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Abstract

Regulatory T cells (Treg cells) are crucial mediators of peripheral self-tolerance, and their specific ablation causes catastrophic autoimmunity. The protein Foxp3 has been described as the key transcription factor delineating cells of this lineage. In this thesis, we investigate different levels of transcriptional control in Foxp3-expressing Treg cells: At first, we identified CpG rich regions along the Foxp3 gene and investigated them for their epigenetic profile. We were able to validate already-described Treg-specific demethylated regions, but we also identified new Treg-specific demethylation patterns along the Foxp3 gene. These patterns are initiated during thymic Treg development and persist in circulation, and remain intact also in DNA-methyltransferase deficient mice. Next, we explored the epigenetic control of Treg cells on a broader scale. We isolated tissue-resident Treg cells from fat, skin, liver, and lymph nodes and subjected them to whole-genome tagmentation-based methylation analysis in correlation with RNA expression profiling. This enables us to investigate tissue-specific epigenetic patterns that drive and manifest Treg cell adaption to local tissues. Additionally, we observed that the Foxp3 core promoter is completely demethylated also in Foxp3 non-expressing cell types. Therefore, we identified binding partners to the Foxp3 gene promoter in a novel procedure called inverted Chromatin IP. We validated the suppressive nature of these target proteins via luciferase-based screens, tested their differential expression profile in different cell types, and investigated the effect of their virus-mediated overexpression on in-vitro Treg cell induction. Finally, we investigated the role of one specific transcriptional regulator in Treg cells: Rbpj, commonly known as an important co-factor of Notch signaling. While we did not find evidence that Notch signaling was active in bona fide Treg cells, we still measured an upregulation of Rbpj mRNA in Treg cells compared to their conventional T-cell (Tconv) counterparts in many different tissues. Upon Treg-specific Rbpj gene deletion, we observed a steady increase in Treg frequency and number in several anatomical locations, finally leading to autoimmune pathology despite the presence of otherwise functionally-intact Treg cells. We identified the IL7-receptor, an important growth-promoting cytokine receptor, to be specifically upregulated in Rbpj-deficient Treg cells, whereas Dtx-1, a paramount anergy-promoting factor, was strongly downregulated. Furthermore, gene expression profiling and antibody staining revealed that both Treg and Tconv cells gained TH subset lineage profiles, indicating an inability of Treg cells to control (auto) immune responses in-vivo. Our data suggest a novel, Notch-independent function of Rbpj to specifically regulate Treg cell proliferation and functionality.