A SOCS-1 promoter variant is associated with total serum IgE levels

Abstract

SOCS-1 is a critical regulator of multiple signaling pathways, including those activated by cytokines that regulate Ig H 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 compared with those of homozygotes for the G-allele. Analysis demonstrated that the SOCS1+1125G > C SNP was in complete linkage disequilibrium with an 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 decreased IgE. 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.

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

(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.

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

(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.

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

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.

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

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.

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

(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.

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

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.

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

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.