Fig 1.
Molecules involved in FOXP3 induction and stable expression and sequences analyzed.
Enhancer (5’ upstream enhancer, conserved non-coding sequences, CNS1, CNS2 and CNS3 serving as enhancer regions) and promoter region are shown (modified after 36,37,68]. Promoter and enhancer regions are bound by several transcription factors and signals. A restriction in differentiation of nTregs is caused by protein inhibitor of the activated signal transducer and activator of transcription STAT1 which binds to the FOXP3 promoter and recruits DNA methyltransferase [68]. CNS1, an intronic enhancer (enhancer 1) is responsive to Tumor-growth-factor-beta (TGFβ) by Smad2/3 binding sites, close to the NFAT site, essential for differentiation of induced iTregs. CNS2, the T cell receptor (TCR)-responsive enhancer (enhancer 2) contains CpG islands and binding sites for transcription factors, CREB and STAT5. The unstable iTreg phenotype is associated with high methylation of the CNS2 region of Treg-specific demethylated regions (TSDRs). TSDR is a key factor in stability of Tregs [36]. Analyzed CpG regions were located in the 5’ upstream enhancer and the promoter of the FOXP3 gene. Analyzed sequences are shown. The analyzed CpGs are located in the promoter region of FOXP3 which do not contain any CpG island. The FOXP3 enhancer lies in a CpG island. Abbreviations: EKR: extracellular signal regulated kinase, PKA: phosphokinase A, NFAT: Nuclear factor activated T cells, NR4a: Orphan nuclear receptor, RUNX: Runt-related transcription factor, CREB: CAMP responsible element binding protein 1, cRel: Proto-oncogene C-Rel, IL-2: interleukin-2, TCR: T cell receptor.
Table 1.
Demographics.
Table 2.
Primers used for bisulfite pyrosequencing.
Table 3.
Sequences analyzed by bisulfite pyrosequencing.
Table 4.
Methylation of FOXP3 Promoter and Enhancer regions.
Fig 2.
T cell receptor excision circles (TRECs).
T cell receptor excision circles (TRECs) per 1,000 peripheral blood mononuclear cells (PBMCs) were significantly lower including all patients compared to controls (A) and in female patients with panic disorder compared to healthy female controls (B) (Mann-Whitney U test). Relative telomere lengths (RTLs) are shown in patients and compared to controls (C) and in female patients with panic disorder compared to healthy female controls (D) (Mann-Whitney U test). Circles (outliers) and asterisks (extreme values) represent individuals coded with numbers.
Fig 3.
Methylations at specific CpG sites in a representative female patient and a healthy female control person.
Methylations at one specific CpG site in FOXP3 promoter sequence 1 (position 1), and at four independent CpG sites in FOXP3 promoter sequence 2 (positions 1 to 4) of a representative female patient (A, C) and a healthy female control person (B, D) were quantified in a single pyrosequencing run. Position-specific information in the context of the analyzed sequence presents broad-sequence methylation patterns (% methylation). The built-in quality control sites (highlighted in yellow) consisting of cytosines converted to thymines demonstrate full bisulfite conversion of the treated DNA.