 activity of the RORgT promoter. Although the proximal binding 6 6 a RORgT 46.3 11.7 34.6 9.3 sites (E-BOX 1 and E-BOX 2) seemed to mediate a strong up- GATA-3 34.8 6 8.4 46.2 6 14.0 IL-17A 271.1 6 79.2 223.7 6 66.4 regulation of promoter activity by both USF-1 and USF-2, the IL-17 secretion (pg/ml) 1488.4 6 1125.2 570.6 6 272.4 distal binding sites (E-BOX 3 and E-BOX 4) seemed to pref- erentially bind USF-1 and USF-2, respectively, and upregulate Naive CD4+ fraction was isolated from PBMCs obtained from healthy donors using immunomagnetic beads and cultured for 5 d under Th17 polarizing conditions (E-BOX 4) or downregulate (E-BOX 3) promoter activity. This as described in Materials and Methods. Differentiated Th17 cells were nucleofected observation is consistent with the pattern of 59-deletion promoter with control siRNA-A with scrambled sequence and mixture of siRNA duplexes targeting USF-1 and USF-2. After nucleofection, Th17 cells were cultured for addi- construct activities in Jurkat cells, in which the two constructs tional 5 d under optimal conditions and collected for RNA extraction. Expression of containing E-BOX 4 had significantly lower activity compared selected genes was determined by real-time RT-PCR and normalized to the house- with the construct containing only E-BOX 1 and E-BOX 2 (Fig. 1B). keeping gene index (HPRT1, HMBS, and RPLI3A). Data are presented as mean 6 SEM from six independent experiments performed using cultures originated from six Additional experimental data obtained using Jurkat lymphocytes different donors (n = 6). Analysis of IL-17 production was determined using the strongly suggest that high RORgT promoter activity and high Quantikine Human IL-17 Immunoassay kit (R&D Systems). aStatistically significant difference at p , 0.05 as compared with cells nucleo- RORgT mRNA expression depend on USF-1 and USF-2 expression fected with control siRNA-A. in those cells. Thus, Jurkat lymphocytes expressed 2- to 3-fold 8 USFs REGULATE RORgT EXPRESSION more USF-1 and USF-2 than nonlymphatic cells (data not shown). regulated by signaling through the pre-T cell receptor and binds to the TEA promoter. Eur. J. Immunol. 29: 4072–4080. USF-1 and USF-2 preferentially occupied RORgT promoter DNA 3. He, Y. W., M. L. Deftos, E. W. Ojala, and M. J. Bevan. 1998. RORgamma t, (Fig. 5E), and suppression of the expression of USF-1 alone, USF- a novel isoform of an orphan receptor, negatively regulates Fas ligand expression 2 alone, or both USFs significantly decreased the level of RORgT and IL-2 production in T cells. Immunity 9: 797–806. 4. Ruan, Q., V. Kameswaran, Y. Zhang, S. Zheng, J. Sun, J. Wang, J. DeVirgiliis, mRNA (Fig. 6). Taken together, these observations suggest that H. C. Liou, A. A. Beg, and Y. H. Chen. 2011. The Th17 immune response is human RORgT is transcriptionally regulated by USF-1 and USF- controlled by the Rel-RORg-RORg T transcriptional axis. J. Exp. Med. 208: 2. To our knowledge, this represents the first report demonstrating 2321–2333. 5. Jetten, A. M., S. Kurebayashi, and E. Ueda. 2001. The ROR nuclear orphan the engagement of USFs in the regulation of RORgT expression. receptor subfamily: critical regulators of multiple biological processes. Prog. Expression of RORgT is a hallmark of Th17 development (8). Nucleic Acid Res. Mol. Biol. 69: 205–247. Increased RORgT mRNA levels are associated with several hu- 6. Wang, Y., N. Kumar, L. A. Solt, T. I. Richardson, L. M. Helvering, C. Crumbley, R. D. Garcia-Ordonez, K. R. Stayrook, X. Zhang, S. Novick, et al. 2010. man pathologies related to the presence of Th17-driven inflam- Modulation of -related orphan receptor alpha and gamma matory processes (22, 41). Our observations of USF-1– and USF- activity by 7-oxygenated sterol ligands. J. Biol. Chem. 285: 5013–5025. g 7. Wei, L., A. Laurence, K. M. Elias, and J. J. O’Shea. 2007. IL-21 is produced by 2–mediated ROR T expression in human lymphocytes raise the Th17 cells and drives IL-17 production in a STAT3-dependent manner. J. Biol. question of what role these transcription factors may play in Th17 Chem. 282: 34605–34610. development. The hypothesis that USFs regulate RORgT expres- 8. Crome, S. Q., A. Y. Wang, C. Y. Kang, and M. K. Levings. 2009. The role of retinoic acid-related orphan receptor variant 2 and IL-17 in the development and sion in human lymphocytes is further substantiated by the increase function of human CD4+ T cells. Eur. J. Immunol. 39: 1480–1493. in the USF-1 and USF-2 mRNA levels observed upon in vitro 9. Cua, D. J., J. Sherlock, Y. Chen, C. A. Murphy, B. Joyce, B. Seymour, L. Lucian, differentiation of Th17 from naive CD4+ lymphocytes that are W. To, S. Kwan, T. Churakova, et al. 2003. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. paralleled by the increases in RORgT and IL-17 mRNAs (Fig. 7), Nature 421: 744–748. Downloaded from and by the observation that suppression of the expression of USFs 10. Langrish, C. L., Y. Chen, W. M. Blumenschein, J. Mattson, B. Basham, significantly decreased the level of RORgT mRNA in Th17 cells J. D. Sedgwick, T. McClanahan, R. A. Kastelein, and D. J. Cua. 2005. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. (Table I). This inhibition of RORgT expression was not associated Exp. Med. 201: 233–240. with significant inhibition of expression of IL-17 that might be 11. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, and B. 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