Methylation status of a single CpG site in the IL6 promoter is related to IL6 messenger RNA levels and rheumatoid arthritis

Abstract

Objective. Genetic variation in the gene for interleukin-6 (IL-6) contributes to the pathogenesis of inflammatory arthritis, but the role, if any, of epigenetic variability has not been reported. The aims of this study were to compare the DNA methylation status of the IL6 promoter in rheumatoid arthritis (RA) patients and control subjects and to study the effects on gene expression.Methods. Genomic DNA was isolated from peripheral blood mononuclear cells (PBMCs) obtained from RA patients and healthy controls. Macrophages from healthy controls were isolated and stimulated with lipopolysaccharide (LPS). Methylation status was determined using bisulfite genomic sequencing and IL6 messenger RNA (mRNA) levels by quantitative polymerase chain reaction. Gel shift assays were performed with methylated or unmethylated probes and HeLa cell nuclear extract.Results. The proximal CpG motifs (-666 to ؉27) were predominantly unmethylated and the upstream motifs (-1099 to -1001) were highly methylated in PBMCs from patients and controls. Methylation of individual CpG motifs was similar, except at -1099C, which was less methylated in the patients than in the controls (58% versus 98%; P ‫؍‬ 1 ؋ 10 -6 ). To test whether this observation might relate to gene regulation, LPS-stimulated macrophages were grouped according to their IL6 mRNA stimulation index (SI). The level of methylation at -1099C was significantly lower in the group with high (SI >75) compared with the group with low (SI <10) induced mRNA levels (71% versus 93%; P ‫؍‬ 0.007). Gel shift assays revealed decreased protein binding to the -1099C unmethylated probe.Conclusion. These data suggest that methylation of a single CpG in the IL6 promoter region may affect IL6 gene regulation and may play a role in the pathogenesis of RA.

Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis

“…Nile et al [15] found that the differences identified between RA patients and healthy individuals could either be a primary phenomenon, implying that lower methylation at IL6 -1099C is inversely correlated with development of RA or are secondary to the disease process or treatment. It was found in our study that lower methylation of IL10 -145C might lead to similar results.…”

“…Until now, there have been very limited data regarding the epigenetic regulation of genes in RA; only the methylation status of DR3 [14] and IL6 [15] was reported. The histone deacetylase activity in synovial tissues from patients with RA was approximately 2-fold lower than that in synovial tissues from patients with osteoarthritis or from normal controls [33] .…”

“…Some epigenetic clues to RA have been revealed. Global hypomethylation of the T cell genome [13] and methylation of the death receptor 3 gene (DR3) [14] and the IL6 promoter are related to the pathogenesis of RA [15] . Because epigenetic alterations are reversible, they may provide new molecular targets for therapeutic intervention.…”

Inflammatory memories: Is epigenetics the missing link to persistent stromal cell activation in rheumatoid arthritis?

“…Interestingly, also in PBMCs and T cells from RA patients disturbances of DNA methylation could be detected [39]. Particularly, in RA PBMCs loss of methylation in the promoter of the IL-6 and the ephrinB1 gene was found to be connected to increased expression of these genes in RA [40,41].…”

Effects of genetic variation on chromatin structure and the transcriptional machinery: analysis of the IL6 gene locus

“…The in vivo situation may be very different from the in vitro settings, where chromatin folding and epigenetic factors will inevitably have a major role in gene regulation. 14 To increase the understanding of the role of non-coding polymorphism in IL6 gene regulation, we have used a combination of techniques that capture the DNA interactions and chromatin structure in living cells to examine the in vivo effects of IL6 polymorphism.…”