Figure 9. Spacer and zinc finger 1-2 domains of YY1 are essential for inhibition of T_reg differentiation.

(a) Schematic diagram of YY1 domains. (b–d) HEK293T cells were transfected with Smad3-, Smad4- or Foxp3-expression vector together with various FLAG-tagged, YY1 domain mutant expression vectors. Cell lysates were immunoprecipitated with anti-FLAG. Then, proteins were immunoblotted by an anti-Smad3 (b), anti-Smad4 (c), anti-Foxp3 (d) or anti-FLAG antibody, as indicated. IP, immunoprecipitation; WB, immunoblot. (e) Naïve CD4 T cells were transduced with a retroviral vector containing control, YY1 (full length), YY1 ΔS, YY1 ΔZ or YY1 ΔSZ, and differentiated into T_reg cells for 4 days. Expression of Foxp3 was measured by flow cytometry. Numbers in the plots indicate percentage of Foxp3⁺ cells from GFP⁺ cells. (f) GFP⁺ cells from e were sorted, and total RNA was isolated. Relative amounts of the Foxp3 and T_reg signature genes were measured by qRT–PCR. Experiments were performed three times with similar results. Error bars shown in b and f represent s.d. Statistical differences in e and f were analysed by Student's t-test (n=3). *P<0.05. **P<0.01.