Figure 5: IL-27-induced permissive chromatin modification in the Tim-3 locus is both NFIL3 and T-bet dependent.

(a) Mouse Tim-3 locus and the location of PCR primers for ChIP-qPCR analysis. The histogram (derived from ECR Browser: http://www.dcode.org) represents CNSs which stand for 70% or greater identity over at least 100 bp stretches of the genomic DNA sequences between human and mouse Tim-3 locus (blue: coding exons, red: intergenic regions; orange: intronic regions; yellow: UTRs). ChIP-PCR primers were marked according to their relative location in the Tim-3 locus. (b,c) Naïve CD4⁺ T cells from C57BL/6 mice were activated by anti-CD3 and anti-CD28 antibodies under Th0 or IL-27-treated Th1 condition. The cells were restimulated on day 5 for 24 h and were subjected to ChIP-qPCR for detection of H3Ac (b) and H3K4me3 (c) enrichment in the Tim-3 locus. (d,e) In a similar experimental setting, H3Ac enrichment in the Tim-3 locus was examined in NFIL3^−/− CD4⁺ T cells and T-bet^−/− CD4⁺ T cells. (f) NFIL3^−/− and WT CD4⁺ T cells were activated by anti-CD3 and anti-CD28 antibodies under Th1 culture condition in the presence of IL-27. Four days after T-cell activation, the cells were subjected to ChIP-qPCR to analyse NFIL3 enrichment in the Tim-3 locus. NFIL3-enriched areas marked with * represent the potential NFIL3-binding sites in both human and mouse homologous sequences, whereas those marked with ♦ represent the potential NFIL3-binding sites only in mouse sequences. (g) Expression plasmids for NFIL3 and T-bet were transiently transfected into 293T cells. Whole-cell lysates were harvested 48 h after transfection for co-IP to detect the interaction between NFIL3 and T-bet. Data are representative of at least two independent experiments with similar results (a–e: mean±s.d.). IP, immunoprecipitation.