Background Members of the Ikaros family of transcription factors have been implicated in controlling the characteristic phenotype of regulatory T cells (Tregs). Ikaros family zinc finger 4 (IKZF4, Eos) mediates Foxp3-dependent gene silencing and is required to maintain the phenotype of Foxp3-positive Tregs, whereas IKFZ2 (Helios) is involved in the suppression of IL2 gene transcription and upregulation of Foxp3. An important regulation level of tissue-specific gene-expression is DNA methylation. In particular, Treg-specific gene expression is controlled by DNA methylation within characteristic clusters, Treg-specific demethylation regions.
Objectives To analyze the DNA methylation status of Treg-specific demethylated regions of Helios and Eos in T cells from patients with rheumatoid arthritis (RA) and to compare the level of methylation to that in T cells from healthy controls.
Methods In both genes, DNA methylation was assessed by bisulfite sequencing in a CpG island located in an intragenic conserved non-coding sequence (CNS). In addition, a CpG-rich region in exon 6 of the Helios gene was also analyzed.
Results All analyzed regions were completely methylated in effector T cells from RA patients and controls. In contrast, considerable degrees of demethylation were detected in the regions of interest in CD25+CD127- T cells, in line with the hypothesis of an important regulatory mechanism facilitating Treg-specific gene expression. Importantly, the methylation rate of the CNS CpG island in the Helios gene was significantly higher in Tregs from RA patients than in those from controls (55% vs. 40%, p<0.05). Similarly, the CpGs within exon 6 of the Helios gene were demethylated to a significantly different level between RA patients and controls (50% vs. 40% methylation, respectively; p<0.05). Finally, in the CNS CpG island of the Eos gene, the methylation level in Tregs from RA patients was also significantly higher as compared to healthy control Tregs (68% vs. 53%, p<0.01).
Conclusions The data are consistent with the hypothesis of Treg specific expression of Helios and Eos regulated by DNA methylation within Treg-specific demethylation regions. Furthermore, the data suggest that impaired function Tregs in RA might be related to an altered expression of Helios and Eos as a consequence of increased DNA methylation. This mechanism might provide a molecular epigenetic insight into the pathogenesis of autoimmune diseases.
Disclosure of Interest None declared
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