An IFNG SNP with an estrogen-like response element ... - Nature

Genes and Immunity (2006) 7, 342–351 & 2006 Nature Publishing Group All rights reserved 1466-4879/06 $30.00 www.nature.com/gene

ORIGINAL ARTICLE

An IFNG SNP with an estrogen-like response element selectively enhances promoter expression in peripheral but not lamina propria T cells R Gonsky1, RL Deem1, JH Bream2, HA Young3 and SR Targan1 Inflammatory Bowel Disease Research Center, Cedars-Sinai Medical Center, Los Angeles, CA, USA; 2Disease Prevention and Control Program, Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA and 3 Cellular and Molecular Immunology Section, Laboratory of Experimental Immunology, National Cancer Institute, Frederick, MD, USA 1

This study examines mucosa-specific regulatory pathways involved in modulation of interferon-g (IFN-g) in lamina propria T cells. Previous studies identified mucosa-specific CD2 cis-elements within the 204 to 108 bp IFNG promoter. Within this region, a single-site nucleotide polymorphism, 179G/T, imparts tumor necrosis factor-a stimulation of IFNG in peripheral blood lymphocytes, and is linked with accelerated AIDS progression. We discovered a putative estrogen response element (ERE) introduced by the 179T, which displays selective activation in peripheral blood mononuclear cells (PBMC) vs lamina propria mononuclear cells (LPMC). Transfection of PBMC with constructs containing the 179G or 179T site revealed CD2-mediated enhancement of the 179T compared to 179G allele, although, in LPMC, a similar level of expression was detected. Electrophoretic mobility shift assay (EMSA) analysis demonstrated CD2-mediated nucleoprotein binding to the 179T but not the 179G in PBMC. In LPMC, binding is constitutive to both 179G and 179T regions. Sequence and EMSA analysis suggests that the 179T allele creates an ERE-like binding site capable of binding recombinant estrogen receptor. Estrogen response element transactivation is enhanced by CD2 signaling, but inhibited by estrogen in PBMC but not in LPMC, although expression of estrogen receptor was similar. This is the first report to describe a potential molecular mechanism responsible for selectively controlling IFN-g production in LPMC. Genes and Immunity (2006) 7, 342–351. doi:10.1038/sj.gene.6364305; published online 25 May 2006 Keywords: human; mucosa; T lymphocytes; cellular activation; transcription factors

Introduction Interferon-g (IFN-g) plays an important role in host immune and inflammatory responses. Secretion of IFN-g by activated T cells and natural killer cells is tightly controlled,1,2 yet the regulatory mechanisms controlling expression are not fully understood. The capacity of T cells to produce IFN-g is determined primarily at the transcriptional level. Therefore, the gene structure and promoter response elements of IFNG have been intensely investigated. The structure of human IFNG consists of four exons and three introns,3 with a remarkable degree of conserved structure across human and rodent species.3 A number of enhancer and repressor cis-regulatory regions have been identified within the 50 promoter region. It is believed that the cooperative interaction of multiple cis-acting elements ultimately determines the level of transcriptional activation. Recent published reports have suggested the existence of regulatory sequences at regions 50 kb upstream, and downstream Correspondence: Dr SR Targan, Inflammatory Bowel Disease Research Center, Cedars-Sinai Medical Center, 8700 Beverly Blvd, D4063, Los Angeles, CA 90048, USA. E-mail: [email protected] Received 16 December 2005; revised 28 March 2006; accepted 28 March 2006; published online 25 May 2006

beyond the region of IFNG itself – far more distal than what has been previously assumed.4 In addition, histone hyperacetylation patterns and alterations in chromatin structure have been demonstrated within 24 kb of IFNG, as cells progress from naive T helper (Th) 0 to IFN-gproducing Th1 cells.4,5 Inappropriate overexpression of IFN-g by activated mucosal T cells is an important contributory factor in the pathogenesis of inflammatory bowel disease (IBD).6–8 In fact, disease severity is correlated with the level of IFN-g expression.9 The pathways leading to activation of mucosal lamina propria T cells are different from those of peripheral T cells.10,11 Lamina propria T cells are particularly sensitive to activation through the CD2 pathway.11,12 The CD2 pathway dominance is believed to result from a T-cell activation process inherent to the mucosal immune compartment. Despite much effort devoted to elucidating these differences, the molecular mechanisms involved remain poorly defined. We have previously identified unique regulatory mechanisms in the mucosa that are distinct from those in peripheral blood lymphocytes (PBL) and T-cell lines.13–15 In PBL, CD2 activation elements have been mapped mainly to the 108 to þ 64 bp region, encompassing the proximal and distal activator protein 1 (AP-1) binding sites.14 In contrast, in lamina propria

Novel ERE-like cis-element in IFNG promoter R Gonsky et al

mononuclear cells (LPMC), a significant CD2 response element resides in the 204 and 108 bp region, a region previously reported in PBL and T-cell lines to possess an essential AP-1 binding site, but CD2-mediated activation of IFNG expression is not altered by deletion of this AP-1 site.14 More recently, we have shown that CD2 stimulation activates the Janus protein tyrosine kinase/ signal transducers and activators of transcription (STAT) pathway in PBMC differently than in LPMC. In LPMC, CD2 signaling results in enhanced phosphotyrosine STAT1 and STAT4 and phosphoserine STAT1. In contrast, in PBMC, phosphorylation is largely restricted to phosphotyrosine STAT1. Moreover, in LPMC (but not in PBMC), CD2 signaling results in enhanced promoter activity through STAT protein binding to an IFNG intronic enhancer element.15 These results suggest that not only are there differences between the cis and trans regulatory elements used in PBMC and those used in LPMC, but additional differences probably exist leading to selective pathway accessibility in these cell populations. In further defining the key regulatory elements responsible for mucosa-specific activation of IFNG expression, we focused on the region between 204 and 108 bp. The CD2 response element in LPMC overlaps with a region (225 to 110 bp) that demonstrates a strong silencing effect in Jurkat T cells or in mouse splenocytes.15,16 Recently, a single-site nucleotide polymorphism (SNP) has been identified within that region.17 The variant SNP allele involves a 179G/T substitution. Enhanced tumor necrosis factor-a (TNF-a) stimulation of IFN-g expression can be detected in highly activated T cells transfected with the 179T allele.17 Interestingly, TNF-a and IFN-g expressions have been reported to synergistically enhance AIDS progression, and the presence of the 179T allele is associated with acceleration of AIDS progression, as measured by CD4 þ count.18 The 179T allele creates a binding site with similarity to, but with differences from, an AP-1 binding element. In fact, although a novel nucleoprotein complex binding to the 179T was identified, the nature of the cis and trans binding elements remains unknown. Moreover, enhancement in IFNG promoter activity followed TNF-a stimulation, even though no concomitant upregulation of nucleoprotein binding to 179T was detected.17 In the process of our studies to further define the correlation between the mucosa-specific IFNG regulatory elements within the 204 and 108 bp and the functional significance of the 179T allele, we discovered a putative non-classic estrogen response element (ERE) introduced by the 179T allele that displays selective regulation in PBMC but not in LPMC.

Materials and methods Isolation of mononuclear cells Peripheral blood mononuclear cells were isolated from normal healthy female volunteers by separation on Ficoll–Hypaque gradients. Intestinal specimens were obtained from female patients undergoing surgical resection of the colon at Cedars-Sinai Medical Center, Los Angeles. Approval for the use of human tissue was granted by the Institutional Review Board at CedarsSinai Medical Center. In this study, all tissue specimens

were taken from an uninvolved area of resected colon from patients with colonic carcinoma (normal), involved areas from patients with ulcerative colitis and uninvolved and involved areas from patients with Crohn’s disease. Lamina propria mononuclear cells were isolated from the resection samples using a technique modified from that described previously.19 Briefly, the intestinal specimen was washed with Hanks’ balanced salt solution (HBSS), and the mucosa was dissected away from the underlying layers. The mucosal layer was incubated in a shaking water bath (100 r.p.m.) in calcium- and magnesium-deficient HBSS, containing 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mg/ml gentamicin, 100 U/ml penicillin, 100 mg/ml streptomycin and 50 mg/ ml fungizone, with the solution changed every 30 min until the supernatant was free of epithelial cells. The remaining LP was minced into 1- to 2-mm pieces and digested for 10 min in RPMI 1640 containing 10% fetal calf serum (FCS), 0.5 mg/ml collagenase B (Roche Applied Science, Indianapolis, IN, USA), 1 mg/ ml hyaluronidase (Sigma, St Louis, MO, USA), 0.1 mg/ ml deoxyribonuclease I (Sigma), 50 mg/ml gentamicin, 100 U/ml penicillin, 100 mg/ml streptomycin and 50 mg/ml fungizone in shaker water bath (100 r.p.m.). The supernatant was collected, filtered through 110-mm nylon mesh (Spectrum Laboratory Products, Houston, TX, USA) and centrifuged at 500 g for 5 min. The cell pellet was resuspended in 15 ml and centrifuged at 30 g for 5 min to remove epithelial and other large cells. The supernatant was removed and lymphocytes were isolated by separation on Ficoll–Hypaque gradients. The cells were then washed three times with HBSS and resuspended in RPMI 1640 containing 10% FCS.

343

Stimulation of mononuclear cells Cells (PBMC or LPMC) were stimulated with anti-CD2 antibodies (clones CB6 and GD10, a gift from Chris Benjamin, Biogen, Cambridge, MA, USA) at 0.1 mg/ 106 cells at 371C for the times indicated for each experiment. Stimulation of LPMC and PBMC resulted in the secretion of an average of 2100 and 1400 ng/ml IFN-g/106 cells, respectively. Gel mobility electrophoretic mobility shift assay Double-stranded oligonucleotide was end-labeled with adenosine 50 -triphosphate, [g-32P] and T4 polynucleotide kinase. A 3-6 mg portion of nuclear extract protein was incubated at 251C with 0.25 mg/ml poly(dI-dC), in 20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM dithiothreitol, 250 mM NaCl and 50 mM Tris pH 7.5 for 10 min. The oligonucleotide was then added (20 000 c.p.m.) and the binding reactions incubated for an additional 30 min. Specificity was determined by the addition of 100-fold excess unlabeled oligonucleotide as competitor. The DNA–protein complexes were separated from unbound probe on a pre-run native 6% polyacrylamide gel in low ionic strength buffer (22.3 mM Tris pH 7.4, 22.3 mM borate, 0.5 mM EDTA pH 8.0). After 2 h, the gel was dried under vacuum and exposed to X-ray film. The 204 bp IFNG wild type (wt) or SNP variant oligonucleotide used was as reported:17 50 -ATC GTC AAA GGA CCC AAG GA-30 (wt) 50 -ATC GTC AAA TGA CCC AAG GA-30 (SNP). Consensus ERE, RAR, CREB, c/EBP, AP-1, NF-kB, Ets and OCT oligonucleotides were obtained from Santa Genes and Immunity


344

Cruz Biotechnology (Santa Cruz, CA, USA) and were as follows: ERE, 50 -GGATCTAGGTCACTGTGACCCCGGATC-30 RAR, 50 -AGGGTAGGGTTCACCGAAAGTTCACTC-30 CREB, 50 -AGAGATTGCCTGACGTCAGAGAGCTAG-30 c/EBP, 50 -TGCAGATTGCGCAATCTGCA-30 AP-1, 50 -CGCTTGATGACTCAGCCGGAA-30 NF-kB, 50 -AGTTGAGGGGACTTTCCCAGGC-30 Ets, 50 -GGGCTGCTTGAGGAAGTA TAAGAAT-30 OCT, 50 -TGTCGAATGCAAATCACTAGAA-30 Antibodies to estrogen receptor a (ERa) and ERb were purchased from Affinity BioReagents (Golden, CO, USA). Recombinant ERa and ERb proteins were purchased from Invitrogen (Carlsbad, CA, USA) The direct interaction between ERa and ERb and the 204 bp IFNG ERE introduced by the SNP was initiated by adding ERa and ERb protein to nuclear extract from unstimulated cells and electrophoretic mobility shift assay (EMSA) was performed as usual. Transfection 179G (wt) or variant 179T allele IFNG reporter constructs have been previously described.17 Multimeric consensus ERE reporter construct was a gift from Peter Kushner (University of San Francisco). Freshly isolated PBMC or LPMC were transfected following overnight culture in RPMI 1640 medium containing 10% FCS. Cells were then washed and resuspended in 250 ml fresh medium at 2 107 cells/ml and electroporated in the presence of 50 mg of reporter construct (600 V, for nine pulses of 500 ms, with 100 ms between pulses) using 4 mm (gap width) cuvettes in a BTX Electro Square Porator ECM 830 (Genetronics Inc., San Diego, CA, USA). A control plasmid containing the b-actin promoter driving a Renilla luciferase (provided by Dr Christopher Wilson, University of Washington) was co-transfected as an internal standard and values were normalized to correct for transfection efficiency with normalized fold increase calculated from the empty pGL3 vector. After electroporation, the cells were diluted in fresh medium, allowed to rest for 1 h before plating and then stimulated with anti-CD2 monoclonal antibodies for 4 h. Luminescence was measured using a Promega (Madison, WI, USA) luciferase assay kit and counted on a 6-detector Perkin Elmer Life Sciences (Gaithersberg, MD, USA) 1450 Microbeta liquid scintillation counter with coincidence counting deactivated. Flow cytometric staining of ERa Freshly isolated PBMC or LPMC were rested overnight and then stained with mouse anti-human CD3 (Caltag Laboratories, Burlingame, CA, USA) for 15 min at room temperature. Cells were fixed and permeabilized using IntraPrept permeabilization reagents (Beckman Coulter Inc., Fullerton, CA, USA) and then stained with rabbit anti-human ERa (HC-20, Santa Cruz Biotechnology) or control rabbit IgG (Caltag Laboratories) and detected with donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). Following a final wash, the cells were analyzed using a BD FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). Statistical analysis Tests for statistical significance were carried out using JMP Statistical Software (SAS Institute GmbH, Heidelberg, Germany).

Genes and Immunity

Results Allele-specific enhanced expression of IFNG 179T construct in PBMC but not in LPMC To further define the functional significance of the 179T allele and its relationship to selected mechanisms of regulation of IFN-g expression in the mucosa, PBMC and LPMC were transfected with 204 bp constructs containing identical sequences, except for a 1 bp substitution of the common 179G or variant 179T regions. CD2mediated signaling of PBMC resulted in transactivation of both the 179G and 179T allele constructs with a significant allele-specific enhancement in expression displayed for the 179T as compared to the 179G allele (Po0.01, paired t-test) (Figure 1). In contrast, when LPMC were transfected with the 179G and 179T allele constructs, no allele-specific difference in the level of expression was detected (P40.05). CD2 stimulation results in allele-specific upregulation of nuclear protein binding to the 179T region in PBMC but not in LPMC To evaluate the events involved in allele-specific functional activation of IFN-g expression, nuclear proteins were isolated from PBMC and LPMC, and EMSA analysis was performed to measure DNA binding activity to the 179G or 179T oligonucleotide probes. CD2 activation of PBMC resulted in a rapid (within 30 min) and sustained upregulation of trans-acting factors binding to the 179T, but no binding was detected utilizing the same extracts to the 179G (Figure 2a). In contrast, LPMC demonstrated constitutive binding to both the 179G and 179T, regardless of activation by CD2 (Figure 2b). The nucleoprotein complex of LPMC displayed a similar migration pattern compared to that of PBMC (Figure 2c). A difference was detected between PBMC and LPMC in the character of the binding to the 179T. Binding to the 179T oligonucleotide probe was specific, as excess unlabeled 179T but not 179G

Figure 1 Role of the 179T single-site nucleotide polymorphism (SNP) allele in CD2-mediated activation of IFNG. Promoter/ reporter constructs containing the common 179G (wild type, wt) or 179T (SNP) IFNG alleles were transfected into (a) human peripheral blood mononuclear cells (PBMC) or (b) human lamina propria mononuclear cells (LPMC). Symbols used: (&) unstimulated cells; (’) cells stimulated with anti-CD2. Bars represent means7s.e.m. of 17 (PBMC) and 11 (LPMC) experiments.


oligonucleotide probe competed for the binding (Figure 2d). Binding to the 179T region in PBMC was competed by as little as fivefold excess oligonucleotide. In LPMC, however, a 500-fold excess unlabeled oligonucleotide was required to partially compete for binding, suggesting nonspecific binding interactions.

345

The nucleotides flanking the SNP 179T participate in nucleoprotein complex formation The data presented above suggest a selective role for CD2 in the regulation of nucleoprotein binding to the 179T region in PBMC but not in LPMC. To further define the region involved in CD2-mediated upregulation of binding to the 179T in PBMC, mutational analysis was carried out utilizing a series of mutant oligonucleotides, in which substitutions were introduced outside of the polymorphic 179 base (Figure 3a). These mutant oligonucleotides were used in cold competition experiments to compete for binding to the variant SNP allele. Mutation of the region flanking the 179T results in oligonucleotides no longer capable of competing for nucleoprotein complex binding to the 179T region (Figure 3b). These results suggest that in addition to the

Figure 2 Electrophoretic mobility shift assay analysis of nuclear protein binding to the 179G (wild type, wt) or 179T allele. Peripheral blood mononuclear cells (PBMC) (a) or lamina propria mononuclear cells (LPMC) (b) were stimulated with 1 mg anti-CD2/ 106 cells for the indicated periods of time and nuclear proteins extracted for nuclear protein binding activity to the 179T or 179G IFNG promoter element. (c) The mobility of protein–DNA complexes in PBMC and LPMC was compared on the same gel. (d) Specificity of binding to the 179T allele was analyzed by adding increasing amounts of excess unlabeled 179T or 179G allele oligonucleotides. Representative results of 2–5 experiments with similar results are shown.

Figure 3 Nucleotides flanking the 179T nucleotide participate in nucleoprotein complex formation. (a) The sequence of mutant oligonucleotides within the region flanking the 179T used in competition experiments. Base changes are underlined. (b) Competition of nuclear protein binding activity to the 179T by mutant oligonucleotides. Representative results of two experiments with similar results are shown. Genes and Immunity


346

Table 1 Competition with consensus oligonucleotides to various potential cis-acting elements Element

Sequence

Competitiona

wt SNP SNPm1 SNPm2 SNPm12 SNPm4 ER RAR CREB c/EBP AP-1 NF-kB Ets OCT

ATCGTCA AAGGA CCCAAGGA ATCGTCA AATGA CCCAAGGA ATCGTTC AATGA CCCAAGGA ATCGTCA AATGA ACCAAGGA ATCGT T C AATGA AC C AAGGA ATCGTCA AATCA CCCAAGGA GGATCTAGGTCACTGTGA CCCCGGATC CGAGT GAAC T T T C GGT GAACCC T ACCCTAGA AGAGATTGCCTGA CGTCAGAGAGCTAG T GC AGAT T GC GC AAT C T G CA CGCTTGATGA CTCAGCCGGAA AGTGA GGGGACTTTCCCAGGC GGGCTGCTTGA GGAAGTATAAGAAT TGTCGAATGCAAATCA CTAGAA

+ +

a

Representative results of 2–3 experiments with similar results are shown.

polymorphic G/T base, additional sequences in the flanking region contribute to the stability of nucleoprotein complex formation. The 179T SNP allele sequence creates a potential ERE binding site Although a previous study had suggested that the 179T sequence created a new potential AP-1 binding site, competition and supershift analysis failed to support this hypothesis.17 Detailed sequence analysis was carried out to identify potential cis- and trans-acting factors capable of binding to this region, which differ from that seen for the common sequence. Emphasis was placed on including an expanded region flanking the 179T nucleotide, which appears to actively participate in stabilizing nucleoprotein complex formation. Table 1 displays a number of putative binding sites in addition to AP-1 that are located within this region, including one for c/EBP, which is believed to play a central role in chromatin restructuring to allow for transcriptional activation. Surprisingly, two putative hormone response elements, retinoic acid and estrogen receptor binding motifs, which belong to the nuclear receptor superfamily of hormoneinducible transcription factors, had also been generated by the 179T allele. These were of particular interest in light of early studies, which had identified regulation of IFNG promoter expression by glucocorticoids, as well as vitamins A and D.20,21 Competition experiments were carried out with consensus oligonucleotides to the various potential cis-acting elements (Figure 4). Table 1 summarizes the results of these experiments. Aside from the 179T oligonucleotide, only a consensus oligonucleotide to the ERE successfully competed for binding to the 179T allele oligonucleotide (Figure 4). Next, we examined the possibility that the 179T introduces an ERE element, which serves as a site for CD2-mediated upregulation of promoter activation. Examination of the sequence flanking the 179T allele reveals imperfect homology to the palindromic consensus ERE sequence 50 -GGTCAnnnTGACC-30 . The 179T allele corresponds with complete homology to the right half-site of the ERE binding region, whereas the 50 flanking sequence displays partial homology. However, many genes that contain EREs have been found to vary from the consensus sequence by one or more nucleotides. Genes and Immunity

To further explore whether CD2-mediated activation results in binding to an ERE element, nuclear proteins were isolated from PBMC, and EMSA analysis was performed. CD2 activation of PBMC resulted in upregulation of trans-acting factors binding to a consensus ERE (Figure 5a). To further define the nature of this interaction, nuclear extracts were prepared from PBMC stimulated with CD2 for 60 min, and nucleoprotein binding was carried out to a consensus ERE in the presence of excess competing oligonucleotide. Binding to the consensus ERE was competed by both a consensus ERE and the 179T allele oligonucleotide (Figure 5b). Two mutant oligonucleotides were designed to disrupt within each half of the palindromic consensus ERE sequence. Mutation of the 179T region abolished its capability to compete for the binding of the protein complex, whereas mutation of the region flanking the 179T, which exhibited only partial homology to the ERE binding sequence, did not affect its ability to successfully compete for binding. These results suggest that nucleoprotein complex generated following CD2 activation requires the homology to the 179T region for functional binding to the ERE. Supershift analysis was performed by preincubating nuclear protein extracts with antibodies specific to ERE-a and ERE-b. The analysis revealed supershift of the nucleoprotein complex binding to the 179T region by anti-ERE-a but not anti-ERE-b (Figure 5c). Furthermore, a direct interaction between ERa and ERb and the 179T region was analyzed by EMSA analysis using recombinant ERa and ERb proteins and DNA oligonucleotide probe corresponding to the 179T region, in the absence of exogenous estrogen. As seen in Figure 5d, no binding was detected in extract from unstimulated PBMC. However, binding was initiated by addition of recombinant ERa and ERb proteins to these extracts, with enhanced binding revealed when comparing ERa with ERb proteins. These data demonstrate that although both ERa and ERb can bind onto the 179T ERE region, there is enhanced preference of binding of ERa. Enhanced expression of a consensus ERE reporter construct in PBMC compared to LPMC In order to establish CD2-mediated functional activation of an ERE, transfection experiments were carried out


347

Figure 4 Competition of nuclear protein binding activity to the 179T allele. Binding to the 179T allele was measured in the presence of increasing amounts of the excess unlabeled consensus estrogen response element (ERE), retinoic acid receptor (RAR), activator protein 1 (AP-1), Oct1 and Ets oligonucleotide binding elements. Representative results of two experiments with similar results are shown.

utilizing a multimeric consensus ERE reporter construct. CD2 ligation resulted in transactivation of a consensus ERE construct in both PBMC and LPMC (Figure 6). The increased promoter activity induced in PBMC was ninefold, whereas only a modest twofold response was seen in LPMC. Moreover, as seen in Figure 6, CD2mediated expression of the ERE was effectively blocked in the presence of estradiol in PBMC (Figure 6a) (Po0.01, Wilcoxon sign-rank), whereas expression in LPMC remained virtually unchanged (Figure 6b) (P40.05, Wilcoxon sign-rank). Likewise, a parallel decrease in the CD2-mediated nucleoprotein complex binding to the 179T probe was detected following treatment with estrogen (Figure 6c). These results suggest that ERE-like elements within the IFNG promoter may function as targets for estrogen-mediated regulation of IFN-g expression. Comparative levels of ERa expressed on both CD3 þ PB and LP T cells To determine whether differences in response between PBMC and LPMC were attributed to the level of estrogen receptor expression, CD3 þ PB T and LP T cells were analyzed by flow cytometry for expression of ERa. As seen in Figure 7, similar level of expression was detected in both PB T- and LP T-cell populations, suggesting that modulation of promoter expression by estrogen was not merely a function of receptor availability.

Discussion In this study, we explored the hypothesis that a variant SNP within the IFNG promoter may participate in the

selective and distinct control mechanisms regulating IFN-g expression in the mucosa. Mucosa-specific IFNG response elements have previously been identified within the 204 and 108 bp region.14 In the present study, we show that an SNP (179G/T) recently detected within this region17 exhibits selective allele-specific enhanced expression compared to the common allele in PBMC, but not in LPMC. In PBMC transfected with reporter constructs containing the polymorphic 179T, there is a CD2-mediated doubling of promoter activity compared to the common 179G allele. In contrast, in LPMC, no allele-specific differences in expression were detected. Signaling through the CD2 pathway in PBMC, but not in LPMC, leads to upregulation of nucleoprotein binding to the 179 sequence. Sequence and binding analysis suggests that a putative non-classic ERE is introduced by the 179T allele. Following CD2 signaling, PBMC exhibit enhanced activation of ERE transactivation compared to LPMC, although LPMC express similar, if not greater, number of estrogen receptors. Moreover, ERE transactivation is selectively sensitive to estrogen downregulation in PBMC but not in LPMC. Previous research has demonstrated the importance of dysregulated T-cell production of IFN-g in the mucosal inflammatory process.6–8 We have previously demonstrated that there are mucosa-specific mechanisms for T-cell cytokine gene regulation of IFN-g expression, which differ from those seen in PBL.11,13–15,22 Studies by other groups have indicated that regulation of IFN-g gene expression in primary T cells differs from that observed in tumor T-cell lines, and likewise, differs in naı¨ve compared to memory T-cell subsets.23–25 Differential cytokine gene regulation may be controlled, in part, Genes and Immunity


348

Figure 6 Effect of estradiol on CD2-mediated activation of estrogen response elements (ERE). Consensus ERE and pGL3 promoter/ reporter constructs were transfected into (a) human peripheral blood mononuclear cells (PBMC) or (b) human lamina propria mononuclear cells (LPMC). Symbols used: (&) control unstimulated cells; (’) cells stimulated with anti-CD2 with or without addition of estradiol. Bars represent means7s.e.m. of 10 (PBMC) and five (LPMC) experiments. (c) Electrophoretic mobility shift assay analysis of nuclear protein binding to the 179T allele. Cells were stimulated with anti-CD2 with or without addition of estradiol. Representative results of three experiments are shown.

Figure 5 Electrophoretic mobility shift assay (EMSA) analysis of CD2-mediated nuclear protein binding to a consensus estrogen response element (ERE). (a) Peripheral blood mononuclear cells (PBMC) were stimulated with 1 mg anti-CD2/106 cells for the indicated periods of time and nuclear proteins extracted for nuclear protein binding activity to a consensus ERE promoter element. (b) Competition of nuclear protein binding activity to a consensus ERE was measured following the addition of increasing amounts of the excess unlabeled 179T, consensus ERE or mutant oligonucleotides. (c) Supershift analysis was performed by preincubating nuclear protein extracts with antibodies specific to ERE-a and ERE-b. (d) Direct interaction between estrogen receptor a (ERa) and ERb proteins and the 179T region exhibiting ERE homology. Extracts of unstimulated PBMC, in the absence of exogenous estrogen, were utilized for EMSA. Nucleoprotein complex formation was initiated by the addition of recombinant ERa and ERb proteins to DNA probes corresponding to the 179T region.

Genes and Immunity

by DNA methylation patterns. The increased IFN-g expression in Th0 or Th1, compared to Th2, cells is correlated with the hypomethylated status of IFNG.26 Considering that the 179T has been linked with accelerated AIDS progression, it is of interest to note that upregulation of DNA methyltransferase and concomitant methylation of the IFNG promoter has been reported following human immunodeficiency virus (HIV) infection.27 Moreover, the 179T is flanked by two previously noted methylation sites at 169 and 186 bp,25 and the CpG methylation site at 186 bp lies within the region of the ERE homology. We have previously reported that intrinsic differences in IFNG promoter exist between PBL and LPL.13–15,22 Our studies defined mucosal-specific functional divergence within the enhancer and repressor regions of the IFNG promoter. The data presented here expand on these findings. The single base-pair change at 179 bp reveals the existence of intrinsic regulatory mechanisms in the mucosa. Intrinsic distinctions of an epigenetic nature exist in the transcriptional regulation of T cells, which are dependent upon their origin and mode of activation, contributing to differential regulation of IFN-g expression. The functional significance of the 179T remains ambiguous. When highly activated PBMC were transfected


Figure 7 Expression of estrogen receptor a (ERa) in CD3 þ peripheral blood mononuclear cells (PBMC) and lamina propria mononuclear cells (LPMC). (a) Human PBMC or (b) human LPMC, gated for CD3 þ expression, were analyzed for expression of intracellular ERa. Symbols used: (. . . . . . ) isotype control; (—) ERa. Representative results of two experiments are shown.

with 179G or 179T allele constructs, they exhibited TNF-a-mediated activation of expression for the 179T construct compared to the 179G. This finding, however, was confounded by the fact that the novel DNA–protein complex binding to the 179T was constitutive and unchanged following TNF-a activation. Further studies suggested a genetic association with the 179T in HIV sero-converters with enhanced progression of AIDS.18 Although it would be of interest to assess whether the allele frequency of 179T is higher in patients with IBD, these studies are extremely difficult to perform. The 179T was originally reported as a rare polymorphism carried by 0.02% of Americans of European descent and 4% of the African-American population,17 a population shown to have restricted access to health care, therefore less likely to be studied.28 Inflammatory bowel disease in African Americans is marked by more severe disease and a higher incidence of extra-intestinal manifestations such as arthritis and primary sclerosing cholangitis.29 However, epidemiological studies have suggested that prevalence of IBD in the African-American population is lower than among European Americans,29 further impeding the study examining the clinical relevance of the 179T allele. IFNG contains a number of additional and more common SNPs and microsatellite polymorphisms. Two SNPs within the gene (at 760 bp and the 30 region) combine with an intronic CA repeat (761 bp) to form a haplotype associated with high levels of IFN-g production.30 An increase in this high IFN-g producer genotype has been noticed in the African-American population. In the initial report,17 it was suggested that the 179T polymorphism produced homology to an AP-1 binding site. However, cold competition experiments failed to support this hypothesis. The sequence analysis was extended in the present study. A number of candidate cis binding sites were introduced by the 179T base-pair transition, including an ERE binding site. Early studies have established transcriptional binding sites modulating IFN-g expression through members of the nuclear hormone receptor family such as glucocorticoid, vitamin D3 and retinoic acid.20,21,31 The estrogen receptor is an inducible trans-activating protein, which initiates tran-

scription through the binding to DNA response elements defined as EREs, following exposure to estrogen. Hormone response elements for the various hormones, such as glucocorticoids, retinoic acid and estrogen, share sequence homology similarities, although each possesses distinct DNA binding sequence requirements. The consensus ERE is defined as a palindromic 50 -GGTCA NNNTGACCC-30 sequence.32 However, many well-characterized estrogen responsive genes exhibit imperfect homology to this consensus sequence. The ERE sequence homology introduced by the 179T exhibits perfect homology to an ERE half-site and significant palindromic, but imperfect, homology to the entire ERE motif. Studies of other imperfect ERE sequences have determined that composition of the sequences adjacent to the ERE and binding of cofactors appear to have a critical role in enhancing transcriptional activation in regions with half-palindromic ERE symmetry.33–36 An adjacent Sp-1 binding site has been identified within the gelatinase A promoter as an independent additive region regulating ER-mediated expression.37 Further analysis of the regions adjacent to the 179 bp SNP will be of interest, considering that an Sp-1 binding site exists downstream of the 179 bp region, and several potential but imperfect regions of ERE homology are present further upstream. Recent studies utilizing animal models of experimental colitis have suggested a role for estrogen in modulating disease. 17b-Estradiol or a highly selective ERb agonist demonstrated antiinflammatory properties in the DNB or HLA-B27 models of colitis, respectively.38–40 However, immunomodulation by estrogen is complex, with proinflammatory effects elicited following estrogen treatment in the DSS model of colitis,38 suggesting that regulation of intestinal inflammation involves multiple levels of immunomodulation. In summary, the data presented in this study add to that which suggests that differential modulation of IFN-g expression exhibited by PBMC vs LPMC encompasses multiple regulatory mechanisms. Previous studies have identified distinct regulatory regions that are preferentially activated in PBMC vs LPMC, suggesting that differences in the composition of DNA–nucleoprotein complexes contribute to this process. The selective allelespecific enhancement of activation in PBMC but not LPMC of the 179T vs the 179G allele sequence points to selective availability of nuclear complexes, and suggests that inherent differences can be seen in the transcriptional machinery between peripheral and mucosal T-cell populations. The functional significance of the 179T in relationship to its capacity to act as an ERE has yet to be determined, but may be of practical importance in designing effective and selective therapeutic agents in the management of IFN-g-mediated inflammatory diseases.

349

Acknowledgements We thank Grace Kim and Jacyln Zhou for providing cultured LPMC. This work was supported by United States Public Health Service Grants DK-43211 and DK46763 and Cedars-Sinai Medical Center Inflammatory Bowel Disease Research Funds. Genes and Immunity


350

References 1 Young HA, Hardy KJ. Role of interferon-gamma in immune cell regulation. J Leukoc Biol 1995; 58: 373–381. 2 Lewis DB, Wilson CB. Gamma-interferon: an immunoregulatory lymphokine with immunotherapeutic potential. Pediatr Infect Dis J 1990; 9: 642–651. 3 Sehgal PB, May LT, Landsberger FR. Primary structural repeats in human and in murine gamma-interferon. J Interferon Res 1986; 6: 39–42. 4 Zhou W, Chang S, Aune TM. Long-range histone acetylation of the Ifng gene is an essential feature of T cell differentiation. Proc Natl Acad Sci USA 2004; 101: 2440–2445. 5 Eivazova ER, Aune TM. Dynamic alterations in the conformation of the Ifng gene region during T helper cell differentiation. Proc Natl Acad Sci USA 2004; 101: 251–256. 6 Powrie F. T cells in inflammatory bowel disease: protective and pathogenic roles. Immunity 1995; 3: 171–174. 7 Strober W, Kelsall B, Fuss I, Marth T, Ludviksson B, Ehrhardt R et al. Reciprocal IFN-gamma and TGF-beta responses regulate the occurrence of mucosal inflammation. Immunol Today 1997; 18: 61–64. 8 Sartor RB. Cytokines in intestinal inflammation: pathophysiological and clinical considerations. Gastroenterology 1994; 106: 533–539. 9 Monteleone G, MacDonald TT, Wathen NC, Pallone F, Pender SL. Enhancing lamina propria Th1 cell responses with interleukin 12 produces severe tissue injury. Gastroenterology 1999; 117: 1069–1077. 10 Pirzer UC, Schurmann G, Post S, Betzler M, Meuer SC. Differential responsiveness to CD3-Ti vs. CD2-dependent activation of human intestinal T lymphocytes. Eur J Immunol 1990; 20: 2339–2342. 11 Targan SR, Deem RL, Liu M, Wang S, Nel A. Definition of a lamina propria T cell responsive state. Enhanced cytokine responsiveness of T cells stimulated through the CD2 pathway. J Immunol 1995; 154: 664–675. 12 Boirivant M, Fuss I, Fiocchi C, Klein JS, Strong SA, Strober W. Hypoproliferative human lamina propria T cells retain the capacity to secrete lymphokines when stimulated via CD2/CD28 pathways. Proc Assoc Am Physicians 1996; 108: 55–67. 13 Gonsky R, Deem RL, Hughes CC, Targan SR. Activation of the CD2 pathway in lamina propria T cells up-regulates functionally active AP-1 binding to the IL-2 promoter, resulting in messenger RNA transcription and IL-2 secretion. J Immunol 1998; 160: 4914–4922. 14 Gonsky R, Deem RL, Bream JH, Lee DH, Young HA, Targan SR. Mucosa-specific targets for regulation of IFN-gamma expression: lamina propria T cells use different cis-elements than peripheral blood T cells to regulate transactivation of IFN-gamma expression. J Immunol 2000; 164: 1399–1407. 15 Gonsky R, Deem RL, Young HA, Targan SR. CD2 mediates activation of the IFN-gamma intronic STAT binding region in mucosal T cells. Eur J Immunol 2003; 33: 1152–1162. 16 Penix LA, Sweetser MT, Weaver WM, Hoeffler JP, Kerppola TK, Wilson CB. The proximal regulatory element of the interferon-gamma promoter mediates selective expression in T cells. J Biol Chem 1996; 271: 31964–31972. 17 Bream JH, Ping A, Zhang X, Winkler C, Young HA. A single nucleotide polymorphism in the proximal IFN-gamma promoter alters control of gene transcription. Genes Immun 2002; 3: 165–169. 18 An P, Vlahov D, Margolick JB, Phair J, O’Brien TR, Lautenberger J et al. A tumor necrosis factor-alpha-inducible promoter variant of interferon-gamma accelerates CD4+ T cell depletion in human immunodeficiency virus-1-infected individuals. J Infect Dis 2003; 188: 228–231. 19 Shanahan F, Brogan M, Targan S. Human mucosal cytotoxic effector cells. Gastroenterology 1987; 92: 1951–1957.

Genes and Immunity

20 Cippitelli M, Santoni A. Vitamin D3: a transcriptional modulator of the interferon-gamma gene. Eur J Immunol 1998; 28: 3017–3030. 21 Cippitelli M, Ye J, Viggiano V, Sica A, Ghosh P, Gulino A et al. Retinoic acid-induced transcriptional modulation of the human interferon-gamma promoter. J Biol Chem 1996; 271: 26783– 26793. 22 Gonsky R, Deem RL, Lee DH, Chen A, Targan SR. CD28 costimulation augments IL-2 secretion of activated lamina propria T cells by increasing mRNA stability without enhancing IL-2 gene transactivation. J Immunol 1999; 162: 6621–6629. 23 Aune TM, Penix LA, Rincon MR, Flavell RA. Differential transcription directed by discrete gamma interferon promoter elements in naive and memory (effector) CD4T cells and CD8T cells. Mol Cell Biol 1997; 17: 199–208. 24 Melvin AJ, McGurn ME, Bort SJ, Gibson C, Lewis DB. Hypomethylation of the interferon-gamma gene correlates with its expression by primary T-lineage cells. Eur J Immunol 1995; 25: 426–430. 25 White GP, Watt PM, Holt BJ, Holt PG. Differential patterns of methylation of the IFN-gamma promoter at CpG and nonCpG sites underlie differences in IFN-gamma gene expression between human neonatal and adult CD45RO T cells. J Immunol 2002; 168: 2820–2827. 26 Yano S, Ghosh P, Kusaba H, Buchholz M, Longo DL. Effect of promoter methylation on the regulation of IFN-gamma gene during in vitro differentiation of human peripheral blood T cells into a Th2 population. J Immunol 2003; 171: 2510–2516. 27 Mikovits JA, Young HA, Vertino P, Issa JP, Pitha PM, TurcoskiCorrales S et al. Infection with human immunodeficiency virus type 1 upregulates DNA methyltransferase, resulting in de novo methylation of the gamma interferon (IFN-gamma) promoter and subsequent downregulation of IFN-gamma production. Mol Cell Biol 1998; 18: 5166–5177. 28 Straus WL, Eisen GM, Sandler RS, Murray SC, Sessions JT. Crohn’s disease: does race matter? The mid-atlantic crohn’s disease study group. Am J Gastroenterol 2000; 95: 479–483. 29 Reddy SI, Burakoff R. Inflammatory bowel disease in African Americans. Inflamm Bowel Dis 2003; 9: 380–385. 30 Vandenbroeck K, Goris A. Cytokine gene polymorphisms in multifactorial diseases: gateways to novel targets for immunotherapy? Trends Pharmacol Sci 2003; 24: 284–289. 31 Cippitelli M, Sica A, Viggiano V, Ye J, Ghosh P, Birrer MJ et al. Negative transcriptional regulation of the interferon-gamma promoter by glucocorticoids and dominant negative mutants of c-Jun. J Biol Chem 1995; 270: 12548–12556. 32 Klein-Hitpass L, Schorpp M, Wagner U, Ryffel GU. An estrogen-responsive element derived from the 50 flanking region of the Xenopus vitellogenin A2 gene functions in transfected human cells. Cell 1986; 46: 1053–1061. 33 Curtis SW, Korach KS. Uterine estrogen receptor–DNA complexes: effects of different ERE sequences, ligands, and receptor forms. Mol Endocrinol 1991; 5: 959–966. 34 Klinge CM, Peale Jr FV, Hilf R, Bambara RA, Zain S. Cooperative estrogen receptor interaction with consensus or variant estrogen responsive elements in vitro. Cancer Res 1992; 52: 1073–1081. 35 Driscoll MD, Sathya G, Muyan M, Klinge CM, Hilf R, Bambara RA. Sequence requirements for estrogen receptor binding to estrogen response elements. J Biol Chem 1998; 273: 29321–29330. 36 Hall JM, McDonnell DP, Korach KS. Allosteric regulation of estrogen receptor structure, function, and coactivator recruitment by different estrogen response elements. Mol Endocrinol 2002; 16: 469–486. 37 Qin H, Sun Y, Benveniste EN. The transcription factors Sp1, Sp3, and AP-2 are required for constitutive matrix metalloproteinase-2 gene expression in astroglioma cells. J Biol Chem 1999; 274: 29130–29137.


351 38 Verdu EF, Deng Y, Bercik P, Collins SM. Modulatory effects of estrogen in two murine models of experimental colitis. Am J Physiol Gastrointest Liver Physiol 2002; 283: G27–G36. 39 Harris HA, Albert LM, Leathurby Y, Malamas MS, Mewshaw RE, Miller CP et al. Evaluation of an estrogen receptor-beta

agonist in animal models of human disease. Endocrinology 2003; 144: 4241–4249. 40 Harnish DC, Albert LM, Leathurby Y, Eckert AM, Ciarletta A, Kasaian M et al. Beneficial effects of estrogen treatment in the HLA-B27 transgenic rat model of inflammatory bowel disease. Am J Physiol Gastrointest Liver Physiol 2004; 286: G118–G125.

Genes and Immunity