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. 2007 Mar 7;104(11):4571–4576. doi: 10.1073/pnas.0700298104

Fig. 4.

Fig. 4.

FOXP3 mediates transcriptional repression via the forkhead domain as part of an ensemble with HDAC7 and TIP60. (A) Schematic representation of GAL4-FOXP3 binding to 5x GAL4-binding sites luciferase reporter construct (pG5Luc) used in luciferase reporter assay. (B) Overexpression of TIP60 promotes FOXP3-mediated transcriptional repression. 293T cells were transfected with the control pBIND empty vector (pBIND), pBIND-FOXP3a, pBIND-FOXP3a and pFLAG-TIP60 or, pBIND-FOXP3qa and the HAT-deficient TIP60 expressing construct (pFLAG-MUT-TIP60), plus pG5Luc luciferase reporter and control MSV-β-Gal as indicated, then analyzed by means of luciferase assay normalized with β-Gal activity. Results are means of three separate experiments with SD. (C) Knockdown of endogenous TIP60 relieves FOXP3-mediated transcriptional repression. 293T cells were transfected with indicated vectors and cell lysates were analyzed by means of luciferase assay normalized with β-Gal activity. Results are means of 3 separated experiments with SD. The knockdown efficiency of TIP60 shRNA was evaluated by Western blotting with Rabbit anti-TIP60, and reprobed with anti-α-tubulin (Inset). (D) A role of FOXP3-TIP60-HDAC7 ensemble in the repression of IL-2 production. Transfected Jurkat E6.1 T cells with vectors as indicated were stimulated respectively with plate-bound TCR Vβ 8.1 plus soluble anti-CD28. IL-2 production in cultured medium was measured with IL-2 ELISA kit (eBioscience). The repression efficiency of the empty vector transfected sample was defined as zero, and the one with 10 μg each of FOXP3a, FOXP3b, TIP60, and HDAC7 plasmids transfected was defined as 100%. The result is the average ± standard error by mean of three independent experiments. (Inset) One representative result of three independent experiments showing the actual amount of IL-2 production after TCR plus CD28 stimulation in Jurkat T cells, which were cotransfected with either 10 μg each of FOXP3a, TIP60 and HDAC7 expressing plasmids, or equal amounts of empty vectors. Transfection of the HAT-deficient TIP60 or HDAC deficient HDAC7 species led to less inhibition of IL2 production.