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C-C Chemokine RANTESFunctional Mutation in the Promoter of the Atopic Dermatitis Is Associated with a

Terri H. Beaty and Shau-Ku HuangSchleimer, Luis Caraballo, Raana P. Naidu, Paul N. Levett, Freidhoff, Claudia Sengler, James R. Plitt, Robert P.Beyer, Kathleen C. Barnes, Beverly S. Plunkett, Linda R. Renate G. Nickel, Vincenzo Casolaro, Ulrich Wahn, Kirsten

http://www.jimmunol.org/content/164/3/1612doi: 10.4049/jimmunol.164.3.1612

2000; 164:1612-1616; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/164/3/1612.full#ref-list-1

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Atopic Dermatitis Is Associated with a Functional Mutation inthe Promoter of the C-C Chemokine RANTES1

Renate G. Nickel,*† Vincenzo Casolaro,* Ulrich Wahn,† Kirsten Beyer,*† Kathleen C. Barnes,*Beverly S. Plunkett,* Linda R. Freidhoff,* Claudia Sengler,* James R. Plitt,*Robert P. Schleimer,* Luis Caraballo,‡ Raana P. Naidu,§ Paul N. Levett,§ Terri H. Beaty,¶ andShau-Ku Huang2*

Up-regulation of C-C chemokine expression characterizes allergic inflammation and atopic diseases. A functional mutation in theproximal promoter of the RANTES gene has been identified, which results in a new consensus binding site for the GATAtranscription factor family. A higher frequency of this allele was observed in individuals of African descent compared withCaucasian subjects (p< 0.00001). The mutant allele was associated with atopic dermatitis in children of the German MulticenterAllergy Study (MAS-90; p < 0.037), but not with asthma. Transient transfections of the human mast cell line HMC-1 and the Tcell line Jurkat with reporter vectors driven by either the mutant or wild-type RANTES promoter showed an up to 8-fold higherconstitutive transcriptional activity of the mutant promoter. This is the first report to our knowledge of a functional mutation ina chemokine gene promoter. Our findings suggest that the mutation contributes to the development of atopic dermatitis. Itspotential role in other inflammatory and infectious disorders, particularly among individuals of African ancestry, remains to bedetermined. The Journal of Immunology,2000, 164: 1612–1616.

A topic dermatitis (AD)3 is a chronic inflammatory skindisease that affects 10–15% of the population duringinfancy. While the molecular basis is currently unclear,

infiltration of the skin with eosinophils, T cells, and monocytes hasbeen well documented (1). Several studies have suggested a crucialrole of C-C chemokines in the pathogenesis of allergic inflamma-tion. The C-C chemokines preferentially activate and attract eo-sinophils, basophils, monocytes, and T cell subsets (2). Tissue eo-sinophilia is pronounced after allergen exposure and is acharacteristic finding in allergic inflammation seen in variousatopic disorders including AD and asthma (1, 3). Evidence forlinkage of asthma to chromosome 17p12-17q11.2 (C-C chemokinecluster) in African American sib pairs has been reported (4), sug-gesting that functional mutations in C-C chemokine-encodinggenes or their regulatory elements may contribute to the pathogen-esis of asthma or associated phenotypes.

RANTES is one of the most extensively studied C-C chemo-kines in allergic and infectious disease. It is a potent chemoattrac-

tant for eosinophils, lymphocytes, monocytes, and basophils (2, 5,6). RANTES is expressed in activated T lymphocytes, airway ep-ithelial cells, platelets, fibroblasts, and renal epithelial and mesan-gial cells (reviewed in Ref. 7). Glucocorticoids, the most effectivedrugs in the treatment of allergic inflammation, decrease the ex-pression of RANTES and other eosinophil-active chemokines invivo and in vitro (8–11). RANTES has also been studied in thecontext of HIV infection, because CCR5, a receptor for RANTES,macrophage inflammatory protein (MIP)-1a, and MIP-1b, hasbeen identified as a coreceptor for macrophage-tropic HIV type 1strains (12, 13).

Because of the potential role of RANTES in allergic and infec-tious diseases, we screened populations of both African and Cau-casian ancestry for mutations in the proximal promoter region ofthe RANTES gene that may affect transcriptional activity and sub-sequently RANTES expression in various cell types. In this com-munication, we demonstrate a novel functional mutation in theproximal promoter region of the RANTES gene. The mutation issignificantly more frequent in individuals of African ancestry thanin Caucasian subjects. Moreover, we could demonstrate an asso-ciation with AD, but not with asthma, in our study populations.

Materials and MethodsStudy populations

German population. Two hundred eighty-six unrelated children of theGerman Multicenter Allergy Study (MAS-90) (14) were genotyped; 188subjects had been diagnosed with AD at two or more of five visits duringthe first 3 years of life and were compared with 98 control subjects (50allergic, 48 nonallergic based on serum IgE Abs.0.35 kU/L to food orinhalant allergens). AD was defined as previously described (15). The def-inition of AD was based on 1) physician’s diagnosis, 2) manifestation of“dry skin,” and 3) three or more symptoms of AD at three or more areas.Based on these criteria, children were examined once a year (every birth-day6 4 wk), and those who were diagnosed with AD at two or more timepoints were selected for the present study.Afro-Caribbean population. Thirty-three nuclear and extended Afro-Ca-ribbean families (n5 713), described in detail previously (16), were re-cruited in Barbados, West Indies.

*Division of Clinical Immunology, Department of Medicine, Johns Hopkins Univer-sity School of Medicine, Baltimore, MD 21224;†Department of Pediatric Pneumol-ogy and Immunology, Charite, Berlin, Germany;‡University of Cartagena, Carta-gena, Colombia;§University of West Indies, Barbados; and¶Department ofEpidemiology, Johns Hopkins University School of Hygiene and Public Health, Bal-timore, MD 21224

Received for publication August 17, 1999. Accepted for publication November16, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants AI-40274, AI-20059, AR-31891, and HL/AI-49612 and the German Ministry of Research and Tech-nology Grant 01EE9406.2 Address correspondence and reprint requests to Dr. Shau-Ku Huang, Johns HopkinsAsthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-6801. E-mail address: skhuang@mail.jhmi.edu3 Abbreviations used in this paper: AD, atopic dermatitis; MIP, macrophage inflam-matory protein; CGSA, Collaborative Study of the Genetics of Asthma; SSCP, single-stranded conformation polymorphism; HMC-1, human mast cell line.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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Colombian population. Forty-nine nuclear families (302 individuals) re-cruited in Cartagena, Colombia, were ascertained through at least one asth-matic proband. Phenotype assessments and definition of asthma were con-ducted as described for the Afro-Caribbean population (16).African American and Caucasian American populations.Sixty-threepredominantly nuclear African American families and 48 nuclear and ex-tended Caucasian families were recruited at the Johns Hopkins Asthma andAllergy Center as part of the Collaborative Study on the Genetics ofAsthma (CSGA) (4). No valid assessment of AD has been available for anyof our populations of African ancestry. The definition of asthma for Cau-casian and African American study populations was based on strict criteriaaccording to the study protocol of the CSGA (4), which included 1) phy-sician’s diagnosis of asthma, 2) a fall in baseline FEV1 by$20% at#25mg/ml methacholine or$15% increase in FEV1 after bronchodilator use,3) more than or equal to two asthmatic symptoms (cough, wheeze, dys-pnea), and 4)#5 pack years of smoking cigarettes. The clinical phenotypesof affected subjects, including their mean age and gender distributions,were described previously in Ref. 4. For subjects of Afro-Caribbean pop-ulation, asthma was defined as having 1) a reported history of asthma usinga standardized questionnaire adapted from the CSGA study (4), and 2)confirmation of asthma by a physician. The asthmatic populations were notdescribed in full detail, because no significant association of the identifiedmutation with asthma was found in any of the three large populationsgenotyped.

Single-stranded conformation polymorphism (SSCP)

Fifty nanograms of genomic DNA were used for 5-ml PCR. Oligonucle-otides (sense, 59-TAAATAACATCCTTCCAT-39; antisense, 59-ATTTCTCTGCTGACATCC-39) were designed to amplify base pairs2435 to2220 of the RANTES promoter. PCR was performed for 35 cycles of 45 sat 94°C, 45 s at 50°C, and 45 s at 72°C. PCR products were renderedsingle-stranded by heating (5 min, 94°C) in denaturing buffer. Electro-phoresis was conducted at room temperature (6W for 18 h; gel composi-tion: 6% acrylamide, 10% glycerol, 0.53TBE). Oligonucleotides for PCR,site-directed mutagenesis, and EMSAs (see below) were custom synthe-sized by Genosys Biotechnologies (The Woodlands, Texas).

DNA sequencing

For sequencing, PCR products from unrelated individuals homozygous forthe 2401A (n 5 3) or 2401G allele (n5 3) were first cloned into a TAvector (Original TA Cloning Kit; Invitrogen, San Diego, CA) according tothe manufacturer’s directions. Plasmid DNA templates were sequencedusing the fluorescent dideoxy terminator method of cycle sequencing on aPerkin-Elmer, Applied Biosystems Division 373A following Applied Bio-systems protocols (Perkin-Elmer, Norwalk, CT).

Transient transfections and cell culture

Two reporter constructs containing either the2401A- or the2401G-ex-pressing RANTES promoter fused to the luciferase structural gene wereused for transient transfections of human mast cell line HMC-1, Jurkat, andBEAS-2B cells. Base pairs2885 to164 of the RANTES gene (2401A,a generous gift of Dr. T. Schall) were cloned into theKpnI site of pGL2basic vector (Promega, Madison, WI). Site-directed mutagenesis at posi-tion 2401 (A to G) was performed using the QuikChange Site-DirectedMutagenesis Kit (Stratagene, La Jolla, CA) according to the manufactur-er’s instructions with oligonucleotides containing the region between basepairs 2416 and2386 of the RANTES promoter (2416-GAGGGAAAGGAGGAGgTAAGATCTGTAATG-2386 and its complement; bold indi-cated where the base pair exchange occurs). Plasmid DNA was obtainedwith double-cesium chloride purification (BioServe Biotechnologies, Lau-rel, MD). HMC-1 cells were cultured at 0.53 106/ml in RPMI 1640 in thepresence of 10% FCS 24 h before transient transfections. Jurkat cells werecultured at 0.53 106/ml in RPMI 1640 containing 2% FCS before trans-fection. BEAS-2B cells were cultured as previously described (8). TheSuperFect reagent (Qiagen, Santa Clarita, CA) was used for transient trans-fections of Jurkat and HMC-1 cells according to the manufacturer’s direc-tions. Two micrograms of plasmid DNA and 8ml SuperFect reagent wereused for transfection of 13 106 HMC-1 cells. One microgram of plasmidDNA and 4 ml SuperFect reagent were used for transfection of 13 106

Jurkat cells. Three microliters of Fugene reagent (Boehringer Mannheim,Mannheim, Germany) and 1mg plasmid DNA were used for transienttransfection of BEAS-2B. Transfections with the promoterless pGL2 basic,RANTES 2401A/pGL2, and RANTES2401G/pGL2 were always per-formed in duplicates.

Luciferase assay

Luciferase expression was monitored by chemiluminescence of cell lysates12–72 h after transfections using the Enhanced Luciferase Assay Kit (An-alytical Luminescence Laboratory, Ann Arbor, MI) as recommended bythe manufacturer. Total protein content of cell lysates was determined withBio-Rad protein assays (Bio-Rad, Hercules, CA). Luciferase activity wasmeasured in a luminometer analyzer (Monolight 3010; Analytical Lumi-nescence Laboratory).

EMSA

Protein extraction from the nuclei of Jurkat and HMC-1 cells and EMSAswere performed as previously described (17). The probes for EMSAs weretwo 18-bp double-stranded oligonucleotides containing the RANTES pro-moter sequence between base pairs2409 and2392 of the2401A (59-GAAAGGAGATAAGATCTG-39 and its complement) or2401G (59-GAAAGGAGGTAAGATCTG-39 and its complement). Abs to GATA-1,GATA-2, and GATA-3 proteins and competitor oligonucleotides (GATA,OCTA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Statistical analyses

Pearsonx2 tests were used to compare genotype frequencies. Wilcoxonnonparametric tests were performed to assess differences in transcriptionalactivities of 2401A and2401G RANTES promoter-driven constructs.Sib-pair analysis and the transmission disequilibrium test for qualitativetraits were conducted as described before (16).

ResultsMutational analysis of RANTES promoter

Using SSCP and DNA sequencing, we identified a point mutationin the RANTES promoter at base pair2401 (A 2401G) that re-sults in an additional consensus binding site for the GATA tran-scription factor family (Fig. 1).

Frequency of mutant allele and association with AD

We screened three populations of African descent with varyingdegrees of Caucasian admixture and three Caucasian populationsfor both promoter alleles using SSCP. Significant ethnic differ-ences in genotype frequencies were observed: 15% of Afro-Car-ibbeans and African Americans were homozygous for the2401Aallele compared with#2.1% in either Caucasian population (p ,0.00001; Table I). Although excess allele sharing at this two-allele

FIGURE 1. SSCP of PCR amplified 215-bp fragment of the RANTESpromoter (base pairs2435 to 2220). Two different allelic forms weredetected in Afro-Caribbean individuals. Cloning and sequencing of PCRproducts from unrelated individuals (three homozygous for one, three ho-mozygous for the other allele) showed a consistent point mutation (G to A)at base pair2401 resulting in a new consensus binding element(AGATAA; underline indicating the position where nucleotide substitutionoccurs) for GATA transcription factors. No other polymorphisms in theproximal RANTES promoter were detected in overlapping PCR productscovering base pairs2888 to167 of the RANTES gene in 40 asthmatic and40 nonasthmatic Afro-Caribbean subjects.

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marker was observed among both African American and Afro-Caribbean asthmatic sib pairs (mean identical-by-descent0.54/0.27 for 143/27 full/half sibs;p 5 0.016), no higher trans-mission rates of the mutant allele to asthmatic offspring were de-tected using the transmission disequilibrium test. These findingssuggest that a mutation different from, but in close proximity to,the RANTES gene may contribute to asthma pathogenesis in pop-ulations of African descent.

However, the2401A allele was more frequent in 188 Germanchildren with AD compared with 98 control subjects (p , 0.037,Table II). The distributions of2401A allele frequency in the con-trol populations were consistent with the expected Hardy-Wein-berg equilibrium. All tested children were participants of the Ger-man Multicenter Allergy Study (MAS-90), a noninterventional,prospective, birth cohort study (14). Valid assessments of AD havenot yet been performed in any of the other genotyped studypopulations.

Functional analysis of RANTES promoter polymorphism

To test for functional differences between the two promoter vari-ants, we performed transient transfections in two human cell lineswith luciferase-reporter constructs driven by either the2401A- or2401G-expressing RANTES promoter. Both mast cells and Tlymphocytes express GATA-binding transcription factors (18).HMC-1 and Jurkat cells express RANTES mRNA (7, 19) and weretherefore selected for comparative studies of transcriptional activ-ities of the two RANTES promoter constructs. In both cell lines,we detected significantly higher constitutive transcriptional activ-ities of the 2401A-expressing promoter (Fig. 2,A and B). Wewere not able to identify stimuli that differentially up-regulatedtranscriptional activity from the two reporter constructs, possiblydue to the either large number of othercis-regulatory elements inthe RANTES promoter or to constitutively elevated nuclear ex-pression of GATA family members in these cells. Transient trans-fections of the bronchial epithelial cell line BEAS-2B with the

same promoter constructs did not show differences in transcriptionalactivities (n5 4, mean fold difference 1.1,p 5 0.47; Fig. 2C).

EMSAs showed clear cut differences in binding of nuclear pro-teins extracted from both HMC-1 and Jurkat cells to probes con-taining the sequence between base pairs2409 and2392 of thewild-type and the mutant RANTES promoter. Using HMC-1 ex-tracts, treatment of the complexes with Abs directed againstGATA-1 or -2 resulted in supershifts of two DNA-protein com-plexes, formed exclusively with the mutant (2401A) probe,whereas anti-GATA 3 had no effect on the pattern of complexformation (Fig. 3). Using Jurkat extracts, supershifts were ob-served when complexes (2401A probe) were treated with anti-GATA-3, whereas anti-GATA -1 and -2 had no effect on the pat-tern of complex formation (Fig. 4). Taken together, these findingsare consistent with published GATA binding protein expressionpatterns in T lymphoid cells and mast cells (18). As expected, veryweak binding, and no difference in the pattern of complex forma-tion with the two probes, was observed using nuclear extracts fromBEAS-2B (data not shown).

DiscussionThe genetic basis of AD is currently unclear. It is thought to con-tain highly interactive and polygenetic components, each withvarying degree of genetic influence. In this study, we identified anovel, functional mutation in the proximal promoter region of theRANTES gene. The point mutation at base pair2401 results in anew consensus binding site for the GATA transcription factor fam-ily and is strikingly more frequent in individuals of Africandescent compared with Caucasian subjects. We observed an asso-ciation of the mutation with AD in a longitudinally well-charac-terized German cohort, but no association with asthma in any studypopulation, suggesting that the mutation contributes, in part, to thedevelopment of AD in Caucasian populations. Additional studies,

FIGURE 2. Transient transfections of HMC-1 cells (A), Jurkat (B), andBEAS-2B (C) with pGl2/RANTES2401A and 2401G reporter con-structs. Cells were lysed 18 h (HMC-1) or 48 h (Jurkat, BEAS-2B) aftertransfection. Luciferase activity was measured and adjusted for total pro-tein concentration in cell lysates. Each column represents the mean ofduplicate assays (CV, 10%). The illustration is representative of five(HMC-1), seven (Jurkat), and four (BEAS-2B) separate experiments.Using HMC-1 cells, we observed a mean 3.9-fold, 12–24 h after transfec-tion (n 5 5, p 5 0.043), and a mean 3.5-fold higher constitutive transcrip-tional activity of the mutant promoter 36–48 h after transfection (n 5 10,p 5 0.0051); using Jurkat cells we observed a mean 4.1-fold higher activity48–72 h after transfection (n5 7, p 5 0.018). No differences (p5 0.47)were observed in BEAS-2B cells (n5 4).

Table I. Distribution of RANTES promoter genotypes in five ethnicgroupsa

Subjects

Genotype

Frequency of AAA AG GG

African American (n5 94) 14 52 28 0.43Afro-Caribbean (n5 85) 13 48 24 0.44Colombian (n5 87) 6 41 40 0.30Caucasian (German;n 5 286) 6 100 180 0.20Caucasian (American;n 5 152) 1 44 107 0.15

a Pearsonx2 statistics of RANTES promoter genotype distributions for wild-type(–401G) and mutant (–401A) alleles in unrelated individuals (founders) of Africandescent (varying degrees of Caucasian admixture) and Caucasians. Genotype frequen-cies were strikingly different between African American and Afro-Caribbean subjectsand either Caucasian population (p , 0.00001).

Table II. Distribution of genotypes in children with AD vs unaffectedcontrol subjectsa

Subjects

Genotype

Frequency of AAA AG GG

AD (n 5 188) 6 72 110 0.22Control subjects (n5 98) 0 28 70 0.14

a The RANTES promoter mutant allele was significantly more frequent in partic-ipants of the MAS-90 with AD than in control subjects. All children tested were ofbilateral German ethnicity (Pearsonx2; p 5 0.037).

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particularly in populations of African ancestry that have been wellcharacterized for AD, are needed to confirm this finding.

GATA DNA binding proteins (GATA 1–4) are expressed in abroad range of hemopoietic and nonhemopoietic tissues (reviewedin Refs. 18 and 20). GATA binding motifs are present in the pro-moter regions of all Th2 cytokines (21) as well as in the regulatoryregions of numerous mast cell- and endothelial cell-expressedgenes (20). GATA-3 is expressed in T lymphocytes, eosinophils,basophils, and mast cells and has been shown to be essential for theactivation of Th2 cytokine genes (21–23). GATA-1 and GATA-2

are expressed in eosinophils (21), mast cells, basophils, andmegakaryocytes (22, 24).

The expression of RANTES is differentially regulated in variouscell types, and a large number of putativecis-acting elements havebeen described in the promoter region (7). A recent study showedthat disruption of any of four putative binding sites for NF-kB inthe upstream region of the RANTES gene resulted in markedlyreduced promoter activity in T cell and monocytic cell lines (25),indicating that multiple binding sites for a specific transcriptionfactor may cooperate in the enhancement of promoter activity.Therefore, it is of interest that in addition to the GATA bindingmotif that we identified at base pair2401, two additional GATAconsensus elements are located 755 and 786 bp upstream of theRANTES transcription start site (7), further implicating GATA-binding transcription factors in the regulation of RANTES geneexpression. Furthermore, functional analyses of the mutant andwild-type alleles in HMC and Jurkat T-cell lines, both of whichexpress RANTES and GATA binding proteins, showed that 1) asignificantly higher transcriptional activity of the2401A mutantallele is found in both cell lines, and 2) differential binding ofGATA transcriptional factor family is seen between HMC andJurkat cells.

Airway epithelial cells are a major source of RANTES (8).However, no significant differences in transcriptional activity be-tween wild-type and mutant RANTES promoters were observed intransiently transfected BEAS-2B cells. GATA expression in epi-thelial cells has not been reported, which may explain these in vitrofindings. In contrast, increased RANTES production in T cells,mast cells, or megakaryocytes, cell types that constitutively ex-press GATA binding proteins (18, 20) and RANTES (6, 26, 27),may contribute to the pathogenesis of AD. Up-regulation ofRANTES expression in the skin could enhance the recruitment ofeosinophils, lymphocytes, and monocytes to the sites of allergicinflammation. Severe pruritus and excoriations are hallmarks ofAD associated with bleeding and subsequent activation of plate-lets. Because megakaryocytes express GATA binding proteins andsignificant quantities of RANTES, increased RANTES content ofplatelets of carriers of the2401A mutation could further explainthe association of the mutant allele with AD.

A strikingly higher frequency of the RANTES2401A allelewas observed in individuals of African ancestry compared withCaucasian subjects. In light of these ethnic differences, it is in-triguing that basic differences in C-C chemokine receptor biologyhave been described in individuals of African vs Caucasian de-scent. First, a 32-bp deletion in the CCR5 receptor, a commonreceptor for RANTES, MIP-1a, and MIP-1b, and a coentry factorfor macrophage-tropic HIV strains (28), is found in.10% of Cau-casians, whereas this mutation is absent in African populations(29). Second, in contrast to Caucasian individuals, the Duffy Agreceptor for chemokines (DARC) is not expressed on RBC in thevast majority of African people, while it is uniformly expressed onRBC in Caucasians. Lack of DARC on RBC confers an evolu-tionary advantage because DARC-negative RBC are resistant toinfection byPlasmodium vivax(30).

Therefore, it would be of interest to determine the biologicalrole of the2401A-expressing RANTES promoter in AD and otherinflammatory and infectious disorders in populations of Africandescent.

AcknowledgmentsWe thank all families in Germany, Cartagena, Barbados, and the UnitedStates for their generous participation in this study; we are grateful to Lun

FIGURE 3. Binding of HMC-1 nuclear proteins to the GATA bindingelement in the2401A RANTES promoter. Radiolabeled oligonucleotidesincorporating the region from base pairs2409 to2392 of the2401A and2401G RANTES promoter alleles were used in EMSAs with nuclear ex-tracts from unstimulated HMC-1 cells. Clear cut differences in binding tothe two probes were observed (lanes 1and 2). Furthermore, addition ofanti-GATA-1 and -2 Abs resulted in supershifts of two DNA-protein com-plexes (lanes 3and4).

FIGURE 4. Binding of Jurkat nuclear proteins to the GATA bindingelement in the2401A RANTES promoter. Radiolabeled oligonucleotidesincorporating the region from base pairs2409 to2392 of the2401A (A)and 2401G (B) RANTES promoter alleles were used in EMSAs withnuclear extracts from unstimulated Jurkat cells. Clear cut differences inbinding to the two probes were observed (lane 1in A andB). Furthermore,addition of anti-GATA-3 Abs resulted in a supershift of the specific DNA-protein complex (lane 4,A). Nonspecific binding (NS) was observed inboth assays. Reduction or elimination of the bands resulting from nonspe-cific binding was seen after competing with nonradiolabeled DNA frag-ments (100-fold molar excess) that were specific (GATA;lanes 6, AandB)or nonspecific (OCTA;lanes 7, AandB) for GATA binding proteins.

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Xue and Eva Ehrlich for excellent technical support. We express our grat-itude to all of the members of the German MAS-90 group and the CSGAfor their contribution to this project.

Note added in proof.A recent publication by Liu et al. (1999Proc. Natl. Acad. Sci USA 96:4581) showed an identical polymor-phism as we identified in our study populations. However, thenumbering of the polymorphic residue is different due to the factthat two additional nucleotides were found in the promoter regionof RANTES in their study population. Our numbering was basedon the original publication by Nelson et al. (7) and by GenBankdatabase (accession no. S64885).

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