Supplementary Figure 5: Loss of Tcf1 and Lef1 does not cause severe dysregulation of histone acetyltransferase and HDAC genes.

(a-b) The RNA-Seq data from control, Tcf7^–/–, and Tcf7^–/–Lef1^–/– CD8⁺ mature thymocytes were analyzed for expression of known histone acetyltransferase (a) and HDAC (b) genes. Gene expression was quantified by FPKM values from the RNA-Seq analysis. Shown in the heatmap are the log2-transformed expression values.

Note that there was an ~1.5-2 fold increase in Kat2b and Ncoa3 transcripts (a) and an ~1.5 fold decrease in the transcripts of Hdac2, Hdac6, Hdac10, Sirt3 and Sirt4 (b) in Tcf7^–/–Lef1^–/– CD8⁺ T cells, based on FPKM values from the RNA-Seq analysis. These moderate gene expression changes less likely accounted for the substantial increase of acetylation at H3K27 and H3K9 in Tcf7^–/–Lef1^–/– CD8⁺ T cells.

(c) Validated expression of selected HDAC genes. CD8⁺ mature thymocytes were sorted from control and Tcf7^–/–Lef1^–/– mice, and mature CD4⁺ thymocytes were from control mice. The expression of indicated HDAC genes were determined by quantitative RT-PCR. All genes were normalized to Gapba in each sample. The expression of each gene in control CD8⁺ thymocytes was set as 1, and its expression in control CD4⁺ and Tcf7^–/–Lef1^–/– CD8⁺ thymocytes were calculated accordingly. Data are means ± s.d. from 2 independent experiments (n = 4). **, p<0.01; ***, p<0.001 by Student’s t-test.

It is of interest to note that Hdac1 and Hdac2, which are known for critical regulation of CD4⁺ lineage integrity, are expressed at similar levels between mature CD4⁺ and CD8⁺ T cells. On the other hand, CD4⁺ T cells express moderately higher levels of Hdac6 and Hdac10, but lower levels of Hdac7 than CD8⁺ T cells. Consistent with the RNA-Seq results, among the validated HDAC genes, only Hdac7 showed a modestly increased expression in Tcf7^–/–Lef1^–/– CD8⁺ T cells.