Background/Purpose: Recently, genome-wide studies identified several Treg-specific genes containing hypomethylated regions that are critical for their expression, so called Treg-specific demethylated regions (TSDR). For example, stable expression of the master transcription factor of Tregs, Foxp3, and expression of the Treg-specific surface receptor, GARP, are regulated by epigenetic modifications of intragenic TSDRs. We hypothesized that impaired Treg function in patients with autoimmune diseases, such as rheumatoid arthritis (RA), might be caused by changes in DNA methylation of Treg-signature gene loci. 

Methods: In the current study, we investigated the methylation status of the TSDRs of the FOXP3 and GARPgenes in RA patients. CD25+ CD127- CD4 Tregs and CD25- CD4 T cells were isolated from the peripheral blood of therapy-naive RA patients as well as of age- and sex-matched healthy volunteers using magnetic cell sorting. Genomic DNA was isolated and processed by bisulfite conversion. The TSDRs were amplified by PCR using primers specific for bisulfite converted DNA. The PCR products were cloned and subsequently sequenced to compare the CpG methylation level and pattern between healthy individuals and RA patients.

Results: No differences in FOXP3 TSDR methylation were found between Tregs from RA patients and controls. In marked contrast, the TSDRs in the intragenic promoter and in the first intron of GARP were surprisingly demethylated to a greater extent in Tregs from RA patients than in Tregs from healthy controls. The higher degree of the demethylation of the GARP gene locus in RA was coincident with lower GARP mRNA expression upon Treg activation and with higher disease activity. As expected, decreased methylation and decreased GARP mRNA expression were reflected by diminished GARP protein expression at the cell surface. In depth molecular analysis revealed that GARP expression in Tregs is tightly regulated by two alternative promoters. Binding of the transcriptional machinery to the demethylated intragenic promoter and the intronic TSDR acts like a roadblock resulting in Treg-specific attenuation of GARP transcription. 

Conclusion: These data indicate that altered methylation in Treg-specific genes, such as GARP, in RA might facilitate impaired Treg function and, thus, contribute to disease pathogenesis.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/methylation-dependent-interference-of-two-promoters-for-the-treg-specific-protein-garp-contributes-to-altered-treg-function-in-rheumatoid-arthritis/