Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: J Immunol. 2011 Jul 27;187(5):2794–2802. doi: 10.4049/jimmunol.0902569

A SOCS-1 Promoter Variant is Associated with Total Serum IgE Levels1

Justin Mostecki *,2, Suzanne L Cassel †,2, Walter T Klimecki ‡, Debra A Stern ‡, Judit Knisz †, Sachiyo Iwashita †, Penelope Graves ‡, Rachel L Miller §, Maartje van Peer *, Marilyn Halonen ‡, Fernando D Martinez ‡, Donata Vercelli ‡, Paul B Rothman †
PMCID: PMC3159751  NIHMSID: NIHMS307019  PMID: 21795592

Abstract

SOCS-1 is a critical regulator of multiple signaling pathways, including those activated by cytokines that regulate immunoglobulin heavy chain class switching to IgE. Analysis of mice with mutations in the SOCS-1 gene demonstrated that IgE levels increase with loss of SOCS-1 alleles. This suggested that overall SOCS-1 acts as an inhibitor of IgE expression in vivo. A genetic association study was performed in 474 children enrolled in the Tucson Children’s Respiratory Study to determine if genetic variation in the SOCS-1 locus correlates with altered levels of IgE. Carriers of the C-allele for a novel, 3′ genomic single nucleotide polymorphism (SNP) in the SOCS-1 gene (SOCS1+1125G>C; rs33932899) were found to have significantly lower levels of serum IgE as compared with homozygotes for the G-allele. Analysis demonstrated that the SOCS1+1125G>C SNP was in complete linkage disequilibrium with a SNP at position SOCS1−820G>T (rs33977706) of the SOCS-1 promoter. Carriers of the T-allele at the SOCS1−820G>T were also found to be associated with. The promoter SNP increased transcriptional activity of the SOCS-1 promoter in reporter assays and human B cells. Consistent with this observation, the presence of this polymorphism within the promoter abolished binding of Yin-Yang-1, which is identified as a negative regulator of SOCS-1 transcriptional activity. These data suggest that genetic variation in the SOCS-1 promoter may affect IgE production.

Introduction

Asthma and other allergic diseases are increasingly prevalent in both developing and westernized nations (1). The immunological hallmark of allergic diseases is elevated total and allergen specific IgE levels along with inflammation. The immune response underlying the inflammation found in allergic states is extremely complex, involving interplay between cytokines, chemokines and cells within the innate and adaptive immune system. Current models invoke activation of T helper (Th) 2-type memory/effector CD4+ T cells by allergen as a major initiator of allergic inflammation (reviewed in (2)). The ensuing production of the Th2 cytokines IL-4, IL-5 and IL-13 by activated CD4+ T cells promotes the migration and activation of inflammatory cell types such as basophils, eosinophils and mast cells within the affected tissue. These cells in turn release soluble factors that promote and exacerbate the inflammatory state. In addition to cells within the immune system, IL-4 and IL-13 also interact with receptors expressed on non-hematopoietic cells, thus contributing to allergic inflammation by inducing functional responses in these populations. In addition to elevated levels of Th2 cytokines, atopic individuals can exhibit increased “base line” IgE levels and IgE-reactive cells even prior to an allergic reaction. As with allergic inflammation, IL-4 and IL-13 are inducers of class switching to IgE by B cells (3). Conversely, IFN-γ can suppress class switching to IgE (4, 5).

One of the essential properties of cytokines is their limited duration of action. This property leads to the effective curtailment of immune responses once an antigen or allergen is removed from the responding organ. Recent studies have demonstrated that cytokine signaling is limited by several mechanisms. A family of proteins, termed Suppressors of Cytokines Signaling (SOCS), appears to be essential for the normal control of cytokine action in vivo. The eight members of the SOCS gene family are characterized by a C-terminal SOCS-Box region and a central SH2 domain (6). SOCS-1, also called JAK binding protein (JAB)-1 or STAT-induced STAT inhibitor (SSI)-1, is an important regulator of many cytokine signaling pathways including those activated by cytokines that regulate IgE class switching (7–9). The SOCS-Box region is important for the recruitment of the ubiquitin transferase apparatus that targets SOCS-1-interacting proteins for proteasome-mediated degradation (10, 11). SOCS-1 has been shown to interact with all four JAK kinases through its SH2 domain and inhibit their tyrosine kinase activity in vitro (8, 9, 12). Thus, SOCS-1 negatively regulates the action of IL-4, which promotes IgE production, as well as IFN-γ, which suppresses it.

SOCS-1−/− mice, although normal at birth, display stunted growth with a multi-organ disease that is characterized by lymphopenia, fatty acid degeneration of the liver and macrophage infiltration of various tissues, followed by death prior to three weeks of age (13). Lethality can be significantly delayed on the RAG2−/−, IFN-γ−/−, STAT1−/− and STAT6−/− backgrounds, thus implicating SOCS-1 as a critical regulator of both the IFN-γ and IL-4 signaling pathways (14, 15). Many of the phenotypes associated with SOCS-1 deficiency can be reconstituted by the transfer of SOCS-1−/− bone marrow into JAK3−/− mice, suggesting that the pathology is mediated by hematopoietic cells (15). However, specific deletion of SOCS-1 in the thymocyte/T/NKT cell compartment is not sufficient to result in lethal multiorgan disease, although it leads to abnormalities including both elevated levels of CD8+ T cells and increased sensitivity to common γ-chain cytokines (16). Consistent with this observation, SOCS-1−/− dendritic cells induced increased IFN-γ- and IL-4-mediated responses, suggesting an important role for SOCS-1 in non-lymphoid cell function as well (17). Furthermore, altered transcriptional activity of SOCS-1 may have important ramifications for the cytokine unresponsiveness demonstrated by many tumors, as the gene has been shown to be silenced by CpG methylation in hepatocellular carcinoma (18) and in multiple myeloma (19).

Cytokines and their downstream signaling pathways are critical regulators of the immune response. Human genetic studies have demonstrated that polymorphisms affecting genes encoding cytokines or components of cytokine signaling pathways are strongly associated with allergic diseases phenotypes (reviewed in (20)). One of the most replicated findings is the linkage of atopy with polymorphisms within the human chromosome 5q31–33, which contains genes encoding the cytokines IL-4, IL-5, and IL-13 (21–23). In addition, polymorphisms in the signaling molecule STAT6, that is activated downstream of IL-4 and IL-13, have also been associated with an increased risk of asthma (24–28). Considered together with functional studies establishing that IL-4 and IL-13 are central mediators of allergic inflammation, these data argue that the IL-4 and IL-13 signaling pathways have a critical and perhaps predominant role in atopic disease development. Our analysis showed that the loss of one copy of SOCS-1 results in increased total and antigen-specific IgE production in mice. This suggests that alteration in the SOCS-1 locus may alter IgE levels in human as well. Indeed, our study identified a single nucleotide polymorphism (SNP) within the SOCS-1 locus (SOCS1+1125G>C, rs33932899) that is associated with lower levels of total serum IgE. An additional SNP (SOCS1−820G>T, rs33977706) is in strong linkage disequilibrium with the SOCS1+1125G>C SNP, also associated with lower levels of IgE and increases the transcriptional activity of the SOCS-1 promoter in reporter assays and in human CD19+ cells. Further, the SOCS1−820T SNP decreases the ability of YY1, a negative regulator of SOCS-1 transcriptional activity, to bind SOCS-1 promoter sequences in vitro. These data suggest that genetic variation in the SOCS-1 locus may affect IgE levels in humans through loss of negative regulation of SOCS-1 with resultant increased SOCS-1 expression blocking the IL-4 mediated class switch to IgE.

Materials and Methods

Tucson Children’s Respiratory Subjects

Anonymous DNA samples from healthy individuals of self-reported ancestry were obtained from the Coriell Cell Repositories (Camden, New Jersey). Twenty-three samples from individuals of European ancestry (EA) and 24 samples from individuals of African ancestry (AA) were studied. Repository Numbers: African American DNA: NA17101, NA17102, NA17103, NA17104, NA17105, NA17106, NA17107, NA17108, NA17109, NA17110, NA17111, NA17112, NA17113, NA17114, NA17115, NA17116, NA17133, NA17134, NA17135, NA17136, NA17137, NA17138, NA17139 and NA17140. CEPH DNA: 12560, NA12547, NA10845, NA10853, NA10860, NA10830, NA10842, NA10851, NA07349, NA10857, NA10858, NA10848, NA12548, NA10844, NA10854, NA10861, NA10831, NA10843, NA10850, NA07348, NA10852, NA06990 and NA07019.

The characteristics of the children enrolled in the Tucson Children’s Respiratory Study, comparisons of participants with and without available DNA samples, and methods used to assess total serum IgE levels have been described in detail elsewhere (46).

Tucson Children’s Respiratory Subjects PCR

Genomic sequences for SOCS-1 were accessed from the USC Human Genome Browser, November 2002 freeze. PCR amplicons were designed to completely traverse the gene, such that each amplicon was approximately 900 base-pairs (bp) in length, and consecutive amplicons overlapped each other by approximately 200 bp. PCR reactions contained 20 ng genomic DNA, 1 pmole of each primer, 0.2 U Platinum Taq polymerase (Invitrogen) and 0.1 μM dNTPs in a total volume of 10 μl. Specific reaction conditions, including primer sequences are available from the authors.

Tucson Children’s Respiratory Subjects Direct PCR Sequencing

PCR amplicons were prepared for cycle sequencing by diluting them with water using a dilution range of 1:3 to 1:6, depending on the reaction yield as determined by agarose gel electrophoresis. Cycle sequencing reactions were assembled using 0.4 μl of cycle sequencing premix (BigDye V3.0), 1 pmol sequencing primer, 1.8 μl 5X sequencing dilution buffer and 5 μl PCR product in a final volume of 10 μl. Cycle sequencing reactions were purified using DNA-affinity magnetic beads (Agencourt Biosciences). Purified sequencing reactions were electrophoretically analyzed using a DNA Analyzer 3730 (Applied Biosystems).

Tucson Children’s Respiratory Subjects Polymorphism Identification and Analysis

Sequence chromatograms were processed for base calling and assembly using the Phred, Phrap and Consed suite of software programs (47, 48). Initial polymorphism tagging was performed using Polyphred, with a minimum sequence quality of phred 25 (49). Potential polymorphic sites that were initially identified by Polyphred were individually confirmed by visual inspection of sequence traces. A criterion of this visual inspection-confirmation was that the polymorphism must be observed in multiple chromatograms from singleton polymorphisms (polymorphisms occurring in only one subject), or in multiple subjects. For these confirmed polymorphic sites, each genotype for each subject was also confirmed by visual inspection of chromatograms. Polymorphic sites and associated subject-identified genotypes were automatically output to a relational database for further analysis, which included the automated generation of ethnicity-specific genotype frequencies, allele frequencies, and goodness of fit tests for Hardy-Weinberg equilibrium. Haplotypes were inferred using a Gibbs-sampling algorithm as implemented in the Phase software program (50). Because the accuracy of statistically inferred haplotypes has been shown to increase with increasing haplotype frequency, we used polymorphisms with a minor allele frequency (MAF) ≥0.10 in order to define relatively common haplotypes (51). Pair-wise linkage disequilibrium was calculated as r2, a measure of the product-moment correlation coefficient (52).

Tucson Children’s Respiratory Subjects Gene Context of Polymorphisms

Each gene was annotated graphically using the Artemis software program (53). Annotations were based on mapping cDNA accession number AB000734 to the human genomic sequence. This produced a gene structure for SOCS1 containing two exons, the first of which is untranslated, followed by a 552 bp intron. The translation initiation site is 49 bp from the 5′ aspect of exon 2.

Human B cells

Whole blood samples from anonymous human donors were obtained from DeGowin Blood Center, University of Iowa Hospitals and Clinics. Samples were diluted with an equal volume of sterile PBS and mononuclear cells isolated using Histopaque 1077 (Sigma) following the manufacturer’s instructions. CD19+ cells were positively selected using CD19 microbeads (Miltenyi Biotech) following the manufacturers instructions. Cells were cultured at 4 × 106 cells / ml for 24 hours after which cells were harvested and genomic DNA and RNA isolated.

Human B cells Real-time PCR

RNA was isolated from cultured CD19+ cells using TRI®zol (Invitrogen) following the manufacturer’s protocol. mRNA was transcribed to cDNA using Superscript III 1st Strand Synthesis Supermix (Invitrogen) following the manufacturer’s protocol. Real time PCR was performed with SYBR® Green and SOCS1 primers (Forward 5′ GAA CTG CTT TTT CGC CCT TA 3′ Reverse 5′ CTC GAA GAG GCA GTC GAA G 3′), and β-Actin primers (Forward 5′ GGC ATC CTC ACC CTG AAG TA 3′ Reverse 5′ AGG TGT GGT GCC AGA TTT TC 3′) according to the manufacturer’s instructions (Applied Biosystems). PCR was performed using Applied Biosystems Model 7000 and analyzed with Applied Biosystems Sequence detection software version 1.2.3.

Human B cells SNP genotyping

Genomic DNA was isolated from human CD19+ cells using Qiagen DNA Blood mini kit (Qiagen) according to the manufacturer’s protocol. Genotype at the −820 SNP was determined using TaqMan® SNP Genotyping Assays ID C__60261090_10 and C___3189845_20 according to the manufacturer’s protocol. End point analysis was performed using Applied Biosystems Model 7000 and analyzed with Applied Biosystems Sequence detection software version 1.2.3.

Plasmids

Regions of the SOCS-1 promoter were cloned into the BglII and MluI sites of the pGL3 Basic Vector (Promega) using primers with the appropriate restriction sites. The common sequence was cloned from the NA12547 DNA sample and the sequence containing a T at the −820 position was cloned from the NA17135 DNA sample. All plasmids were confirmed by sequencing. The pRL-TK Renilla luciferase plasmid was purchased from Promega.

Cell Lines and Transfection

The RAW/FPR.10 clone derived from the RAW 264.7 murine macrophage cell line was obtained from Dr. Steve Greenberg (54). Transient transfections were performed using the Lipofectamine reagent (Invitrogen) as per the manufacturer’s instructions with 1ug of reporter plasmid and 100ng of Renilla luciferase control plasmid (Promega) for 4 hours. Following this period, the transfection mixture was removed and replaced with media ± 100 ng/ml LPS for 20 hours. Cell extracts were subsequently prepared and assayed using the Dual Luciferase Kit (Promega) as per the manufacturer’s instructions.

Mice

The B6.129S7-Ifngtm1Ts/J mice were purchased from Jackson Laboratories and the SOCS-1−/− mice were provided by Dr. James Ihle.

Assay of Serum Immunoglobulin Levels and allergen-specific responses

Total serum Ig levels were assessed by ELISA using the anti-Ig antibodies (BD Biosciences) per the manufacturer’s protocol. Antigen-specific responses were assessed by injecting mice i.p. with 50 μg OVA and 2 mg alum at 4 weeks of age and measuring OVA-specific IgE serum levels at 6 weeks of age. Levels of OVA-specific IgE were determined by ELISA, pre-coating plates with 50 μg/ml OVA instead of the anti-IgE capture antibody. An anti-OVA IgE monoclonal antibody standard (2C6) (55), was provided by Dr. Lester Kobzik.

Electrophoretic Mobility Shift Assay (EMSA)

Preparation of nuclear extracts has been described previously (56). Binding reactions were performed for 15 minutes at RTº in 20μl buffer containing 5 mM HEPES pH 7.9, 20% glycerol, 2.5 mM MgCl2, 2.5 mM DTT, 500 ng dIdC and 40 μg BSA. Competitions and supershift experiments included a 15 minute RTº incubation step with unlabeled competitor oligonucleotides (10, 50, and 100 fold molar excess) or antibody (0.8μg), respectively, prior to the addition of the labeled probe. Supershift analyses were performed using anti-YY1 monoclonal antibody (Active Motif). Probe sequences are as follows: SOCS-1−820G (−827/−813): 5′-gatcAAAAGTGGAGCTGGG-3′ & 5′-gatcCCCAGCTCCACTTTT-3′; SOCS-1−820T (−827/−813): 5′-gatcAAAAGTGTAGCTGGG-3′ & 5′-gatcCCCAGCTACACTTTT-3′. The polymorphic −820 position is underlined.

Results

IgE Levels Increase as Levels of SOCS-1 Decrease in Mice

Given the importance of SOCS-1 in regulating both IL-4 and IFN-γ signaling, it is difficult to predict how alterations in SOCS-1 expression may influence IgE levels. To determine the importance of SOCS-1 in regulating immunoglobulin (Ig) production, two animal model systems (SOCS-1+/+ vs. SOCS-1+/− and IFN-γ−/−, SOCS-1+/+ vs. IFN-γ−/−, SOCS-1−/− mice) were compared. A comparison between SOCS-1+/+ and SOCS-1−/− mice was not performed because the SOCS-1−/− mice suffer from a severe multi-organ pathology due most significantly to a failure to properly regulate IFN-γ signaling. Instead, we examined the levels of immunoglobulins in SOCS-1+/− mice that do not demonstrate any of these severe pathologies (13, 29). In this effort, we were encouraged by a previous study showing elevated IgE production in SOCS-1+/−, but not SOCS-1+/+ control mice after challenge with N. brasiliensis, a pathogen that elicits Th2 responses (30). This suggests that the lack of one copy of SOCS-1 can alter antibody responses in vivo. We examined levels of IgM, IgG2a, IgG1, and IgE in WT and SOCS-1+/− mice. Figure 1A shows that basal levels of IgE were 2.5-fold higher (P=0.023) among SOCS-1+/− mice than in SOCS-1+/+ littermates. In contrast, the levels of the other Ig isotypes were not different. These data demonstrate an important role for SOCS-1 in the regulation of basal IgE levels. In order to examine mice that lack both copies of SOCS-1, SOCS-1-deficient mice were crossed onto the IFN-γ−/− background (129SV) and immunoglobulin production was assessed (Figure 1B). Consistent with the findings discussed above, basal levels of serum IgE were 11.5-fold higher among IFN-γ−/−SOCS-1−/− mice when compared to the IFN-γ−/−SOCS-1+/+ littermate controls (P=0.002). The IgE levels in the IFN-γ−/−SOCS-1+/− littermates were intermediate and significantly different from both IFN-γ−/−, SOCS-1+/+ (P=0.001) and IFN-γ−/−, SOCS-1−/− mice (P=0.007), corroborating the importance of SOCS-1 gene dosage in IgE regulation. No other significant alterations in immunoglobulin levels were observed for IgM, IgG1 or IgG2a in these mice, implying that the effects of SOCS-1 dysregulation were specific to IgE (Figure 1A & B).

Figure 1. Elevated IgE levels in mice lacking SOCS-1.

Figure 1

Open in a new tab

(a) The total baseline unstimulated IgM, IgE, and IgG1 serum concentrations in four week old SOCS-1+/− mice (n=7) and SOCS-1+/+ littermate controls (n=6) were assessed by ELISA. (b) The total baseline unstimulated Ig serum concentrations were compared in 6 week old IFN-γ−/−, SOCS-1+/+ (n=5), IFN-γ−/−, SOCS-1+/− (n=7) and IFN-γ−/−, SOCS-1−/− (n=5) littermate mice by ELISA. The total baseline unstimulated IgE serum concentrations were compared in IFN-γ−/−, SOCS-1+/+ (n=9), IFN-γ−/−, SOCS-1+/− (n=22) and IFN-γ−/−, SOCS-1−/− (n=6) littermate mice by ELISA. (c) IFN-γ−/−, SOCS-1+/+ (n=10), IFN-γ−/−, SOCS-1+/− (n=26) and IFN-γ−/−, SOCS-1−/− (n=6) littermate mice were injected at four weeks of age with OVA (50 μg) /alum (2 mg) and OVA-specific IgE serum levels were determined at 14 days post-injection by ELISA. Each shape indicates the concentration from 1 mouse with the mean noted by a line. The statistical significance (P) was determined using a student’s t test.

To determine whether SOCS-1 is important for the regulation of antigen-specific IgE responses, serum levels of ovalbumin (OVA)-specific IgE were assessed two weeks after immunization with a single OVA-alum i.p.-injection (Figure 1C). The IFN-γ−/−, SOCS-1+/− mice demonstrated a 2.1-fold increase in mean OVA-specific IgE levels in comparison to IFN-γ−/−, SOCS-1+/+ controls (P=0.029), confirming that SOCS-1 is an important regulator of antigen-specific IgE responses in vivo. The IFN-γ−/−, SOCS-1−/− mice showed an even higher (4.1-fold) increase in mean anti-OVA IgE relative to the IFN-γ−/−, SOCS-1+/+ mice (P=0.045), whereas OVA-specific IgE levels in IFN-γ−/−, SOCS-1+/− were not significantly different. Collectively, these data demonstrate that SOCS-1 is an important regulator of IgE levels in vivo, and deletion of a single SOCS-1 allele is sufficient to significantly alter both basal and antigen-specific IgE responses.

Genetic Variants and Haplotypic Structure in the Human SOCS-1 Locus

Our results from the SOCS-1 deficient mice prompted us to assess whether variants within the SOCS-1 locus might be associated with altered IgE levels. To this purpose, we started by analyzing genetic variation in the SOCS-1 genomic locus. Sequencing of DNA samples of both African (AA) and European ancestry (EA) identified 21 polymorphisms which were observed in the ~3.5 kb region that contains SOCS-1 (Figure 2A). Of these polymorphisms, 20 were observed in African ancestry samples and 9 in European ancestry samples (Figure 2B). Although one polymorphism was unique to Europeans, and 12 SNPs were observed only in African ancestry samples, most high frequency polymorphisms were shared by the two ethnic groups. Interestingly, although overall nucleotide variability was high, no polymorphic sites were found within the SOCS-1 coding region. Given the relatively small genomic region re-sequenced in this study, the full extent of linkage disequilibrium (LD) among high frequency polymorphic sites (here arbitrarily defined as having a minor allele frequency in excess of 10%) could not be determined.

Figure 2. The SOCS-1 genomic locus and SNP LD groups.

Figure 2

Open in a new tab

(a) The SOCS-1 genomic locus is shown with exons depicted as boxed regions. The shaded coding region is entirely within the second exon; the translational start and stop codons are illustrated above. The transcriptional start site as defined by the cDNA sequence of SOCS-1 (GenBank Accession Number NM_003745) is denoted with the bent arrow. SNP locations are shown with an ‘X’ and numbered relative to the A(+1) of the translation ATG start codon (GenBank Accession Number DQ086801). (b) SNPs from the sequencing of 23 EA and 24 AA patients are listed with the minor allele frequency shown on the right.

Decreased Levels of Serum IgE in Children with the SOCS1+1125C SNP

Three SNPs (SOCS1−1630C>T, SOCS1−343G>A, and SOCS1+1125G>C), which tagged the three bins most frequent in Europeans, were genotyped in 488 unselected participants in the Tucson Children’s Respiratory Study who had serum IgE levels measured at 11 years of age. Sample sizes varied slightly for each SNP and all four SNPs were in Hardy-Weinberg equilibrium (not shown). There was no association between total serum IgE and SOCS1−1630C>T or SOCS1−343G>A (not shown). In contrast, genotypes for SOCS1+1125 were significantly associated by total serum IgE (ANOVA with 2 degrees of freedom: p=0.035 (Figure 3). No genotype could be obtained in 19 subjects). Inspection of the results showed that carriers of the SOCS1+1125CC and SOCS1+1125CG genotypes had significantly lower total serum IgE levels than SOCS1+1125GG homozygotes, indicating a dominant effect of the C allele, and the corresponding test (CC+CG vs. GG) was highly significant (p=0.009). Results remained significant after adjusting for ethnic background and limiting the analysis to White, non-Hispanic children (p=0.04). There was no significant association between any SOCS-1 polymorphism and specific IgE to common aeroallergens (data not shown).

Figure 3. Decreased levels of IgE in non-selected children with the SOCS1+1125C allele.

Figure 3

Open in a new tab

Total serum IgE levels were measured at 11 years of age in the Tucson Children’s Respiratory Study. IgE levels for the three SOCS1+1125 genotypes are shown along with the number of subjects with each genotype (n). Number of subjects, geometric means (in I.U.), and 95% confidence intervals were: 260, 75.0 (59.7–94.2); 193, 47.7 (36.4–62.5); and 16, 45.4 (15.0–138.0) for the GG, CG, and CC genotypes, respectively. Genotype could not be determined in 19 patients.

A SOCS-1 Promoter with a T at the −820 Position Demonstrates Increased Activity

SOCS1+1125G>C is in near complete LD with SOCS1−820G>T (noted by the arrow in Figure 4), a SNP located within a region of the SOCS-1 gene known to be important for the regulation of SOCS-1 promoter activity. The SOCS1−820G>T region is adjacent to an IRF-binding element (IRF-E) that regulates SOCS-1 promoter in response to LPS (31). This suggested the possibility that an alteration in the ability of LPS to regulate SOCS-1 due to the SOCS1−820T SNP might factor into the lower levels of IgE observed in the population study. Additionally, there is a Sp1-/Sp3-binding GC-Box located next to the SOCS1−820 region (Figure 4). Alignment of the human and murine SOCS-1 promoter demonstrates that both of these sites are conserved (data not shown). The proximity to these two functional elements suggested the possibility that SOCS1−820G>T might influence the transcriptional activity of the SOCS-1 promoter.

Figure 4. Structure of the Human SOCS-1 Promoter Region.

Figure 4

Open in a new tab

The promoter sequence is numbered relative to the translation ATG start codon. The region that is important for the regulation of the SOCS-1 promoter is noted by an arrow. The SOCS1−820G>T SNP is marked by red color.

To confirm the association of the SOCS1−820G>T SNP with IgE levels as implied by its linkage analysis with SOCS1+1125G>C, the genotypes at SOCS1−820 were determined for the patient samples analyzed above and compared with serum IgE levels. As shown in Supplemental Figure 1, significantly lower total serum IgE levels were associated with a dominant effect of the T allele for SOCS1−820. However, the relation was not statistically significant after adjusting for ethnic background (p=0.063) and limiting the analysis to White, non-Hispanic children (p=0.12), though the effect size for both was similar to that seen in the whole population. The decreased significance observed for the SOCS1−820/IgE association likely resulted from still unexplained phenotypic fluctuations within the population. Even though SOCS1+1125G>C and SOCS1−820G>T were in high linkage disequilibrium, a small group of children (n=17) with a geometric mean total serum IgE of 42.4 IU/ml were GG homozygous at SOCS1−820 and heterozygous at SOCS1+1125G>C. IgE levels placed these subjects in the low IgE group for SOCS1+1125, but in the high IgE group for SOCS1−820. Therefore, data from these 17 children appeared to contribute to the decreased significance observed for the relationship between IgE levels and SOCS1−820. Indeed, excluding this group from the analysis, carriers of SOCS1−820 GT and TT had significantly lower total serum IgE levels than SOCS1−820GG homozygotes (p=0.018).

To test the possibility that SOCS1−820G>T could modify the activity of the SOCS-1 promoter, SOCS-1 promoter reporter constructs carrying either a G or T at the −820 position were generated and transiently transfected into the RAW 264.7 macrophage cell line. Previous experimental evidence demonstrated that LPS is capable of inducing endogenous SOCS-1 expression in the RAW 264.7 cell line (32), and SOCS-1 overexpression inhibits LPS signaling in these cells (33). Previous experimental evidence showed that the −882/−659 region of SOCS-1 contains important cis-acting elements within the SOCS-1 promoter and is sufficient to drive maximal basal and LPS-induced transcriptional activity (31). These promoter constructs contain an IRF-E (position −839/−824) and an Egr-1 binding sites (−849/−841) that are required for the full LPS and IFN-γ response of the promoter (31).

In an average of three experiments, the −820T (−882/−659) promoter construct containing the minor −820T allele demonstrated a 2.4-fold increase in transcriptional activity in comparison to the −820G (−882/−659) construct (Figure 5A). Interestingly, the basal promoter activity of the −820T construct was also increased compared to the basal activity of −820G −882/−659 and −945/−659 constructs. Furthermore, a shorter construct (−819/−659) lacking the −820 site exhibited the lowest increase among all constructs tested in both basal and LPS induced transcriptional activity. These data suggest that the SOCS1−820 site has a crucial role in regulating the SOCS1 promoter and the presence of the SOCS1−820T allele in the SOCS-1 promoter increases SOCS-1 transcription.

Figure 5. Definition of Human SOCS-1 Promoter using Luciferase Reporters and the expression of SOCS-1 in human CD19+ cells.

Figure 5

Open in a new tab

(a) The luciferase activities were tested with pGL3 basic promoter constructs containing the −882/−659 (relative to the A (+1) of the ATG start codon) region of the SOCS-1 promoter with either a G or T at the −820 position as indicated. Additionally, a longer construct (−945/−659) containing G at the −820 position and a shorter construct (−819/−659) lacking the −820 site were tested as well for reporter activity. RAW cells were transfected for 4 hours, after which time the transfection solution was removed and replaced with medium for 20 h. Luciferase activities were first normalized to the Renilla transfection control and then subsequently graphed relative to the value of the −882/−659 region. Values are given as the mean of three independent experiments ± SD. (b) Expression of SOCS-1 mRNA in human CD19+ cells by genotype at SOCS1−820. Each point represents a single sample, bars represent mean ± SEM.

To confirm the relevance of these in vitro findings the expression of SOCS-1 in human cells was analyzed. The association of the genotype with modifications in IgE levels suggested the effect of the SNP would be found in B cells. Human CD19+ cells were purified and cultured in vitro. Expression of SOCS-1 was measured via real time PCR and found to be, in accordance with our in vitro promoter analysis, significantly higher in samples heterozygous or homozygous for the −820T SNP (Figure 5B).

The SOCS1−820T Allele Abolishes the Ability of YY1 to Bind to the SOCS-1 Promoter

To determine the trans-acting factor(s) responsible for the altered transcriptional activity demonstrated by the SOCS1−820T promoter, DNA/protein interactions occurring at the SOCS-1 promoter were studied by electrophoretic mobility shift assay (EMSA) using probes containing either a G (Figure 6 lanes 3–15) or a T (Figure 6 lanes 1&2) at the −820 position (Figure 6). A specific complex (complex 1) was observed with thymocyte nuclear extracts and a labeled −827/−813 oligonucleotide carrying the common −820G allele (Figure 6 lane 4). This complex was not observed using the −820T variant of the same probe (Figure 6 lane 2). Cold competition assays demonstrated that 10, 50, and 100 fold molar excess of unlabeled −827/−813 oligonucleotide carrying the −820T sequence failed to compete complex 1, indicating that the G/T substitution is sufficient to abolish binding activity (Figure 6 lanes 8–10). In contrast, a 100 fold molar excess of the −820G competitor was sufficient to virtually abrogate complex 1 formation. (Figure 6 lanes 5–7). These results, combined with the data from reporter assays, suggest that the SOCS1−820G allele of the SOCS-1 promoter binds a transcriptional repressor, while the SOCS1−820T SNP prevents this interaction, thereby increasing SOCS-1 transcriptional activity.

Figure 6. A T at the −820 position abolishes the ability of YY1 to bind the SOCS-1 promoter by EMSA.

Figure 6

Open in a new tab

Nuclear extracts from Balb/c thymocytes were used in EMSA with sequences from the SOCS-1 promoter. Antibodies (0.8 μg) for the supershift assay and excess unlabeled probe for the cold competition were added for a 10 minute RTº incubation period prior to the addition of labeled probe. The −820G (−827/−813) or −820T (−827/−813) labeled probes were incubated for 15 minutes at RTº. The cold competitions included a 10, 50 and 100 fold molar excess of the indicated unlabeled probe. The control unlabeled probe includes sequence from the YY1 binding site in the α-actin promoter.

An analysis of the SOCS-1 promoter sequence surrounding SOCS1−820G>T was performed to determine the identity of the protein(s) present within this complex. The −820/−830 sequence of the SOCS-1 promoter (5′-CCACTTTTGGT-3′) resembled the YY1 consensus binding motif for repressor sites: 5′-CCATNTT/ANNNA/T-3′ (34). Further, the −818/−826 region of SOCS-1 (5′-CTCCACTTT-3′) bears considerable homology with the adeno-associated virus (AAV) P5 initiator sequence (5′-CTCCATTTT-3′) that makes base contacts with the DNA binding domain of YY1 in the co-crystal structure (35). YY1 is a constitutively expressed member of the GLI-Krüppel family of zinc finger transcription factors that has been shown to function as a transcriptional repressor, activator and initiator of various viral and cellular promoters (34, 36). Addition of an anti-YY1 antibody generated a supershift of the −820G sequence-specific complex, identifying the unknown binding factor as YY1 (Figure 6 lane 15). The slower migrating complexes that bind to both the −820T and −820G probes were not affected by the addition of the anti-YY1 antibody. An unlabeled probe that corresponds to the YY1 consensus sequence within the α-actin promoter competed with the formation of this complex, further supporting the identification of YY1 as the factor binding the SOCS-1 promoter (Figure 6 lanes 11–13). This complex was observed with nuclear extracts from thymocytes and monocytic cell lines, consistent with the ubiquitous expression pattern of YY1 (data not shown). Together, these data suggest that SOCS1−820T inhibits the binding of YY1 to the SOCS-1 promoter, thereby altering promoter activity.

A T at the -820 Position Reduces the Ability of Overexpressed YY1 to Repress the SOCS-1 Promoter

The interaction of YY1 with SOCS-1 promoter sequence and the lack of binding to the promoter sequence containing the −820T alteration suggest that YY1 may act as a transcriptional repressor of SOCS-1. Co-transfection of 10 ng of YY1 expression plasmid effectively repressed the −820G (−882/−659) promoter construct that demonstrated only 0.28 relative activity to the empty vector control (1.0) (Figure 7). The −820T construct showed slightly higher levels of basal transcription (1.28) and was not as effectively repressed by YY1 overexpression demonstrating a relative activity of 0.57. These data indicate that the overexpression of YY1 inhibits SOCS-1 promoter activity and that a T at the −820 position reduces the ability of YY1 to inhibit this activity.

Figure 7. Overexpression of YY1 Represses the SOCS-1 Promoter.

Figure 7

Open in a new tab

The −882/+659 constructs with either a G or T at the −820 position were used in luciferase experiments in RAW cells. 10 ng of expression vector encoding YY1 or empty expression vector were transfected along with 500 ng of luciferase and 100 ng Renilla construct per transfection. Luciferase activities were first normalized to the Renilla transfection control and then subsequently graphed relative to the value of the −882/−659 region. Values are given as the mean of 3 independent experiments ± s.d.

Discussion

A genetic predisposition for the increased production of IgE has been established in patients with atopic conditions (37). The importance of the regulation of cytokines that mediate the balance between Th1 and Th2 development made SOCS-1 an attractive candidate gene for study with atopy and asthma. Our studies demonstrate that SOCS-1 is an important regulator of IgE levels in vivo. The loss of one allele of SOCS-1 in SOCS-1-deficient mice increased IgE levels, suggesting that SOCS-1 has important functional roles in maintaining both the normal basal IgE levels and limiting antigen-specific IgE responses. Another group also described polymorphisms in the promoter region of the SOCS-1 gene; although they did not find association between the SNPs and total serum IgE level, they showed significant association between a SNP at position −1478 and adult asthma susceptibility in Japanese population as well as increased SOCS-1 transcription in vitro (38).

The results obtained from mouse studies prompted us to assess whether alterations in the SOCS-1 locus might be associated with altered IgE level in children. We identified 21 single nucleotide polymorphisms in the approximately 3.5 kb genomic region of human chromosome 16 that contains SOCS-1. No association with either the prevalence or severity of asthma was found with the 21 SNPs; however, a significant association was found between lower mean IgE levels and the SOCS1+1125G>C SNP. Moreover, this SNP was found to be in high LD with the SOCS1−269C>T (LD=0.8) and in very high LD with the SOCS1−820G>T SNP.

The SOCS1−820T is located in the promoter region of SOCS-1 and has been identified as a ‘protective’ variant. Characterization of this polymorphism identified YY1 as a transcriptional regulator of SOCS-1. YY1 is a transcription factor that is capable of regulating a plethora of cellular and viral genes as either a transcriptional activator or repressor. YY1 is highly conserved among humans, mice and Xenopus (39); the polycomb group protein pleiohomeotic, the Drosophila homolog of YY1, is necessary for the maintenance of homeotic gene repression (40, 41). YY1 deletion in mice is embryonically lethal due to a peri-implantation defect, consistent with the presumed role of YY1 in the regulation of developmental genes (42). Published evidence has demonstrated that the regulation by YY1 depends on the promoter context that in turn influences the interaction of co-activators and co-repressors with other transcription factors or constituents of the basal transcription machinery. The −820 position in the YY1 binding site of the SOCS-1 promoter corresponds to an important base in the sequence of the AAV P5 initiator sequence that makes contacts with the DNA binding domain of YY1 in the co-crystal structure (35). Based upon this homology, the SOCS1−820T SNP is therefore unlikely to be tolerated and is expected to prevent the interaction of the SOCS-1 promoter with YY1. This prediction was confirmed by a failure of the SOCS1−820T allelic variant to interact with YY1 in vitro. Further, the SOCS1−820T alteration within the YY1 response element suggested a decrease in the ability of YY1 to inhibit the SOCS-1 promoter as indicated by the relative increase in transcriptional activity shown by luciferase experiments. YY1 can also interact with a promoter variant (IL13-1112T) of the IL-13 gene (43). This SNP was shown to be associated with high serum IgE levels and bronchial hyperresponsiveness (44). The T at position −1112 creates an YY1 binding site and results in increased promoter activity in primary human and murine CD4+ Th2 lymphocytes due to an overlap with a STAT motif, thus attenuating STAT6-mediated repression of the IL-13 gene (43). Together with our findings these results point to the importance of YY1 as an important regulator of IgE levels.

The increased transcriptional activity observed with the SOCS1−820T SNP is consistent with the murine data that demonstrated that SOCS-1 has an important in vivo role in limiting IgE levels. Hence, the observed increase in SOCS-1 transcriptional activity suggests a role for the SOCS1−820T variant in the lower levels of IgE observed with patients with the promoter variant. Which pathway is affected by the increased SOCS-1 transcriptional activity, however, remains unclear. SOCS-1 negatively regulates the action of both IL-4 and IFN-γ, thus the lower IgE level observed with the SOCS1−820T variant might suggest that SOCS-1 has a more pronounced effect on the IL-4 pathway. In order to determine if IL-4 regulation was the critical factor, the CD19+ samples described in Figure 5B were also cultured in the presence of IL-4 stimulation, after which mRNA levels of SOCS-1 were measured and compared to the genotype at SOCS1−820. We may have predicted that the samples with the −820T variant would have impaired upregulation of SOCS-1 expression following IL-4 stimulation. This would be consistent with their increased baseline SOCS-1 expression (shown in Figure 5B) serving to block signaling by the IL-4 receptor and hence subsequent upregulation of IL-4 dependent pathways such as SOCS-1. However, our findings were inconclusive as samples showed no significant difference in expression of SOCS-1 following IL-4 stimulation, regardless of genotype at SOCS1−820. There are a number of possible explanations for this finding. First, it may be that the importance of the SOCS1−820T variant is in cells at their resting, not stimulated state. Secondarily, while the decreased levels of IgE found in patients suggests a possible role for B cells in this phenotype, it may be the result of regulation of SOCS-1 expression in other cell types. For example, the increased SOCS1 associated with the TT genotype is likely to also be found in CD4+ T cells. These CD4+ cells may then be less capable of autocrine responses to IL-4, resulting indirectly in decreased IgE production by B cells. IL-13 is another important cytokine that has functions that are separate from as well as overlapping with those of IL-4. Receptors for IL-13 are expressed on B cells and thus it may be the B cell response to IL-13 alone or in combination with IL-4 or other cytokines that finally regulates SOCS-1 expression. In addition, the finding that a mutations in STAT3 are found in patients with hyper-IgE syndrome (45) suggests that other cytokines are also essential in controlling IgE levels in humans. Thus, SOCS-1 may perform a role in regulating all, or some, of these pathways and thereby affect IgE levels and perhaps atopy.

Supplementary Material

1

Abbreviations

SOCS

Suppressor of Cytokine Signaling

SNP

Single Nucleotide Polymorphism

LD

Linkage Disequilibrium

YY1

Ying-Yang-1

AAV

Adeno-Associated Virus

Egr-1

Early growth response-1

Footnotes

1

This work was supported in part by National Institutes of Health Grant R01 AI077516 (P.B.R.) and K08 AI067736 (S.L.C.)

References

  • 1.Braman SS. The global burden of asthma. Chest. 2006;130:4S–12S. doi: 10.1378/chest.130.1_suppl.4S. [DOI] [PubMed] [Google Scholar]
  • 2.Umetsu DT, DeKruyff RH. The regulation of allergy and asthma. Immunol Rev. 2006;212:238–255. doi: 10.1111/j.0105-2896.2006.00413.x. [DOI] [PubMed] [Google Scholar]
  • 3.Coffman RL, Lebman DA, Rothman P. Mechanism and regulation of immunoglobulin isotype switching. Adv Immunol. 1993;54:229–270. doi: 10.1016/s0065-2776(08)60536-2. [DOI] [PubMed] [Google Scholar]
  • 4.Finkelman FD, Holmes J, Katona IM, Urban JF, Jr, Beckmann MP, Park LS, Schooley KA, Coffman RL, Mosmann TR, Paul WE. Lymphokine control of in vivo immunoglobulin isotype selection. Annu Rev Immunol. 1990;8:303–333. doi: 10.1146/annurev.iy.08.040190.001511. [DOI] [PubMed] [Google Scholar]
  • 5.Finkelman FD, I, Katona M, Mosmann TR, Coffman RL. IFN-gamma regulates the isotypes of Ig secreted during in vivo humoral immune responses. J Immunol. 1988;140:1022–1027. [PubMed] [Google Scholar]
  • 6.Hilton DJ, Richardson RT, Alexander WS, Viney EM, Willson TA, Sprigg NS, Starr R, Nicholson SE, Metcalf D, Nicola NA. Twenty proteins containing a C-terminal SOCS box form five structural classes. Proc Natl Acad Sci U S A. 1998;95:114–119. doi: 10.1073/pnas.95.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Starr R, Willson TA, Viney EM, Murray LJ, Rayner JR, Jenkins BJ, Gonda TJ, Alexander WS, Metcalf D, Nicola NA, Hilton DJ. A family of cytokine-inducible inhibitors of signalling. Nature. 1997;387:917–921. doi: 10.1038/43206. [DOI] [PubMed] [Google Scholar]
  • 8.Endo TA, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, Mitsui K, Matsumoto A, Tanimura S, Ohtsubo M, Misawa H, Miyazaki T, Leonor N, Taniguchi T, Fujita T, Kanakura Y, Komiya S, Yoshimura A. A new protein containing an SH2 domain that inhibits JAK kinases. Nature. 1997;387:921–924. doi: 10.1038/43213. [DOI] [PubMed] [Google Scholar]
  • 9.Naka T, Narazaki M, Hirata M, Matsumoto T, Minamoto S, Aono A, Nishimoto N, Kajita T, Taga T, Yoshizaki K, Akira S, Kishimoto T. Structure and function of a new STAT-induced STAT inhibitor. Nature. 1997;387:924–929. doi: 10.1038/43219. [DOI] [PubMed] [Google Scholar]
  • 10.Kamura T, Sato S, Haque D, Liu L, Kaelin WG, Jr, Conaway RC, Conaway JW. The Elongin BC complex interacts with the conserved SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat families. Genes Dev. 1998;12:3872–3881. doi: 10.1101/gad.12.24.3872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ungureanu D, Saharinen P, Junttila I, Hilton DJ, Silvennoinen O. Regulation of Jak2 through the ubiquitin-proteasome pathway involves phosphorylation of Jak2 on Y1007 and interaction with SOCS-1. Mol Cell Biol. 2002;22:3316–3326. doi: 10.1128/MCB.22.10.3316-3326.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Yasukawa H, Misawa H, Sakamoto H, Masuhara M, Sasaki A, Wakioka T, Ohtsuka S, Imaizumi T, Matsuda T, Ihle JN, Yoshimura A. The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop. Embo J. 1999;18:1309–1320. doi: 10.1093/emboj/18.5.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Starr R, Metcalf D, Elefanty AG, Brysha M, Willson TA, Nicola NA, Hilton DJ, Alexander WS. Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1. Proc Natl Acad Sci U S A. 1998;95:14395–14399. doi: 10.1073/pnas.95.24.14395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Naka T, Tsutsui H, Fujimoto M, Kawazoe Y, Kohzaki H, Morita Y, Nakagawa R, Narazaki M, Adachi K, Yoshimoto T, Nakanishi K, Kishimoto T. SOCS-1/SSI-1-deficient NKT cells participate in severe hepatitis through dysregulated crosstalk inhibition of IFN-gamma and IL-4 signaling in vivo. Immunity. 2001;14:535–545. doi: 10.1016/s1074-7613(01)00132-7. [DOI] [PubMed] [Google Scholar]
  • 15.Marine JC, Topham DJ, McKay C, Wang D, Parganas E, Stravopodis D, Yoshimura A, Ihle JN. SOCS1 deficiency causes a lymphocyte-dependent perinatal lethality. Cell. 1999;98:609–616. doi: 10.1016/s0092-8674(00)80048-3. [DOI] [PubMed] [Google Scholar]
  • 16.Chong MM, Cornish AL, Darwiche R, Stanley EG, Purton JF, Godfrey DI, Hilton DJ, Starr R, Alexander WS, Kay TW. Suppressor of cytokine signaling-1 is a critical regulator of interleukin-7-dependent CD8+ T cell differentiation. Immunity. 2003;18:475–487. doi: 10.1016/s1074-7613(03)00078-5. [DOI] [PubMed] [Google Scholar]
  • 17.Hanada T, Yoshida H, Kato S, Tanaka K, Masutani K, Tsukada J, Nomura Y, Mimata H, Kubo M, Yoshimura A. Suppressor of cytokine signaling-1 is essential for suppressing dendritic cell activation and systemic autoimmunity. Immunity. 2003;19:437–450. doi: 10.1016/s1074-7613(03)00240-1. [DOI] [PubMed] [Google Scholar]
  • 18.Yoshikawa H, Matsubara K, Qian GS, Jackson P, Groopman JD, Manning JE, Harris CC, Herman JG. SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet. 2001;28:29–35. doi: 10.1038/ng0501-29. [DOI] [PubMed] [Google Scholar]
  • 19.Galm O, Yoshikawa H, Esteller M, Osieka R, Herman JG. SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma. Blood. 2003;101:2784–2788. doi: 10.1182/blood-2002-06-1735. [DOI] [PubMed] [Google Scholar]
  • 20.Hoffjan S, Nicolae D, Ober C. Association studies for asthma and atopic diseases: a comprehensive review of the literature. Respir Res. 2003;4:14. doi: 10.1186/1465-9921-4-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Beyer K, Nickel R, Freidhoff L, Bjorksten B, Huang SK, Barnes KC, MacDonald S, Forster J, Zepp F, Wahn V, Beaty TH, Marsh DG, Wahn U. Association and linkage of atopic dermatitis with chromosome 13q12–14 and 5q31–33 markers. J Invest Dermatol. 2000;115:906–908. doi: 10.1046/j.1523-1747.2000.00096.x. [DOI] [PubMed] [Google Scholar]
  • 22.Holloway JW, Lonjou C, Beghe B, Peng Q, Gaunt TR, Gomes I, Hall IP, Dewar JC, Wilkinson J, Thomas NS, Holgate ST, Morton NE. Linkage analysis of the 5q31–33 candidate region for asthma in 240 UK families. Genes Immun. 2001;2:20–24. doi: 10.1038/sj.gene.6363723. [DOI] [PubMed] [Google Scholar]
  • 23.Jacobs KB, Burton PR, Iyengar SK, Elston RC, Palmer LJ. Pooling data and linkage analysis in the chromosome 5q candidate region for asthma. Genet Epidemiol. 2001;21(Suppl 1):S103–108. doi: 10.1002/gepi.2001.21.s1.s103. [DOI] [PubMed] [Google Scholar]
  • 24.Gao PS, Mao XQ, Roberts MH, Arinobu Y, Akaiwa M, Enomoto T, Dake Y, Kawai M, Sasaki S, Hamasaki N, Izuhara K, Shirakawa T, Hopkin JM. Variants of STAT6 (signal transducer and activator of transcription 6) in atopic asthma. J Med Genet. 2000;37:380–382. doi: 10.1136/jmg.37.5.380a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Tamura K, Suzuki M, Arakawa H, Tokuyama K, Morikawa A. Linkage and association studies of STAT6 gene polymorphisms and allergic diseases. Int Arch Allergy Immunol. 2003;131:33–38. doi: 10.1159/000070432. [DOI] [PubMed] [Google Scholar]
  • 26.Weidinger S, Klopp N, Wagenpfeil S, Rummler L, Schedel M, Kabesch M, Schafer T, Darsow U, Jakob T, Behrendt H, Wichmann HE, Ring J, Illig T. Association of a STAT 6 haplotype with elevated serum IgE levels in a population based cohort of white adults. J Med Genet. 2004;41:658–663. doi: 10.1136/jmg.2004.020263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Schedel M, Carr D, Klopp N, Woitsch B, Illig T, Stachel D, Schmid I, Fritzsch C, Weiland SK, von Mutius E, Kabesch M. A signal transducer and activator of transcription 6 haplotype influences the regulation of serum IgE levels. J Allergy Clin Immunol. 2004;114:1100–1105. doi: 10.1016/j.jaci.2004.07.048. [DOI] [PubMed] [Google Scholar]
  • 28.Kabesch M, Schedel M, Carr D, Woitsch B, Fritzsch C, Weiland SK, von Mutius E. IL-4/IL-13 pathway genetics strongly influence serum IgE levels and childhood asthma. J Allergy Clin Immunol. 2006;117:269–274. doi: 10.1016/j.jaci.2005.10.024. [DOI] [PubMed] [Google Scholar]
  • 29.Alexander WS, Starr R, Fenner JE, Scott CL, Handman E, Sprigg NS, Corbin JE, Cornish AL, Darwiche R, Owczarek CM, Kay TW, Nicola NA, Hertzog PJ, Metcalf D, Hilton DJ. SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell. 1999;98:597–608. doi: 10.1016/s0092-8674(00)80047-1. [DOI] [PubMed] [Google Scholar]
  • 30.Fujimoto M, Tsutsui H, Yumikura-Futatsugi S, Ueda H, Xingshou O, Abe T, Kawase I, Nakanishi K, Kishimoto T, Naka T. A regulatory role for suppressor of cytokine signaling-1 in T(h) polarization in vivo. Int Immunol. 2002;14:1343–1350. doi: 10.1093/intimm/dxf094. [DOI] [PubMed] [Google Scholar]
  • 31.Mostecki J, Showalter BM, Rothman PB. Early growth response-1 regulates lipopolysaccharide-induced suppressor of cytokine signaling-1 transcription. J Biol Chem. 2005;280:2596–2605. doi: 10.1074/jbc.M408938200. [DOI] [PubMed] [Google Scholar]
  • 32.Nakagawa R, Naka T, Tsutsui H, Fujimoto M, Kimura A, Abe T, Seki E, Sato S, Takeuchi O, Takeda K, Akira S, Yamanishi K, Kawase I, Nakanishi K, Kishimoto T. SOCS-1 participates in negative regulation of LPS responses. Immunity. 2002;17:677–687. doi: 10.1016/s1074-7613(02)00449-1. [DOI] [PubMed] [Google Scholar]
  • 33.Kinjyo I, Hanada T, Inagaki-Ohara K, Mori H, Aki D, Ohishi M, Yoshida H, Kubo M, Yoshimura A. SOCS1/JAB is a negative regulator of LPS-induced macrophage activation. Immunity. 2002;17:583–591. doi: 10.1016/s1074-7613(02)00446-6. [DOI] [PubMed] [Google Scholar]
  • 34.Shrivastava A, Calame K. An analysis of genes regulated by the multi-functional transcriptional regulator Yin Yang-1. Nucleic Acids Res. 1994;22:5151–5155. doi: 10.1093/nar/22.24.5151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Houbaviy HB, Usheva A, Shenk T, Burley SK. Cocrystal structure of YY1 bound to the adeno-associated virus P5 initiator. Proc Natl Acad Sci U S A. 1996;93:13577–13582. doi: 10.1073/pnas.93.24.13577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Thomas MJ, Seto E. Unlocking the mechanisms of transcription factor YY1: are chromatin modifying enzymes the key? Gene. 1999;236:197–208. doi: 10.1016/s0378-1119(99)00261-9. [DOI] [PubMed] [Google Scholar]
  • 37.Ono SJ. Molecular genetics of allergic diseases. Annu Rev Immunol. 2000;18:347–366. doi: 10.1146/annurev.immunol.18.1.347. [DOI] [PubMed] [Google Scholar]
  • 38.Harada M, Nakashima K, Hirota T, Shimizu M, Doi S, Fujita K, Shirakawa T, Enomoto T, Yoshikawa M, Moriyama H, Matsumoto K, Saito H, Suzuki Y, Nakamura Y, Tamari M. Functional polymorphism in the suppressor of cytokine signaling 1 gene associated with adult asthma. Am J Respir Cell Mol Biol. 2007;36:491–496. doi: 10.1165/rcmb.2006-0090OC. [DOI] [PubMed] [Google Scholar]
  • 39.Pisaneschi G, Ceccotti S, Falchetti ML, Fiumicino S, Carnevali F, Beccari E. Characterization of FIII/YY1, a Xenopus laevis conserved zinc-finger protein binding to the first exon of L1 and L14 ribosomal protein genes. Biochem Biophys Res Commun. 1994;205:1236–1242. doi: 10.1006/bbrc.1994.2797. [DOI] [PubMed] [Google Scholar]
  • 40.Brown JL, Mucci D, Whiteley M, Dirksen ML, Kassis JA. The Drosophila Polycomb group gene pleiohomeotic encodes a DNA binding protein with homology to the transcription factor YY1. Mol Cell. 1998;1:1057–1064. doi: 10.1016/s1097-2765(00)80106-9. [DOI] [PubMed] [Google Scholar]
  • 41.Atchison L, Ghias A, Wilkinson F, Bonini N, Atchison ML. Transcription factor YY1 functions as a PcG protein in vivo. Embo J. 2003;22:1347–1358. doi: 10.1093/emboj/cdg124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Donohoe ME, Zhang X, McGinnis L, Biggers J, Li E, Shi Y. Targeted disruption of mouse Yin Yang 1 transcription factor results in peri-implantation lethality. Mol Cell Biol. 1999;19:7237–7244. doi: 10.1128/mcb.19.10.7237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Cameron L, Webster RB, Strempel JM, Kiesler P, Kabesch M, Ramachandran H, Yu L, Stern DA, Graves PE, Lohman IC, Wright AL, Halonen M, Klimecki WT, Vercelli D. Th2 cell-selective enhancement of human IL13 transcription by IL13-1112C>T, a polymorphism associated with allergic inflammation. J Immunol. 2006;177:8633–8642. doi: 10.4049/jimmunol.177.12.8633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Graves PE, Kabesch M, Halonen M, Holberg CJ, Baldini M, Fritzsch C, Weiland SK, Erickson RP, von Mutius E, Martinez FD. A cluster of seven tightly linked polymorphisms in the IL-13 gene is associated with total serum IgE levels in three populations of white children. J Allergy Clin Immunol. 2000;105:506–513. doi: 10.1067/mai.2000.104940. [DOI] [PubMed] [Google Scholar]
  • 45.Minegishi Y, Saito M, Tsuchiya S, Tsuge I, Takada H, Hara T, Kawamura N, Ariga T, Pasic S, Stojkovic O, Metin A, Karasuyama H. Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome. Nature. 2007;448:1058–1062. doi: 10.1038/nature06096. [DOI] [PubMed] [Google Scholar]
  • 46.Graves PE, Siroux V, Guerra S, Klimecki WT, Martinez FD. Association of atopy and eczema with polymorphisms in T-cell immunoglobulin domain and mucin domain-IL-2-inducible T-cell kinase gene cluster in chromosome 5 q 33. J Allergy Clin Immunol. 2005;116:650–656. doi: 10.1016/j.jaci.2005.05.004. [DOI] [PubMed] [Google Scholar]
  • 47.Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998;8:175–185. doi: 10.1101/gr.8.3.175. [DOI] [PubMed] [Google Scholar]
  • 48.Gordon D, Abajian C, Green P. Consed: a graphical tool for sequence finishing. Genome Res. 1998;8:195–202. doi: 10.1101/gr.8.3.195. [DOI] [PubMed] [Google Scholar]
  • 49.Nickerson DA, V, Tobe O, Taylor SL. PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res. 1997;25:2745–2751. doi: 10.1093/nar/25.14.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet. 2001;68:978–989. doi: 10.1086/319501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Tishkoff SA, Pakstis AJ, Ruano G, Kidd KK. The accuracy of statistical methods for estimation of haplotype frequencies: an example from the CD4 locus. Am J Hum Genet. 2000;67:518–522. doi: 10.1086/303000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Devlin B, Risch N. A comparison of linkage disequilibrium measures for fine-scale mapping. Genomics. 1995;29:311–322. doi: 10.1006/geno.1995.9003. [DOI] [PubMed] [Google Scholar]
  • 53.Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B. Artemis: sequence visualization and annotation. Bioinformatics. 2000;16:944–945. doi: 10.1093/bioinformatics/16.10.944. [DOI] [PubMed] [Google Scholar]
  • 54.Cox D, Chang P, Zhang Q, Reddy PG, Bokoch GM, Greenberg S. Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leukocytes. J Exp Med. 1997;186:1487–1494. doi: 10.1084/jem.186.9.1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Hamada K, Suzaki Y, Goldman A, Ning YY, Goldsmith C, Palecanda A, Coull B, Hubeau C, Kobzik L. Allergen-independent maternal transmission of asthma susceptibility. J Immunol. 2003;170:1683–1689. doi: 10.4049/jimmunol.170.4.1683. [DOI] [PubMed] [Google Scholar]
  • 56.Andrews NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991;19:2499. doi: 10.1093/nar/19.9.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS307019-supplement-1.tif (711.3KB, tif)

RESOURCES