Background/Purpose:  The expression of Treg specific genes, such as the master transcription factor of Tregs, FoxP3 or the Treg specific surface molecule, glycoprotein A repetitions predominant (GARP) controls the function of regulatory T cells. As Tregs are functionally impaired in rheumatoid arthritis (RA), we have performed an in depth analysis of GARP promoter regulation in healthy controls and in patients with RA.

Methods:  CD25+ CD127- CD4 Tregs and CD25- CD4 T cells were isolated from the peripheral blood of therapy-naive RA patients and of age- and sex-matched healthy volunteers by magnetic cell sorting. Genomic DNA was isolated and processed by bisulfite conversion. DNA regions of interest were amplified by PCR using primers specific for bisulfite converted DNA. The PCR products were cloned and subsequently sequenced to compare methylation between controls and RA patients.

Results:  Two transcripts transcribed from two alternative promoters were identified for the human GARP gene. While neither transcript was expressed in conventional T cells, both transcripts were detected in Tregs. Luciferase reporter assays with the two isolated promoters in transfected primary CD4 Tregs indicated that both transcripts initiated gene transcription upon T cell specific activation (i.e. mAbs to CD3 and CD28). Treg-specific GARP transcription was initiated by synergistic interaction of Foxp3 with NFAT and was underpinned by permissive chromatin remodeling caused by release of the H3K4 demethylase, PLU-1. The transcript initiated from the up-stream P2 promoter was markedly more abundant, suggesting the P2 promoter to be the main promoter of GARP. Surprisingly, the promoter activity of the full length GARP promoter was markedly reduced when compared to the upstream P2 promoter alone. DNA methylation analysis revealed several CpG island in both promoter regions. While the CpG islands of the P2 promoter were fully demethylated in both, effector and regulatory T cells, they were hypomethylated in the downstream P1 promoter only in activated Tregs. Surprisingly, the transcriptional activity of the strong upstream P2 promoter was down-regulated upon demethylation of the weak downstream P1 promoter, suggesting an inhibitory interaction between both promoters within the full-length promoter sequence. Of great interest, demethylation-induced transcriptional attenuation regulated the magnitude of GARP expression in Tregs. As GARP expression is required for Treg function and Tregs are functionally altered in RA, we finally investigated GARP expression in RA Tregs and found significantly reduced expression in RA. This reduced expression strongly correlated with RA disease activity and was caused by hypomethylation of the downstream P1 promoter in RA Tregs, thereby facilitating increased hindrance of P2-induced GARP transcription.

Conclusion: Our findings describe a novel function of alternative promoters in regulating the extent of gene transcription in human T cells. They further show, that in RA, GARP promoter regions are altered with regard to DNA methylation, which affects GARP expression and, thus, Treg function. Altered promoter methylation of GARP might therefore contribute to RA pathogenesis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/hypomethylation-of-an-intragenic-alternative-promoter-contributes-to-impaired-treg-function-in-rheumatoid-arthritis-by-transcriptional-interference-with-expression-of-the-treg-specific-protein-glycop/