Human Molecular Genetics Advance Access originally published online on April 6, 2006
Human Molecular Genetics 2006 15(9):1475-1481; doi:10.1093/hmg/ddl071
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Context-specific functional effects of IFNGR1 promoter polymorphism
Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK
* To whom correspondence should be addressed at: Kennedy Institute of Rheumatology Division, Imperial College, London W6 8LH, UK. Tel: +44 2083834484; Fax: +44 2083834499; Email: i.udalova{at}imperial.ac.uk
Received February 3, 2006; Accepted March 15, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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We report evidence of a polymorphism in the promoter region of IFNGR1 (encoding interferon-
receptor 1) that has opposite functional effects in different cellular contexts. It is a deletion/insertion polymorphism that is found in Africans but not Europeans or Asians, and has been associated with resistance to severe malaria. We find that the IFNGR1-470del allele acts to suppress binding of nuclear proteins to the IFNGR1 promoter region in a manner that is specific for cell type. In B-lymphocytes, the IFNGR1-470del allele suppresses the binding of a 
35 kDa nuclear protein and acts to increase reporter gene expression. In epithelial cells, the same allele acts to decrease gene expression and suppresses the binding of 90 kDa STAT-1 and STAT-2 proteins. In T-lymphocytes, this allele causes only subtle differences in nuclear protein binding and has no significant effect on gene expression. These findings suggest a mechanism by which a single genetic variant may cause a broad range of phenotypic consequences. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Interferon-
(IFN-) is a key mediator of the host immune response, and the IFN- receptor 1 subunit (encoded by IFNGR1) is essential for IFN- binding and signalling (1
). Rare disruptive mutations in IFNGR1 cause extreme vulnerability to mycobacterial infection and are often fatal (2,3). There is emerging evidence that common IFNGR1 variants may affect susceptibility to infection in the general population, as exemplified by an association of two IFNGR1 promoter polymorphisms with protection against severe malaria in West Africa. Case–control studies of Gambian children with severe malaria showed that in Mandinka, the major Gambian ethnic group, heterozygotes for the IFNGR1-56T/C polymorphism were protected against cerebral malaria and fatal outcome, whereas people who carried the IFNGR1-470ins/del polymorphism were protected against severe malaria in general (4). These polymorphisms have features that suggest a possible role in transcriptional regulation. The sequence around the IFNGR1-56 polymorphism is reminiscent of the activator protein-4 (AP-4) binding site, whereas the sequence around the IFNGR1-470 polymorphisms is similar to the signal transducer and activator of transcription 1 (STAT-1) binding motif. Functional investigations of the IFNGR1 promoter showed that the presence of the IFNGR1-56C allele resulted in lower levels of luciferase reporter gene expression in a B-cell precursor leukaemia cell line (5), but had little effect on promoter activity in myeloid cell lines (6). Here we use B- and T-lymphocytes, monocytes and epithelial cells to examine the functional properties of the IFNGR1-470 and IFNGR1-56 promoter polymorphisms in different cellular contexts. We demonstrate that the IFNGR1-470del allele has a significant effect on both cell-specific nuclear protein binding to the region and the level of gene expression, and has intriguing genetic features which suggest its recent origin in malaria endemic areas.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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The IFNGR1-470 promoter polymorphism is only detected in African population
Previous reports of two malaria-associated variants, IFNGR1-470 and IFNGR1-56, indicated that IFNGR1-56 is polymorphic in both African and Caucasian populations, whereas IFNGR1-470 is found in Africans but not Europeans or Asians (6
). We genotyped the two promoter polymorphisms in 24 unrelated West African and 24 unrelated Caucasian individuals. The IFNGR1-470del allele occurred with the frequency of 10% in Africans, and was not detected in Caucasians. The frequencies of the minor allele IFNGR1-56C were approximately equal in both populations (42% Africans; 33% Caucasians). Consistent with the previously published data (6), this suggests that the IFNGR1-470 polymorphism might have only recently originated in Africa.
Distinct nuclear proteins are recruited to the IFNGR1 promoter region at the 470 polymorphic site
First, we examined the recruitment of nuclear proteins to the DNA sequences that encompassed variant sites IFNGR1-470 and IFNGR1-56 by electrophoretic mobility shift assays (EMSA). We used nuclear extracts from B-cell lines (Raji and RPMI 8226) and the T-cell line Jurkat stimulated with phorbol myristic acetate (PMA) and ionomycin; monocyte cell line MonoMac6, stimulated with lipopolysaccharide (LPS) and lung epithelial cell line A549, stimulated with tumour necrosis factor (TNF). We detected a single nuclear protein complex formed at the IFNGR1-56 site similar to the one observed in Juliger et al. (5), but it was not allele-specific in any of the cells analysed (Fig. 1A).
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In contrast, allele-specific binding was detected for the IFNGR1-470 polymorphism (Fig. 1B). In A549 epithelial cells, a high-molecular weight protein–DNA complex was formed at the probe corresponding to the IFNGR1-470ins allele but not to the IFNGR1-470del allele (complex 1 in Fig. 1B). Stimulation with TNF (Fig. 1B) resulted in a slight increase in complex formation. In Raji, RPMI 8226 and MonoMac6 another low-molecular weight constitutive protein–DNA complex was formed at the IFNGR1-470ins allele but not at the IFNGR1-470del allele (complex 2 in Fig. 1B). In Jurkat cells, multiple complexes were formed at the IFNGR1-470ins allele, including the two complexes detected in A549 and Raji cells. Specificity of differential binding to the IFNGR1-470ins allele was confirmed by competition assays with unlabelled probes corresponding to two variant alleles (data not shown).
Taken together, the binding of two cell-specific protein complexes to the IFNGR1 gene regulatory region is affected by the IFNGR1-470 promoter polymorphism.
IFNGR1-470del has opposite effects on gene expression in different cell types
The effect of altered protein binding on functional activity of the IFNGR1-470del was investigated by gene reporter analysis. Merlin et al. (7) reported that the entire promoter activity of the IFNGR1 gene was encoded in the first 692 nt 5' of the transcription start site. To examine the functional impact of the double deletion at IFNGR1-470, we have generated three luciferase-reporter constructs that span the first –722 nt of the IFNGR1 promoter and carry either the IFNGR1-470ins or the IFNGR1-470del allele. As the IFNGR1-470 polymorphism only occurs on the haplotype that carries the IFNGR1-56C allele, we have also generated the IFNGR1-470del/-56C reporter construct.
In all cells, the presence of the IFNGR1-56C polymorphism caused no effect on the reporter gene activity (Fig. 2). In A549 epithelial cells, expression of a reporter gene driven by IFNGR1-470del alleles was reduced four-fold when compared with the IFNGR1-470ins allele (Fig. 2A). Stimulation of the A549 cells with TNF had little effect on reporter gene expression. In RPMI 8226 B-cells, activity of a reporter gene driven by the IFNGR1-470del allele was increased by 30% when compared with the IFNGR1-470ins allele (Fig. 2B). The effect was statistically significant and observed in both resting cells and cells stimulated with PMA/ionomycin. In Jurkat T-cells, activity of a gene reporter was independent of variants of the promoter and was unaffected by stimulation with PMA/ionomycin (Fig. 2C). However, there was significant variation between the five transfection experiments in Jurkat cells as indicated by the relatively large standard error of the mean for the luciferase activity.
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Thus, modulation of gene expression by the IFNGR1-470del allele was cell type dependent and mirrored the differential recruitment of distinct nuclear factors to the IFNGR1 promoter in different cells.
Protein(s) of low- and high-molecular weight interact with the IFNGR1-470 site in B-cells and epithelial cells, respectively
To determine the molecular weight of the nuclear proteins interacting with the IFNGR1-470ins allele, we separated protein–DNA complexes on EMSA and subjected the gel to UV-light to cross-link protein–DNA complexes. The protein–DNA complexes formed at the IFNGR1-470ins allele were excised, eluted from the gel and run on SDS-PAGE. The low-molecular weight complex 2 detected in Raji cells (Fig. 1B) migrated as 50 kDa species (Fig. 3A). Subtracting the molecular weight of the DNA probe, the molecular weight of the protein bound to IFNGR1-470ins was estimated as 35 kDa. The high-molecular weight complex 1 detected in EMSA with nuclear extract from A549 cells (Fig. 1B) migrated as 105 kDa species (Fig. 3A), corresponding to a protein of 90 kDa. We also observed a minor complex running at 60 kDa, which could correspond to the second protein(s) in the complex with the molecular weight of 45 kDa.
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High-molecular weight complex is also formed at the interferon-stimulated response element (ISRE) binding motif
Sequence analysis of the IFNGR1-470ins allele using the TRANSFAC database indicated that this might be the binding site for proteins of the STAT family (compare TTcctcaAA to the consensus TTN4–6AA), which was disrupted by the deletion of the first two nucleotides. The molecular masses of six currently known members of the STAT family range from 84–113 kDa. STAT1 is a protein of 91 kDa, similar in size to the major nuclear factors interacting with the IFNGR1-470ins allele in A549 cells. We investigated the levels of STAT-1, STAT-2, STAT-3 and STAT-5a/b proteins in the nuclei of A549 cells, in which the formation of the high-molecular weight complex was observed, and compared them with the levels in the nuclei of Raji cells, where the same complex was absent (Fig. 4A). STAT-1 and STAT-2 proteins were significantly more abundant in the nuclei of TNF-activated A549 cells, whereas STAT-3 and STAT-5a/b proteins were detected in the nuclei of both cell types.
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Next, we used binding motifs specific to various STAT homodimers (STAT-1, STAT-3, STAT-4, STAT-5 and STAT5/6), as well as the DNA elements known to bind STAT-containing multimers:
-activated sequence (GAS)/ISRE for STAT-1/STAT-2/interferon regulatory factor-9 (IRF-9) and sis-inducible element (SIE) for STAT-1/STAT-3 (Supplementary Material, Table S2) in competition assay against the IFNGR1-470ins probe. When 100x excess of unlabelled GAS/ISRE probe was used in the binding reaction, it fully competed off the binding of nuclear factors to IFNGR1-470ins allele in A549 cells. A minor competition for binding was also observed with the STAT-5 consensus site, but not with other variants of the STAT binding consensus (Fig. 4B). None of the STAT consensus sites competed with the low-molecular weight complex formed in Raji B-cells (data not shown). Thus, we concluded that the high-molecular complex observed in A549 cells can also be formed at the GAS/ISRE binding motif. Indeed, EMSA analysis of nuclear factor binding to the GAS/ISRE motif in A549 cells revealed three major bands, with the highest of them migrating similarly to the complex formed at the IFNGR1-470ins (Supplementary Material, Fig. S1). An unlabelled IFNGR1-470ins probe efficiently competed for the binding of the highest band (data not shown).
STAT-1 and STAT-2 proteins and IRF-9 interact with the IFNGR1-470 site in epithelial but not B-cells
GAS elements bind STAT-1 homodimers, whereas ISRE elements primarily recruit the interferon-stimulated gene factor 3 (ISGF3), which consists of STAT-1, STAT-2 and IRF-9. We observed high levels of both STAT-1 and STAT-2 proteins in nuclear extracts of stimulated A549 cells (Fig. 4A), but no competition for the complex by the STAT-1 consensus site (Fig. 4B). In addition, the molecular weight of IRF factors range between 45 and 55 kDa, consistent with the minor protein of 45 kDa observed in the complex formed at the IFNGR1-470ins allele in A549 cells. Thus, we speculated that the complex formed at the IFNGR1-470ins allele in epithelial cells may consist of STAT-1, STAT-2 and IRF-9. Of interest, significantly higher concentration of IRF-9 was found in TNF-activated A549 cells compared with Raji cells, whereas another member of the IRF family which is not expected to form multicomponent complexes with STAT proteins, IRF-3, was present in both cells (Fig. 5A).
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To test this hypothesis, we used DNA-binding protein pull-down analysis with a biotin-labelled oligoduplex comprising the IFNGR1-470ins site and attached to streptavidin-coated magnetic beads. We detected binding of STAT-1, STAT-2 and IRF-9 proteins to the site in nuclear extracts from A549 cells, but not from Raji cells (Fig. 5B). As a control of the specificity of the pull-down analysis, we used antibodies to IRF-3, and detected no signal (Fig. 5B).
To estimate the discriminative power of the protein pull-down assays, we also used an oligoduplex comprising the INFGR1-470del site with nuclear extracts from TNF-activated A549 cells and observed a modest decrease in the amount of pulled-down STAT-1 and STAT-2 proteins compared with the IFNGR1-470ins site variant (Fig. 5C). We concluded that compared with the EMSA, the conditions of protein–DNA binding reaction in the pull-down assay resulted in somewhat lower sensitivity of interacting proteins to exact nucleotide compositions of the two IFNGR1-470 site variants, perhaps because of the higher concentrations of DNA molecules in the reaction.
Thus, STAT-1, STAT-2 and IRF-9 proteins can interact with the IFNGR1-470 site in A549 cells but not in Raji cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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The discovery that rare IFNGR1 gene disruptions can cause profound immune deficiency has led to considerable interest in the extent of genetic diversity at this locus, and in the question of whether common IFNGR1 variants affect susceptibility to infection in the general population. Although two promoter polymorphisms IFNGR1-56T/C and IFNGR1-470ins/del have been associated with susceptibility to malaria (4
), thus far there has been limited functional evidence of common regulatory polymorphisms in this gene.
We screened a variety of immune cell types (B-lymphocytes: Raji, RPMI 8226; T-lymphocytes: Jurkat; monocytes: MonoMac6) and epithelial cells (A549) for allele-specific binding of nuclear factors to the two polymorphic sites. In all cell types, we detected a single nuclear protein complex at the IFNGR1-56 site similar to the one observed in Juliger et al. (5), but it was not allele-specific. However, the IFNGR1-470 insertion/deletion polymorphism had a marked effect on the binding of distinct nuclear factors. In B-lymphocytes, the IFNGR1-470del allele suppresses the binding of a 35–40 kDa nuclear protein and acts to increase reporter gene expression by 30%. In epithelial cells, the same allele suppresses the binding of a 90–95 kDa nuclear protein and causes a four-fold reduction in gene expression. In T-lymphocytes, this allele causes only subtle differences in nuclear factor binding and has no significant effect on gene expression. The IFNGR1-470 ins/del polymorphism provides a new example of a functional genetic variant that has opposite effects on gene expression in different cell types.
We identify STAT-1, STAT-2 and IRF-9 proteins as nuclear factors that are likely to contribute to the formation of the high-molecular weight complex at the IFNGR1-470ins allele in epithelial cells. STAT-1/STAT-2 heterodimers are best known for their function in IFN-
/ß-stimulated gene expression (8). However, preformed STAT-1/STAT-2 heterodimeric complexes have also been observed in unstimulated cells (9), and there are some indications that the nuclear translocation of STAT-2 can be triggered by growth factors in epithelial cells (10). STAT-1 also plays a role in resting cells (11), where it is required for efficient constitutive expression of certain genes, including caspases and the low-molecular mass polypeptide 2 (LMP2) (12). Thus, it is possible that STAT-1 and STAT-2 are essential for both constitutive and induced gene expression in A549 epithelial cells. Further investigation is needed to identify the exact composition of the low-molecular weight protein–DNA complexes formed at the IFNGR1-470ins allele in B-cells.
The IFNGR1-470del allele is found in Africans but not in Europeans or in Asian populations that we and others (6) have sampled. In the Gambia, it has been associated with protection against severe malaria (4) and the preliminary analysis of the chromosomal region around the IFNGR1 gene suggests that this allele may be predominately associated with a single haplotype (data not shown). One could speculate that a mechanism for cell-specific modulation of the IFNGR1 expression may have arisen in malaria endemic areas to favour anti-parasite immunity [e.g. by inducing IgG heavy chain switching in B-cells (13)] while reducing the risk of severe complications [e.g. by inhibiting the level of intercellular adhesion molecule-1 expression on endothelium (14,15)].
A growing body of evidence suggests that small nucleotide changes in a promoter may not have obvious and dramatic effects (e.g. loss of gene activity) but may fine tune promoter function, thus resulting in subtle functional differences that may be appreciated only in specific dynamic conditions (16,17). One of the best models for such fine tuning are sequence polymorphisms occurring in transcription factor binding sites that alter the binding of specific transcription factors (18–21). Our finding of a common regulatory polymorphism with opposing effects in different cell types highlights the underlying complexity of gene regulation and illustrates the concept that a single genetic variant may have a broad range of phenotypic consequences.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Genotyping
The IFNGR1-56C/T and IFNGR1-470ins/del polymorphisms were genotyped in 24 unrelated West African and 24 unrelated Caucasian individuals using Sequenom mass-spectrometry genotyping system.
Nuclear extracts and EMSA
Oligonucleotide duplexes corresponding to the IFNGR1-470ins/del alleles, to the IFNGR1-56T/C alleles, or to the GAS/ISRE consensus binding sites were radiolabelled with (-32P) dCTP (Amersham Pharmacia Biotech). Cells were stimulated with either PMA and ionomycin (Raji, Jurkat, RPMI8226), LPS (MonoMac6) or TNF (A549), and nuclear extracts were prepared as described previously (22). For EMSA, binding reaction and electrophoresis were performed essentially as in Udalova et al. (23). Unlabelled oligonucleotide duplexes corresponding to the IFNGR1-470ins site, or to the STAT-1, STAT-3, STAT-4, STAT-5, STAT-5/6, GAS/ISRE and SIE binding motifs (Supplementary Material, Table S1) were used in EMSA competition analysis.
UV crosslinking
The binding reaction was performed with radiolabelled oligoduplex corresponding to the IFNGR1-470 ins allele in which three central dT nucleotides were substituted with bromodeoxyuridine. The EMSA gel was UV-illuminated at 302 nm for 30 min at 4°C and exposed to autoradiography for 2–4 h at the same temperature. The region corresponding to the DNA–protein complex was excised, and the proteins were eluted in 2x SDS buffer (100 mM Tris–Cl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 20% glycerol) at 37°C overnight. The eluted protein complexes were separated on SDS-PAGE gel.
Western blot and protein pull-down
For western blot analysis, 10 µg of nuclear extracts were run on 10% pre-cast SDS-PAGE gel (Invitrogen), transferred to supported PVDF membrane (Hybond-P, Amersham Pharmacia Biotech), and proteins were detected using primary antibodies (Supplementary Material, Table S2), secondary anti-rabbit HRP antibodies (Amersham Pharmacia Biotech) and ECL-PLUS kit (Amersham Pharmacia Biotech). For protein pull-down analysis, 100 pmol of biotin-labelled oligonucleotide duplex comprising either the IFGNR1-470ins or the IFNGR1-470del site were attached to the streptavidin-coated magnetic beads (Dynal) and incubated with 40 µg of nuclear extracts in EMSA binding reaction buffer on ice for 2 h. The protein–DNA complexes were pull-down by a magnet and washed three times in the excess of EMSA binding buffer. The pulled-down proteins were eluted into 1x SDS buffer, separated on SDS-PAGE gel and subjected to western blot analysis as above.
Plasmids, cells and gene-reporter analysis
The human IFNGR1-470ins and IFNGR1-470del/56C promoter constructs were generated by PCR amplification of the first 722 nt of the IFNGR1 promoter with the primers [F: AGC TAG CGA GCA CAA GCG CTG AAG G containing a NheI site; R: AAT AGA TCT CTG CTA CCG ACG GTC GCT, containing a BglII site (underlined)] using genomic DNA from a heterozygote IFNGR1ins/del individual, and subsequent selection by sequencing. The fragments were cloned into NheI/BglII sites of the pGL3-basic luciferase gene-reporter vector (Promega). The IFNGR1-470del/56T construct, that does not exist naturally, was generated by site-directed mutagenesis in the construct IFNGR1-470ins-pGL3 using oligonucleotides bearing -470del nucleotide deletion together with the original F and R primers: -470del F: CTA GCT AAG TCT CAG GCC TCA AAT GAA AAA GC and the primer R; -470del R: GCT TTT TCA TTT GAG GCC TGA GAC TTA GCT AG and the primer F. The two fragments were subsequently used as a template for PCR amplification of the IFNGR1 promoter with the primers F and R, and cloned into NheI/BglII sites of the pGL3-basic vector. All constructs were verified by sequencing.
Except for A549 cells which were cultured in DMEM medium, all cells were cultured in RPMI medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.2 mM L-glutamine (all from Sigma). Transient transfections of luciferase reporter constructs were performed by using Fugene 6 non-liposomal reagent according to the manufacturer's instruction (Roche). After transfection cells were incubated for 24 h prior to activation and another 24 h prior to harvesting, Luciferase and Renilla luciferase activities were measured with the Dual-luciferase kit (Promega) assay using a Turner Designs Luminometer Model 20.
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SUPPLEMENTARY MATERIAL |
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Supplementary Material is available at HMG Online.
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ACKNOWLEDGEMENTS |
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This work was supported by the Medical Research Council, UK. We thank Dr Kirk Rockett (Oxford University), Dr Robin Wait (Imperial College) and Dr Jonathan Dean (Imperial College) for help and valuable suggestions on the manuscript. We also thank all those involved in building the DNA archive used to estimate genotype frequencies, particularly Dr Margaret Pinder and Dr Muminatou Jallow (MRC Unit, The Gambia).
Conflict of Interest statement. None declared.
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