Proc. Natl. Acad. Sci. USA Vol. 94, pp. 3195–3199, April 1997 Immunology

Effects of a polymorphism in the human ␣ promoter on transcriptional activation (genetics͞major histocompatibility complex͞cytokine͞gene regulation͞autoimmune diseases)

ANTHONY G. WILSON†,JULIAN A. SYMONS†,TARRA L. MCDOWELL†,HUGH O. MCDEVITT‡, AND GORDON W. DUFF†§

†Section of Molecular Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, United Kingdom; and ‡Departments of Microbiology and Immunology, and Medicine, Stanford University, Stanford, CA 94305

Contributed by Hugh O. McDevitt, December 23, 1996

ABSTRACT Tumor necrosis factor ␣ (TNF␣) is a potent of TNFA in different strains of mice. Further, variation in the immunomodulator and proinflammatory that has production of TNF␣ from macrophages has been shown to been implicated in the pathogenesis of autoimmune and vary between strains (10). Several of these polymorphisms infectious diseases. For example, plasma levels of TNF␣ are have been correlated with TNF␣ production and with suscep- positively correlated with severity and mortality in malaria tibility to, or severity of, several diseases. The (NZB ϫ and leishmaniasis. We have previously described a polymor- NZW)F1 mouse develops a severe autoimmune disease that is .phism at ؊308 in the TNF␣ promoter and shown that the rare very similar to systemic lupus erythematosus (SLE) in humans allele, TNF2, lies on the extended haplotype HLA-A1-B8-DR3- A restriction fragment length polymorphism in the TNFA DQ2, which is associated with autoimmunity and high TNF␣ is correlated with low production of TNF␣ and with the production. Homozygosity for TNF2 carries a sevenfold in- development of lupus nephritis (11), and replacement therapy creased risk of death from cerebral malaria. Here we dem- with recombinant TNF␣ delays the onset of nephritis with onstrate, with reporter under the control of the two increased survival rate (12). Correlation of TNF␣ polymor- allelic TNF promoters, that TNF2 is a much stronger tran- phism with production of TNF␣ mRNA and resistance to scriptional activator than the common allele (TNF1) in a development of murine Toxoplasma gondii encephalitis has human B cell line. Footprint analysis using DNase I and B cell also been demonstrated in the BALB͞c strain (13). A study of nuclear extract showed the generation of a hypersensitive site Th2 cell-mediated local inflammatory responses has shown at ؊308 and an adjacent area of protection. There was no that the TNF dependence of this phenomenon is related to difference in affinity of the DNA-binding (s) between H2D haplotypes and corresponding TNF␣ production pheno- the two alleles. These results show that this polymorphism has types, suggesting, at least in mice, that differential expression direct effects on TNF␣ gene regulation and may be responsible of TNF␣ from distinct alleles may influence the nature of an for the association of TNF2 with high TNF␣ phenotype and immune response (14). more severe disease in infections such as malaria and leish- TNF has potent biological actions, and control of its pro- maniasis. duction is tightly regulated, occurring both at the transcrip- tional and posttranscriptional levels (15). In response to lipo- Tumor necrosis factor ␣ (TNF␣) is a potent cytokine with a polysaccharide stimulation of macrophages, TNF transcription wide range of proinflammatory activities (1). It is classically increases 3-fold, TNF mRNA increases 50- to 100-fold, and produced by monocytes͞macrophages, although other cell protein secretion increases by a factor of Ϸ10,000-fold (16). types, such as T and B cells, also produce significant amounts. Sequences within the 1100-bp stretch of DNA between the 3Ј The TNFA gene lies in the class III region of the major end of the alpha gene and the 5Ј end of TNFA histocompatibility complex (MHC), Ϸ250 kilobases centro- have been shown to be central to the control of transcription meric of the HLA-B and 850 kilobases telomeric of (17, 18). HLA-DR. In view of its biological effects and gene location it Recently we and others have described two polymorphisms has been speculated that polymorphism within this locus might in the human TNFA promoter at Ϫ308 (19) and Ϫ238 (20), contribute to MHC associations with autoimmune and infec- both involving the substitution of guanine by adenosine in the tious diseases (2), particularly those in which TNF␣ has been uncommon alleles. We showed that the rare allele at Ϫ308 implicated in initiating or sustaining the inflammatory re- (TNF2) is part of an extended MHC haplotype HLA-A1-B8- sponse, such as rheumatoid arthritis (3), or where increasing DR3-DQ2 (21), which is associated with high TNF␣ produc- blood levels have been shown to be predictive of poorer tion (5, 6). Studies in large populations have indicated that outcome, such as malaria (4). This has been supported by the carriage of TNF2 is associated with a worse outcome in association of specific MHC haplotypes with different TNF␣ phenotypes: DR3 and DR4 haplotypes produce higher levels cerebral malaria (22) and in leishmaniasis (23). of TNF␣ (5, 6) while DR2 haplotypes are associated with low To test whether the Ϫ308 polymorphism has a functional production (5, 7), suggesting that functional polymorphism significance, we have investigated its effects on transcription might exist within regions that regulate the TNFA gene. using reporter gene assays. Our results show that the TNF2 Studies in mice have implicated the TNF locus with disease allele is a much more powerful transcriptional activator than phenotype. Polymorphisms have been described in the pro- the common allele. Although we can demonstrate specific moter region (8), the first intron, and 3Ј untranslated region (9) binding of a nuclear protein and DNase I hypersensitivity at the polymorphic site, there was no obvious difference in

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: TNF␣, tumor necrosis factor ␣; TNFA, gene for TNF␣; accordance with 18 U.S.C. §1734 solely to indicate this fact. MHC, major histocompatibility complex; SLE, systemic lupus ery- thematosus; CAT, chloramphenicol acetyltransferase; EMSA, elec- Copyright ᭧ 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA trophoretic mobility-shift assay; IL, ; PMA, phorbol 12- 0027-8424͞97͞943195-5$2.00͞0 myristate 13-acetate. PNAS is available online at http:͞͞www.pnas.org. §To whom reprint requests should be addressed.

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affinity of the protein(s) for the two alleles. These results Extraction of DNA-Binding from Raji Cells. Nu- indicate that this polymorphism may have a direct effect on clear proteins were extracted from Raji cells as described (27). transcriptional activity and may underlie the association of the Prior to use, the protein concentration was determined. HLA-A1-B8-DR3 haplotype with high TNF␣ production and Generation of Radiolabeled TNFA Promoter Fragment. A be directly responsible for the poorer outcome reported in 119-bp fragment (Ϫ345 to Ϫ226) of the TNF␣ promoter was malaria and leishmaniasis with carriage of the TNF2 allele. amplified by PCR from TNF1 and TNF2 homozygous indi- viduals using primers 5Ј-CAAAAGAAATGGAGGCAAT-3Ј and 5Ј-TCCTCCCTGCTCCGATTCCG-3Ј. The two allelic MATERIALS AND METHODS fragments were then cloned into the TA vector, and the Generation and Cloning of TNF␣ Promoter Fragments. A sequences were confirmed as above. Probe was prepared by fragment of 691 bp (Ϫ585 to ϩ106) of the TNFA gene was restriction digestion using NsiI and HindIII, [␣-32P]dCTP amplified by PCR using primers 5Ј-GCTTGTCCCTGCTAC- end-labeled using the Klenow fragment (Promega), and then CCGC-3Ј and 5Ј-GTCAGGGGATGTGGCGTCT-3Ј, and cy- purified with a Sephadex G-50 NICK column (Pharmacia). cles as described (19). The fragments were cloned into the TA The specific activity of the probe was measured with a vector and used to transform Escherichia coli (strain INVFЈ␣) ␤-counter. (Invitrogen, United States Biochemical). Following selection Electrophoretic Mobility Shift Assay (EMSA). Nuclear pro- and propagation, pure plasmid DNA was prepared by standard tein extract (10 ␮g) was incubated with 2 ␮g of poly(dI-dC) methods (24). The TNF promoter alleles were removed from (Pharmacia) in 25 ␮l of buffer composed of 10 mM Tris⅐HCl the TA vector by restriction enzyme digestion with HindIII and (pH 7.5), 75 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, XbaI (Promega) to allow for directional cloning into the 12.5% glycerol, and 0.1% (vol͞vol) Triton X-100 at room pBLCAT3 expression vector (25). The sequences of the inserts temperature for 30 min. After the addition of 2.5 ng of labeled were checked by the dideoxy chain termination method using probe and incubation for a further 30 min, 2.5 ␮l of loading Sequenase (United States Biochemical). buffer consisting of 250 mM Tris⅐HCl (pH 7.5), 0.2% bromo- Transfection of Human B Cells. Experiments were per- phenol blue, 0.2% xylene cyanol, and 40% glycerol was added formed using the human Raji B cell line. Cells were cultured and the samples were electrophoresed in a 0.25 ϫ TBE (90 mM in 1ϫ RPMI 1640 medium adjusted to pH 7.4 with 1 M NaOH, Tris͞64.6 mM boric acid͞2.5 mM EDTA, pH 8.3)͞4% non- buffered with 7.5% (vol͞vol) sodium bicarbonate, and supple- denaturing polyacrylamide gel. Visualization of bands was by autoradiography. Quantification of competitor DNA concen- mented with 5% (vol͞vol) fetal calf serum (GIBCO͞BRL), trations was by measurement of optical density at 260 nm. This penicillin (100 units per ml), streptomycin (100 ␮g͞ml), and glutamine (2 mM) (Northumbria Biologicals, Northumber- was checked by comparing band intensities following agarose land, England). Cultures were incubated at 37ЊC in a humid- gel electrophoresis and ethidium bromide staining. DNase I Footprint. Initially 80 ng of Raji nuclear extract was ified 5% CO ͞95% air atmosphere. Raji cells, which were 2 incubated at 37ЊC for 20 min in 50 ␮l of buffer containing 25 maintained in rapid growth phase by change of medium and mM Hepes (pH 7.8), 50 mM KCl, 0.05 mM EDTA, 0.5 mM passaged 1:10 every 3 days, were centrifuged at 400 ϫ g for 5 DTT, 0.5 mM phenylmethylsulfonyl fluoride, 5% glycerol, and min. The cells were washed once with medium, centrifuged, 100 ng poly(dI-dC). Labeled probe (40,000 cpm) was then and then resuspended at a concentration of 1 ϫ 107 cells per added and incubated for a further 20 min. Digestion was ml of medium; 800 ␮l of this was used for each transfection. In performed at 0ЊC with 0.01 units of DNase I (Boehringer each experiment three different plasmids were transfected: (i) Mannheim) for 1 min following the addition of 1 mM CaCl2 pBLCAT3, (ii) TNF1-pBLCAT3, and (iii) TNF2-pBLCAT3. and 5 mM MgCl . The reactions were then terminated with 100 Each time 80 ␮g of DNA was used. Electroporation was 2 ␮l stop solution consisting of 0.375% (wt͞vol) SDS, 15 mM performed with a single pulse from a gene pulser apparatus EDTA, 100 mM NaCl, and 100 mM Tris⅐HCl (pH 7.6). The (Bio-Rad) with a capacitance extender unit at settings of 300 products were then incubated at 37ЊC for 15 min with 0.18 mg V and 960 ␮Fd. Cells were incubated at room temperature for proteinase K and 10 ␮g of tRNA in a final volume of 163 ␮l, 10 min before and after electroporation. The cell suspension and phenol͞chloroform extracted and ethanol precipitated. At was added to 19.2 ml of medium and incubated for 24 hr. Each the same time a Maxam–Gilbert guanidine ladder was gener- cell culture was then split in half and again resuspended in 20 ated as described (28). The fragments were separated on a 6% ml of medium. Phorbol 12-myristate 13-acetate (PMA) (Sig- denaturing polyacrylamide gel. The gel was then dried and ma) was added to one culture from each duplicate to a final visualized by autoradiography. concentration of 50 ng͞ml and the cultures incubated for a further 48 hr. The cells were then harvested by centrifugation, and the pellets were resuspended in 0.25 M Tris⅐HCl (pH 8.0) RESULTS and stored at Ϫ70ЊC. The cell suspensions were subjected to Induction of CAT Protein by TNFA Promoter Fragments. three episodes of rapid freezing͞thawing to obtain lysates. Raji cells were transfected with three plasmids: a negative Quantification and Normalization of Chloramphenicol control consisting of the pBLCAT3 vector alone and plasmids Acetyltransferase (CAT) Expression. Measurement of CAT containing either of the two TNF allelic promoter fragments. protein production in transfected cells was performed using a The experiments were performed four times using DNA from commercially available enzyme-linked immunosorbant assay different plasmid preparations. Efficiency of transfection was (Boehringer Mannheim). The lower detection limit was 100 assessed by Southern blot analysis of the CAT gene and pg͞ml CAT. Quantification of protein concentrations in cel- quantified by ␤-counting. Total protein was also measured in lular extracts was determined using a modification of the cellular lysates. The CAT protein concentration was corrected micro-Lowry technique (protein assay kit; Sigma). using these results to minimize differences due to transfection To determine transfection efficiencies, a dot-blotting pro- efficiency and cell numbers and was expressed in picograms of cedure was followed (26) using a ␤-counter (Tri-Carb 260-DU, CAT protein per milligram of total protein. The results of this Packard). The probe used was isolated from pBLCAT3 by experiment are shown in Fig. 1. As expected, the negative digesting the plasmid with EcoRI. The fragment, Ϸ1500 bp in control produced very low levels of CAT protein in both the length, spanning the CAT cDNA, was randomly labeled with unstimulated and PMA-stimulated cells. There was a signifi- [␣-32P]dCTP [3000 Ci͞mmol (1 Ci ϭ 37 GBq); Amersham] cantly higher production of CAT from the TNF2-CAT plasmid using a T7 Quickprime kit according to the manufacturer’s compared with TNF1-CAT in both the unstimulated and instructions (Pharmacia). stimulated cells. The TNF1-CAT plasmid showed no evidence Downloaded by guest on September 30, 2021 Immunology: Wilson et al. Proc. Natl. Acad. Sci. USA 94 (1997) 3197

FIG. 2. Specific binding of the TNF promoter fragment by a nuclear protein. Nuclear extract (10 ␮g) from Raji cells was incubated with each of the two TNF promoter fragments with (lanes 3–5 and 8–10) or without (lanes 1, 2, 6, 7) 100-fold molar excess of unlabeled probe. The gel retardation complex is indicated by “Protein͞DNA complex”. Lanes 4 and 9, competition with unlabeled IL-1 DNA fragment did not disrupt the complex.

for a disease association. This is the most polymorphic region of the genome and contains many genes that encode proteins FIG. 1. Induction of CAT protein from reporter gene constructs that are involved in inflammatory and immune responses. transfected into Raji cells. Raji cells (0.8 ϫ 107) were transfected with Another important feature is the strong linkage disequilibrium the pBLCAT3 vector containing either no insert, which served as a between alleles across the MHC. Thus, for example, the negative control, or 691 bp of each allelic promoter. After 24 hr the haplotype HLA-A1-B8-DR3-DQ2 occurs much more fre- cells were split in two, and one flask of each duplicate was stimulated quently than the product of the individual allelic frequencies with PMA (50 ng͞ml). After a further 48 hr incubation the cells were would suggest. Therefore, the association of MHC haplotypes harvested. Results have been corrected for transfection efficiency by with TNF␣ phenotypes might not be due to polymorphism Southern blot analysis of CAT DNA and also for total cell numbers by measuring total protein. The experiments were performed four times within the TNF gene itself, but rather to variation in a linked and means and standard errors are shown. gene that regulates expression of this cytokine. A possible example is the recent cloning of an IkB-like gene within 90 of inducible production of CAT. Although there appeared to kilobases of the TNF locus (29). There are at least three NFkB be some inducibility of the TNF2-CAT plasmid by PMA, this consensus sequences within the TNF␣ promoter and these was not significant. This result was replicated four times with have each been demonstrated specifically to bind a B cell different batches of Raji cells. nuclear protein (17). Therefore, it is important to show a direct EMSA of TNF␣ Fragments. To investigate protein–DNA functional effect of any polymorphism, because the association interactions in the vicinity of the polymorphism we performed with a disease may be entirely due to linkage disequilibrium EMSAs using extract from Raji cells and a 119-bp TNF with the true etiological gene. fragment. The results of this experiment are shown in Fig. 2. Our results demonstrate that the polymorphism at Ϫ308 has Binding of at least one protein is demonstrated with both a significant effect on transcriptional activity in reporter gene allelic fragments (lanes 2 and 7). The specific nature of binding assays and that this could explain the association between the is demonstrated by the disappearance of both DNA–protein high TNF␣ phenotype and the DR3 haplotype. The molecular complexes using competition with a 100-fold excess of unla- mechanism of this difference is not completely clear because beled TNF1 probe (lanes 3 and 8) or TNF2 probe (lanes 5 and there was no evidence of a major difference in affinity of the 10), but not when a similar-sized fragment of the interleukin DNA-binding protein(s) to the two allelic forms of the TNFA 1A (IL-1A) promoter was used (lanes 4 and 9). promoter, at least in Raji cells. This should have shown up in DNase I Footprint of TNF␣ Promoter Region Fragment. the competition experiment if based on protein–DNA inter- The exact site of DNA–protein interaction around the poly- actions. Perhaps as a result of difference in the DNA͞ morphic site was defined in a footprint assay. A hypersensitive chromatin structure at the polymorphic site, the interaction of site was induced at Ϫ308 with an adjacent protected region transcription factors is enhanced leading to stronger transac- (Fig. 3). No other evidence of interaction was seen. tivation of the TNF␣ gene. Our results demonstrating a lack of Competitive EMSA. To examine whether the difference in inducibility of both TNF␣ promoter allelic fragments in human transcriptional activity was due to a difference in affinity of the B cells are in keeping with a previous study which demon- two alleles for the binding protein, a competition EMSA was strated that the minimum promoter fragment required for performed using labeled TNF1 and increasing excesses of cold PMA responsiveness in the 729–6 B cell line extended to oligonucleotides. There was no evidence of a major difference Ϫ1105 bp with a high basal activity and poor inducibility in affinity between the two alleles (Fig. 4). compared with a T cell and monocytic cell line (30), and with the finding of high constitutive levels of TNF␣ mRNA in Raji DISCUSSION cells (18). Although the polymorphic site lies in a consensus sequence for AP2 we found no evidence, using recombinant Several features make it difficult to determine whether a human protein in a gel retardation assay, of AP2 binding to the particular DNA variant within the MHC is directly responsible polymorphic site (data not shown). Interestingly, a homolo- Downloaded by guest on September 30, 2021 3198 Immunology: Wilson et al. Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 4. Competitive EMSA using allelic TNF promoter fragments and Raji nuclear extracts. Labeled TNF2 was used. Lanes: 1 and 7, no competitor; lanes 2–6 and 7–12, increasing excess of unlabeled TNF2 and TNF1, respectively, as competitor. No difference in affinity for nuclear proteins between TNF alleles was apparent.

disease (38). In humans with malaria, the highest levels of TNF are seen in fatal cases of cerebral malaria (4). A study of TNF genotypes in West African malaria patients has shown that homozygosity for TNF2 is associated with a 7-fold increased risk of death or severe neurological complications due to cerebral malaria (22). The TNF2 allele may therefore be responsible for the lower incidence of SLE in Africa as a consequence of endemic malaria causing higher levels of TNF production, while the absence of this stimulant of TNF pro- duction in the United States allows for the increased incidence of lupus seen in Afro-Americans. Despite the adverse effects of homozygosity in malaria, TNF2 is maintained at similar levels in West African and Northern European populations, suggesting that compensatory pressures in Africa exist to maintain the allele. Perhaps it has beneficial effects in other major infectious diseases, such as measles, meningococcal FIG. 3. Detection of a hypersensitive site at Ϫ308. In vitro DNase I footprint analysis of TNF1 allele (coding strand) is shown. The disease, , or tuberculosis. There may also be heterozy- unfilled arrowhead indicates an area of protection, and the filled gous advantages. arrowhead a DNase I hypersensitive site. Lanes: 1, Maxam–Gilbert The associations of HLA-DR4 and -DR2 with high and low guanidine ladder; 2, naked DNA control; 3, plus Raji cell nuclear TNF␣ production, respectively, has not been explained. It extract. seems reasonable to speculate that polymorphism, which may exist in other regulatory regions of the TNFA gene or in linked gous sequence in the TNFA promoter (Ϫ254 to Ϫ230) has genes, plays a role in TNF␣ production. The most important been shown to bind a transcriptional repressor and not AP2 region in the regulation of seems to be the 3Ј (31). It may be, therefore, that a novel protein binds to the untranslated region, which contains a tandem repeat of an polymorphic TNFA Ϫ308 site. A number of other groups have octameric sequence, TTATTTAT (39). The corresponding studied the effects of this polymorphism on gene expression. UA-rich sequence in mRNA binds an inducible cytoplasmic Results similar to ours have been found in one study in both factor (40), resulting in mRNA instability and translational Jurkat cells or U937 cells with evidence of binding of a protein blockade (41). It may be that DNA variants in this region are only to the TNF2 allele (32). Two other groups have been in linkage disequilibrium with HLA-DR4 or -DR2. Microsat- unable to demonstrate a difference in transcriptional activity ellite alleles in the TNF locus can be used to subdivide DR4 between the promoter alleles. This may be because of differ- haplotypes into high and low TNF phenotypes, and this has ences in cell types, stimulants, and reporter gene constructs suggested that functionally important DNA variants do indeed used (33, 34). For example, we found that TNFA fragments exist within, or close to, this locus. TNF␣ is believed to be one shorter than those described here were inactive in reporter of the key mediators of the chronic inflammatory response gene assays (data not shown). However, a recent study of seen in rheumatoid arthritis (42). Severe forms of this disease TNF␣ production from peripheral blood mononuclear cells are associated with homozygosity of the DRB1*0401 allele stimulated with anti-CD3 and anti-CD28 has shown a higher (43). It will therefore be interesting to see if this subtype of TNF␣ production phenotype in TNF2 carriers, and of two DR4 is associated with a high TNF␣ phenotype. TNF haplotypes, differing only at Ϫ308, the TNF2 ϩve A study of the Ϫ238 TNFA promoter polymorphism has haplotype produced significantly more TNF␣ (35). There is, demonstrated strong linkage disequilibrium of the rare allele therefore, evidence that the TNF2 genotype is associated with with two extended MHC haplotypes: B18-DR3 and B57-DR7. increased TNF␣ productions in vitro. However, no genotype–phenotype association could be seen, An interesting observation is the low incidence of SLE in and the high TNF␣ production of the B18-DR3 haplotype areas of West Africa in which malaria is endemic (36), in could not be further differentiated by typing this variant (35), contrast to the high incidence in Afro-American populations suggesting that it does not have a direct effect on gene who are mostly of West African descent (37). Furthermore expression. Polymorphisms may of course also exist in the infection of the (NZB ϫ NZW)F1 mouse with Plasmodium coding region of the gene, as has been found in the nearby Berghei leads to protection from the spontaneous lupus-like gene (44). With regard to Ϫ308 TNFA2, Downloaded by guest on September 30, 2021 Immunology: Wilson et al. Proc. Natl. Acad. Sci. USA 94 (1997) 3199

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