Extended Data Fig. 9. NR4A1 inhibits recruitment of AP-1 components in CD4⁺ T cells.

a, Top, EMSA analysis of AP-1 binding. Using the AP-1 consensus motif as a probe, DNA binding was performed on nuclear extracts from empty vector or RV-Nr4a1-GFP-transduced CD4⁺ T cells stimulated with PMA/ionomycin (PMA/Iono) for the indicated time periods. Super-shift experiments (lane 3) were conducted in the presence of anti-c-Jun antibody. An OCT-1-binding consensus probe was used as a control. Bottom, western blotting (WB) analysis of c-Fos, c-Jun and lamin B1 in nuclear extract. b, Luciferase reporter assay to measure the effect of NR4A1 on AP-1-and NF-AT-mediated transcription. RV-Nr4a1-GFP or empty vector were co-transfected with AP-1 or NF-AT reporter constructs into pre-activated T cells and treated with PMA and ionomycin for 6 h. Cell samples were lysed and luciferase activity was assessed. c, Naive CD4⁺ T cells were sorted from wild-type and Nr4a1^−/− mice and activated with plated anti-CD3 and anti-CD28 for 36 or 72 h. Activated T cells were collected and subjected to ChIP assay using anti-c-Jun antibody, followed by qPCR analysis. Graphs show ChIP–qPCR measurement of c-Jun enrichment at the Jund locus and Pgpep1 and Naf1 promoter regions. Chr, chromosome. d, Distribution of H3K27ac peaks at the Il2 locus in naive and 6-h-activated wild-type and NR4A1 knockout CD4⁺ T cells, as well as RV-vector-and RV-Nr4a1-transduced CD4⁺ T cells; and distribution of NR4A1 peaks at the Il2 locus in RV-Nr4a1-transduced CD4⁺ T cells. e, ChIP–qPCR measurement of c-Jun enrichment at the Il2 locus in wild-type and Nr4a1^{−/ −} CD4⁺ T cells activated with plated anti-CD3 and anti-CD28 for 36 or 72 h. f, H3K27ac and NR4A1 peaks at the Nr4a1, Bach2 , Nt5e and Samhd1 loci in RV-vector-and RV-Nr4a1-transduced CD4⁺ T cells. g, H3K27ac and NR4A1 peaks at the Hivep3 locus in naive and 6-h-activated wild-type and NR4A1 knockout CD4⁺ T cells, as well as RV-vector-and RV-Nr4a1-transduced CD4 ⁺ T cells. h , H3K27ac and NR4A1 peaks at Pdcd1, Havcr2 and Lag3 loci. For d , f–h, SICER v.1.1 was used to identify significant peaks (FDR of 5%) with input DNA (ChIP– seq) as the control. Detailed information for ChIP–seq samples is provided in Supplementary Table 5. n = 3 (b, c, e); n = 1 (d, f–h ). P values were calculated using a two-sided unpaired Student’s t-test; NS, not significant. Data are representative of two individual experiments and graphs show mean ± s.d.