Supplementary Figure 4: Validation of H3K4me3 and H3K27me3 marks at select gene loci.

Splenic WT CD4⁺ and CD8⁺, Tcf7^–/–Lef1^–/– CD8⁺ T cells were sort-purified from the spleens and subjected to ChIP analysis using antibodies to H3K4me3 and H3K27me3 or control IgG. The signals of H3K4me3 and H3K27me3 at the indicated genomic locations were normalized to that obtained from IgG ChIP at the same location to calculate their enriched signals.

It is of note that unlike uniformly increased H3K27Ac signals at the loci of upregulated genes in Tcf7^–/–Lef1^–/– CD8⁺ T cells (compare with Fig. 2e), changes in H3K4me3 and H3K27me3 were more dependent on the gene context. St8sia6 and Fasl showed clear increase in H3K4me3 and decrease in H3K27me3 in Tcf7^–/–Lef1^–/– CD8⁺ T cells compared with WT CD8⁺ T cells. Similar changes were found at the Itgb3 and Prdm1 genes but were less pronounced. On the other hand, at the Cd4 TSS or silencer, both WT and Tcf7^–/–Lef1^–/– CD8⁺ T cells had low levels of H3K4me3 and maintained high levels of H3K27me3 compared with WT CD4⁺ T cells. Consistent with lack of strong derepression of Thpok in Tcf7^–/–Lef1^–/– CD8⁺ T cells, the Thpok gene harbored little H3K4me3 but strong H3K27me3 signals in both WT and Tcf7^–/–Lef1^–/– CD8⁺ T cells. The Cd40lg upstream regulatory region was distal to the TSS or gene body and was used a negative control for H3K4me3 and H3K27me3 signals. Data are pooled results from 2 independent experiments with each sample measured in duplicate.