Figure 4. Direct suppression of SATB1 transcription by Foxp3.

(a) Foxp3 ChIP tiling array data (blue) from human expanded cord-blood T_reg. Data were analyzed with MAT and overlayed to the SATB1 locus to identify binding regions (1–13, p < 10⁻⁵ and FDR < 0.5%). Data are representative of two independent experiments with cells derived from different donors. (b) Schematic representation of Foxp3 binding regions (BR) at the human genomic SATB1 locus identified by in silico prediction within the regions identified in a. (c) ChIP analysis of human expanded cord-blood T_reg cells with a Foxp3-specific antibody and PCR primers specific for Foxp3 binding regions. Relative enrichment of Foxp3 ChIP over input normalized to IgG was calculated. A region −15 kb upstream (−15kb) was used as a negative control. *P < 0.05 (Student’s t-test). Data are representative of three independent experiments (mean and s.d.) with cells derived from different donors. (d) Luciferase assay of FOXP3 binding to the respective binding regions at the SATB1 locus in HEK293T cells, transfected with luciferase constructs containing wild-type (WT) or mutated (Mut) binding regions BR9–14 of the SATB1 locus (with mutation of the Foxp3-binding motifs), together with a FOXP3 encoding expression vector or control vector (Ctrl). *P < 0.05 (Student’s t-test). Data is from one representative experiment of two (mean and s.d. of triplicate wells). Numbers indicate Foxp3-binding motifs within each region. (e) Determination of the K_D-values of Foxp3 binding to a wild-type (WT) or mutated (Mut) Foxp3 binding motif in BR9 and BR10 at the SATB1 locus by filter retention analysis. Data are representative of three independent experiments (mean and s.d. of triplicates).