Thymocyte development requires a complex orchestration of multiple transcription factors. Ablating either TCF-1 or HEB in CD4+CD8+ thymocytes elicits similar developmental outcomes including increased proliferation, decreased survival, and fewer late Tcra rearrangements. Here, we provide a mechanistic explanation for these similarities by showing that TCF-1 and HEB share ~7,000 DNA-binding sites genome wide and promote chromatin accessibility. The binding of both TCF-1 and HEB was required at these shared sites for epigenetic and transcriptional gene regulation. Binding of TCF-1 and HEB to their conserved motifs in the enhancer regions of genes associated with T cell differentiation promoted their expression. Binding to sites lacking conserved motifs in the promoter regions of cell-cycle-associated genes limited proliferation. TCF-1 displaced nucleosomes, allowing for chromatin accessibility. Importantly, TCF-1 inhibited Notch signaling and consequently protected HEB from Notch-mediated proteasomal degradation. Thus, TCF-1 shifts nucleosomes and safeguards HEB, thereby enabling their cooperation in establishing the epigenetic and transcription profiles of CD4+CD8+ thymocytes.
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Sequencing data supporting the findings of this study have been deposited in the Sequence Read Archive (SRA) database under SRA accession number SRP142342. All other relevant data are available from the corresponding authors upon reasonable request.
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We thank H. Kawamoto (RIKEN Research Center for Allergy and Immunology) for reagents; M. Mandal for help with ATAC–seq; B. Kee, M. Clark, A. Khan, and A. Bendelac for advice; and M. Krishnan for helpful suggestions. This work was supported by National Institutes of Health grants R21AI076720, R01AI108682, and U54CA193419, and an ASH bridge grant (to F.G.), and R01CA160436 (to K.K). Further support came from the Chicago Biomedical Consortium (to F.G.) and UL1TR002003 (to M.M.-C.). A.O.E. was supported by an NIH minority supplement; P.S.M. was supported by Institutional NRSA T32 HL07605 and is currently supported as an LLS Fellow; J.Q. was supported by an AAI Careers in Immunology Fellowship; and M.K.O. is a T32HD007009 recipient.
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Integrated supplementary information
Supplementary Figure 1 WT, Cd4-Cre Tcf7fl/+, and Cd4-Cre Tcf12fl/fl DP thymocytes have comparable fractions of CD71+FSChi cells.
a. Representative CD71 expression versus FSC on gated WT, Cd4-Cre Tcf7fl/+, Cd4-Cre Tcf7fl/– and Cd4-Cre Tcf12fl/fl DP thymocytes. Numbers indicate the percent of cells in each quadrant. b. Cumulative data of CD71+FSChi DP thymocytes in indicated mice (number of biologically independent samples; WT = 9, Cd4-Cre Tcf12fl/fl = 6, Cd4-Cre Tcf7fl/–n = 9, Cd4-Cre Tcf7fl/+n = 3). P-values determined by Ordinary one-way Anova test. Error bars, s.d.
a. K-means clustered heatmap centered on HEB binding sites in WT thymocytes ( ± 1.5 kb) showing enrichment of indicated histone modifications, RNA PolII, and accessibility (ATAC-seq) in DP thymocytes. b. Comparative enrichment histograms of permissive histone modifications (H3Ac, H3K4me1, H3K4me2, H3K4me3 and H3K27ac) and chromatin accessibility (ATAC-seq) at HEB binding sites ( ± 1.5 kb) in K-means clusters shown in a. c. Genomic distribution of HEB peaks in wildtype thymocytes. d. Average expression in DP thymocytes of genes in the indicated groups E = enhancer, P = promoter. Numbers are mean log2 FPKM of means and s.d.. (**** = p ≤ 0.0001, Kruskal-Wallis test). All genes n = 23,360; HEB unbound n = 16,689; HEB bound n = 6,320; HEB p n = 3,519 genes; HEB Active e n = 451 genes; HEB Poised e n = 1255 genes; TCF-1 P + E n = 1,095 genes).
a. ChIP-seq tracks of TCF-1 and HEB co-binding to the promoter of E2f5. b. ChIP-seq tracks of TCF-1 and HEB co-binding to the poised enhancer of Ifng. c. ChIP-seq tracks of TCF-1 and HEB co-binding at the active enhancer of Runx3. d. ChIP-seq tracks of TCF-1 and HEB co-binding at the promoter and enhancer of Rag2. Relative enrichments of H3K4Me1 and H3K4Me3, as well as distance from the transcriptional start site were used in all tracks to identify the promoter, poised enhancer, and active enhancer of the associated gene.
Supplementary Figure 4 TCF-1- and HEB-bound super-clusters overlap at genes involved in T cell development.
a. TCF-1 superclusters representing adjacent TCF-1 binding sites in WT thymocytes within 12.5 kb regions (Homer). Regions were ranked (x-axis) based on the number of sequenced tags (y-axis) per region. The top 271 regions and genes annotated to those regions are shown in red. b. HEB superclusters representing adjacent HEB binding sites in WT thymocytes within 12.5 kb regions (Homer). Regions were ranked (x-axis) based on number of sequenced tags (y-axis) per region. The top 213 regions and genes annotated to these regions are shown in red. c. Overlapping TCF-1 and HEB superclusters and pathways enriched within these clusters (pathways and log10 enrichment values determine by Metascape).
Supplementary Figure 5 TCF-1 and HEB promote chromatin accessibility and affect each other’s DNA binding.
a. Heat map of HEB ChIP-seq enrichment (left) and chromatin accessibility (right) in WT and Cd4-Cre Tcf7fl/– thymocytes ( ± 1.5 kb) around indicated WT HEB binding sites. ATAC-seq was performed in sorted DP thymocytes. b. Comparison of total HEB bound sites in WT and Cd4-Cre Tcf7fl/– thymocytes. c. Histogram representation of HEB enrichment (top) and chromatin accessibility (bottom) around WT HEB binding sites in WT and Cd4-Cre Tcf7fl/– thymocytes. d. Heatmap of TCF-1 ChIP-seq enrichment (left) and chromatin accessibility (right) in WT and Cd4-Cre Tcf12fl/fl thymocytes ( ± 1.5 kb) around indicated WT TCF-1 binding sites. ATAC-seq was performed in sorted DP thymocytes. e. Comparison of total TCF-1 bound sites in WT and Cd4-Cre Tcf12fl/– thymocytes. f. Histogram representation of TCF-1 enrichment (top) and chromatin accessibility (bottom) around WT TCF-1 binding sites in WT and Cd4-Cre Tcf12fl/fl thymocytes.
Supplementary Figure 6 TCF-1 and HEB promote accessibility at sites containing their conserved motifs.
a. Motif enrichment analysis (HOMER) indicating the top 5 enriched motifs (based on p-value, HOMER) in chromatin accessible sites (ATAC-seq) in sorted WT DP thymocytes (51, 452 sites, p≤10e-5). b. Motif enrichment analysis indicating the top 5 enriched motifs in sorted Cd4-Cre Tcf12fl/fl DP thymocytes (42,237 sites, p≤10e-5). c. Motif enrichment analysis indicating the top 5 enriched motifs in chromatin accessible sites in sorted Cd4-Cre Tcf7fl/– DP thymocytes (48,479 sites, p≤10e-5). d. Venn diagram indicating commonly accessible (34,866) and uniquely accessible sites in WT, Cd4-Cre Tcf12fl/fl, and Cd4-Cre Tcf7fl/– DP thymocytes. e. Motif enrichment analysis indicating the top 5 enriched motifs in 7,241 sites only accessible in WT DP thymocytes. f. Motif enrichment analysis indicating the top 5 enriched motifs in 3,485 sites only accessible in Cd4-Cre Tcf12fl/fl DP thymocytes. g. Motif enrichment analysis indicating the top 5 enriched motifs in 4,295 sites only accessible in Cd4-Cre Tcf7fl/– DP thymocytes.
Supplementary Figure 7 TCF-1 is highly enriched at genes associated with Notch signaling and protein ubiquitination.
TCF-1 and HEB binding within core Notch Signaling Cascade genes Dtx1 (a), Lfng (b), and Notch1 (c), that are significantly upregulated in Cd4-Cre Tcf7fl/– DP thymocytes in comparison to WT and Cd4-Cre Tcf12fl/fl DP thymocytes. d. ChIP-seq enrichments of TCF-1, H3K27Ac, H3K4Me1, and chromatin accessibility (ATAC-seq) at TCF-1 binding sites in Notch Signaling/Ubiquitination genes compared to TCF-1-HEB co-bound promoters, poised enhancers, and active enhancers. e. Log2 fold change in chromatin accessibility between WT and Cd4-Cre Tcf7fl/– DP thymocytes at genomic regions described in d.
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Emmanuel, A.O., Arnovitz, S., Haghi, L. et al. TCF-1 and HEB cooperate to establish the epigenetic and transcription profiles of CD4+CD8+ thymocytes. Nat Immunol 19, 1366–1378 (2018). https://doi.org/10.1038/s41590-018-0254-4
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