The single nucleotide variant rs12722489 determines differential estrogen receptor binding and enhancer properties of an IL2RA intronic region

Abstract

We studied functional effect of rs12722489 single nucleotide polymorphism located in the first intron of human IL2RA gene on transcriptional regulation. This polymorphism is associated with multiple autoimmune conditions (rheumatoid arthritis, multiple sclerosis, Crohn's disease, and ulcerative colitis). Analysis in silico suggested significant difference in the affinity of estrogen receptor (ER) binding site between alternative allelic variants, with stronger predicted affinity for the risk (G) allele. Electrophoretic mobility shift assay showed that purified human ERα bound only G variant of a 32-bp genomic sequence containing rs12722489. Chromatin immunoprecipitation demonstrated that endogenous human ERα interacted with rs12722489 genomic region in vivo and DNA pull-down assay confirmed differential allelic binding of amplified 189-bp genomic fragments containing rs12722489 with endogenous human ERα. In a luciferase reporter assay, a kilobase-long genomic segment containing G but not A allele of rs12722489 demonstrated enhancer properties in MT-2 cell line, an HTLV-1 transformed human cell line with a regulatory T cell phenotype.

Fig 1. Purified human ERα selectively binds to the genomic sequence containing G variant of rs12722489.

(A) Position of rs12722489 in IL2RA gene. (B) ERα and β binding motifs displayed as motif logos from HOCOMOCO v10 [16]. Aligned sequence from IL2RA gene containing putative ER binding site around rs12722489 is shown underneath. Motif P-values indicated for alternative alleles refer to ERα. The major (risk) allele is shown in red, the minor (protective) allele—in blue. (C) Sequence of DNA probes used for electrophoretic mobility shift assay (EMSA). Genomic sequences are given by the chromosome (-) strand. ER-binding sites are shown in bold. The variable nucleotide is shown in color. (D) EMSA was performed using human recombinant ERα and indicated radiolabeled probes. ERα-DNA complex can only be seen for the control V1B1 oligonucleotide containing estrogen-response element from Xenopus laevis vitellogenin gene B1 5' flanking region and for the 32 bp genomic sequence containing G variant of rs12722489.

Fig 2. Endogenous ERα binds genomic region containing rs12722489 and binding efficiency depends on the allelic variant.

(A) Chromatin immunoprecipitation was performed in Jurkat and MT-2 cells using antibodies to human ERα. Precipitated DNA was analyzed by real-time PCR using primers specific to a 189-bp genomic sequence containing rs12722489. *p<0.05 comparing to isotype control. **p<0.05 comparing to Jurkat cells. (B) DNA pull-down assay was performed using MT-2 nuclear extract, 189-bp amplicons from human IL2RA gene containing rs12722489 allelic variants, and antibodies to human ERα. *p<0.05 comparing to isotype control. **p<0.05 comparing to the G variant. Data from at least 3 independent experiments are represented as mean±SEM.

Fig 3. Enhancer activity of the 1 kb IL2RA intronic region containing rs12722489 depends on the single nucleotide variant and is estrogen-dependent.

(A) Design of the pGL3-based vectors used for luciferase reporter assay. The constructs contained either G or A variant of the 1kb fragment of IL2RA first intron downstream of the luciferase gene. The control vector (not shown on the figure) contained an irrelevant 1 kb sequence (see Materials and methods section for details). Position of rs12722489 within the 1kb intronic region is indicated. (B) MT-2 or Jurkat cells grown in complete RPMI medium were transiently transfected with the luciferase reporter constructs. *p<0.05 comparing to control or 1kb(A) construct. (C) MT-2 cells transfected with the constructs indicated in the legend were placed in steroid-free medium and 17β-estradiol (E2) was added 24h later at the specified concentrations. Luciferase signal was assessed 18h after E2 addition. *p<0.05 comparing to cells without E2. Data from at least 3 independent experiments are represented as mean±SEM.