Figure 8. YY1 inhibits expression of Foxp3-target genes.

(a) HEK293T cells were transfected with a Foxp3- or YY1-expression vector. Cell lysates were immunoprecipitated with an anti-Foxp3, anti-YY1 or control IgG antibody. Then, proteins were immunoblotted by an anti-Foxp3 or anti-YY1 antibody, as indicated. IP, immunoprecipitation; WB, immunoblot. (b) Transactivation activity of YY1 and Foxp3 on the Foxp3-target genes was measured by transient reporter assay. EL4 cells were transfected with CD25P-, IcosP-, GitrP- or Ctla4P-luciferase reporter construct in combination with Foxp3 or YY1 expression vector. (c,d) Binding of Foxp3 (c) and YY1-Flag (d) to the promoters of the Foxp3-target genes in control or YY1-Flag-expressing T_reg cells was measured by ChIP assay. (e) Binding of YY1 to YY1-binding sites (YBS) in the promoters of the CD25, Gitr and Clta4 genes was detected by EMSA using control or YY1-expressing T_reg nuclear extracts. Competition assay was performed with YY1 consensus sequence (x80). (f) Binding of YY1 to the promoters of the Foxp3-target genes in T_conv or T_reg cells was measured by ChIP assay. Error bars shown in b–e represent s.d. Statistical significance of differences of b–e) were determined by Student's t-test (n=3). *P<0.05. **P<0.01. Data are representative of three independent experiments with similar results.