Fig. 6

Ctla-4 is a functional target gene of Zfp281, and Zfp281 inhibits the transcriptional activity of Ctla-4 by binding its promoter. a Zfp281 inhibits the luciferase activity of the Ctla-4 promoter. (Left) Schematic of the Ctla-4 promoter with the indicated constructs. (Right) HEK293 cells were infected for 48 h with luciferase reporter plasmids as shown on the left as well as a construct that encodes Zfp281 or the empty vector (Mock), and then, cells were harvested to detect luciferase activity. ChIP analysis of Zfp281 (b), H3K4me3 (c), and H3K27me3 (d) in wild-type and Zfp281-deficient CD4⁺ T cells unstimulated (Top) and stimulated with the anti-CD3 mAb (Bottom), followed by q-PCR detection of the specific enrichment of each modification at the Ctla-4 promoter. e Naive CD4⁺ T cells sorted from WT and cKO mice were stimulated with 1 μg/mL coated anti-CD3 mAb for 24 h and then infected with the shCtla-4 or shNC retrovirus. After 48 h, the cells were restimulated with the anti-CD3 mAb for another 24 h, and the supernatant was collected for IL-2 detection by ELISA. The IL-2 level in the shCtla-4 supernatant was normalized to that in the shNC supernatant. f CTLA-4 blockade assay. Data are representative of three independent experiments (mean ± s.d.). A two-tailed, unpaired t-test was used. NS not significant. *P < 0.05, **P < 0.01