Global Regulator SATB1 Recruits β-Catenin and Regulates TH2 Differentiation in Wnt-Dependent Manner (original) (raw)
Figure 8
Recruitment of β-catenin and p300 by SATB1 at its binding site in GATA-3 promoter is Wnt-dependent.
(A) SATB1 recruits β-catenin at the SBS within GATA-3 promoter. Naïve CD4+ T cells were differentiated to TH2 subtype by adding IL-4 for 24, 48, and 72 h in the presence or absence of siSATB1 or scrambled RNA (Scr) as described in Materials and Methods. Occupancy of the GATA-3 promoter by SATB1 and β-catenin during TH2 differentiation was monitored by quantitative real-time PCR of the SBS (upper graph) and an upstream non-SBS region (lower graph) using chromatin immunoprecipitated from ex vivo differentiated TH cells as described in Materials and Methods. Data represent relative occupancy of ChIP products as compared to the corresponding IgG controls, after normalizing for the input chromatin. Each error bar depicts standard deviation calculated from triplicates. (B) Naïve CD4+ T cells were differentiated to TH2 subtype by adding IL-4 for 72 h in the presence or absence of the Wnt inhibitor Dkk1 as described in Materials and Methods. Differential occupancy of the GATA-3 promoter by SATB1, β-catenin, and p300 during TH2 differentiation was monitored by quantitative real-time PCR of the SBS (upper graph) and an upstream non-SBS regions (lower graph) using chromatin immunoprecipitated from ex vivo differentiated TH cells. Vehicle (1×PBS) treated cells were used as controls. The occupancy of these three proteins on the two selected regions within GATA-3 promoter was determined in TH0 and TH2 subsets. Data represent relative occupancy of ChIP products as compared to the corresponding IgG controls, after normalizing for the input chromatin. Error bars depict standard deviation calculated from triplicates. Insets depict schematic representation of the upstream 2 kb region of the GATA-3 promoter showing relative positions of the SBS (star) and non-SBS (circle). Arrows depict positions of regions corresponding to which PCR primers were designed.