Regulation und funktionelle Rolle des murinen Transkriptionsfaktors Foxp3 in T-Zellen

Abstract

The aim of the study was to analyze the function and regulation of the transcription factor Foxp3. In a first step we designed a BAC-transgenic mouse with eYFP under the control of the Foxp3 promoter. For creating these mice we use the ETcloning method. The step of homologous recombination of the target vector into the BAC failed. Because of that, we decided to work in cooperation with the group of Tim Sparwasser from Munich and their BACtransgenic mouse called DEREGmouse. This mouse expresses the coding region of eGFP fused to the diphtheriatoxinreceptor under the control of the Foxp3 promoter. Therefore Foxp3 T cells can be easily detected by eGFP expression and can even be depleted by diphtheriatoxinapplication. We confirmed the coexpression of Foxp3 and eGFP and furthermore tested the functionality of the depletionprocess of Foxp3 T cells by treatment with diphtheriatoxin. In a second study, we analyzed the stability of Foxp3 expressing cells in vivo. Therefore we transferred Foxp3 T cells in syngenic mice and analyzed these cells after 14 days for their Foxp3expression. Furthermore, we tested the induction of Foxp3 expression through TGF-beta and the suppressive activity of these cells. We also analyzed those cells for their methylation pattern, comparing cells, which showed an induction of Foxp3expression after one week of culture with TGF-beta to cells, which received TGF-beta for one week and were then restimulated in the absence of TGF-beta. The stability of Foxp3 expression seems to correlate with the demethylated state of the TSDR (Treg Specific Demethylated Region). To get a closer look on the region called TSDR in the murine foxp3 locus, we decided to analyze this region under different aspects. First, we checked for putative binding sites of transcription factors by database analysis of the TSDR. We also analysed histon modifications, such as acetylation of histon H3 and H4 and trimethylation of lysine 4 at histon3, in this region. Presence of these modifications hinted an epigenetic regulation of Foxp3 involving the TSDR. In a last step, the transcriptional activity of TSDR was tested to delineate whether the TSDR serves as an alternative promoter or acts as a regulative element like an enhancer. Luciferase assays showed that TSDR is a regulative enhancer element, which loses transcriptional activity when methylated. Deletion mutants determined the most important fragment of the TSDR. Schlagworter: Transkriptionsfactor Foxp3; regulatory Tcells ; TSDR = Treg specific demethylated region; epigentic control