Deciphering the role of histone modifications in memory and exhausted CD8 T cells

Abstract

Exhausted CD8 T cells (T_EX) arising during chronic infections and cancer have reduced functional capacity and limited fate flexibility that prevents optimal disease control and response to immunotherapies. Compared to memory (T_MEM) cells, T_EX have a unique open chromatin landscape underlying a distinct gene expression program. How T_EX transcriptional and epigenetic landscapes are regulated through histone post-translational modifications (hPTMs) remains unclear. Here, we profiled key activating (H3K27ac and H3K4me3) and repressive (H3K27me3 and H3K9me3) histone modifications in naive CD8 T cells (T_N), T_MEM and T_EX. We identified H3K27ac-associated super-enhancers that distinguish T_N, T_MEM and T_EX, along with key transcription factor networks predicted to regulate these different transcriptional landscapes. Promoters of some key genes were poised in T_N, but activated in T_MEM or T_EX whereas other genes poised in T_N were repressed in T_MEM or T_EX, indicating that both repression and activation of poised genes may enforce these distinct cell states. Moreover, narrow peaks of repressive H3K9me3 were associated with increased gene expression in T_EX, suggesting an atypical role for this modification. These data indicate that beyond chromatin accessibility, hPTMs differentially regulate specific gene expression programs of T_EX compared to T_MEM through both activating and repressive pathways.

Fig. 1

Histone modifications act in concert to regulate gene expression in T_EX and T_MEM. (a) Experimental design. (b) PCA of H3K27ac, H3K4me3, H3K27me3 and H3K9me3 data for T_N, T_MEM and T_EX. For T_MEM and T_EX, n = 4 biological replicates; for T_N, n = 8 biological replicates. (c) Correlation of change in RNA expression between T_MEM and T_EX and change in H3K27ac (top), or H3K27me3 (bottom). R and associated P-value represent Pearson correlation. Each dot represents one gene with one associated peak. (d-e) Genome tracks showing RNA-seq, ATAC-seq and hPTM data. Differentially modified regions between T_MEM and T_EX for each hPTM are highlighted in black boxes. (f) Heatmap of DEGs showing K-mean clusters for all pairwise comparisons between T_N, T_MEM, and T_EX. (g-h) Meta plot (top) and heatmap plot (bottom) of H3K27ac at TSS for DEGs (g) cluster 1 (C1) and (h) cluster 5 (C5). (i-j) Meta plot (top) and heatmap plot (bottom) of H3K27me3 at TSS for DEGs (i) cluster 1 (C1) and (j) cluster 1 (C5). (k-l) Comparison of hPTM patterns between T_MEM and T_EX for (k) genes with increased expression in T_EX compared to T_MEM or (l) genes with increased expression in T_MEM compared to T_EX. Top six most frequent groups for each set of genes plotted.

Fig. 2

Identification of predicted TF binding motifs under H3K27ac identities role for ZEB1 in T_EX. (a) Heatmap of normalized Taiji PageRank scores determined using RNA-seq and H3K27ac data. (b) Correlation plot comparing Taiji PageRank scores from H3K27ac to ATAC-seq data. Axes represent log2 fold change in Taiji PageRank scores between T_EX and T_MEM. (c) Venn diagram comparing number of ZEB1 motifs in regions with increased H3K27ac in T_EX to regions with increased chromatin accessibility (ATAC) in T_EX. (d) Heatmap showing DEGs between T_MEM and T_EX associated with regions with increased H3K27ac in T_EX that contain ZEB1 motifs. (e) Bar graph showing cell type expression of DEGs associated with ZEB1 motifs in regions with H3K27ac enriched in T_EX without concurrent increases in chromatin accessibility. (f) Genome tracks highlighting ZEB1 motifs in regions with increased H3K27ac levels in T_EX without changing chromatin accessibility. Differentially modified regions for H3K27ac and open chromatin are highlighted in boxes under tracks. Predicted ZEB1 binding sites are shown in red. (g) Number of differentially expressed TFs between T_MEM and T_EX, with representative TFs indicated. (h) Comparison of hPTMs between T_EX and T_MEM for TFs with increased expression in T_EX. Top six most frequent groups plotted. (i) Genome tracks showing RNA-seq, ATAC-seq and hPTM data for TFs with increased expression in T_EX. Differentially modified regions for each modification are highlighted in black bars.

Fig. 3

Super enhancers drive transcriptional phenotype of T_EX. (a) Distribution of H3K27ac signal across stitched enhancer regions in T_N, T_MEM and T_EX. Top 10 ranked putative SEs plus selected SEs are highlighted. N indicates the number of putative SEs identified for each cell type. Stitched enhancers above horizontal dashed lines are associated with putative SEs; enhancers below horizontal dashed lines are typical enhancers (TE). (b) Comparison of SE ranks identified using H3K27ac signal (x-axis) and ATAC signal (y-axis). Selected SEs are labeled. (c) Genome tracks showing RNA-seq, ATAC-seq and H3K27ac data. Putative SE regions identified using H3K27ac data but not ATAC-seq data are highlighted in black boxes below tracks. Individual enhancers within SE regions are highlighted in grey. Promoter regions (± 2,500 bp of TSS) are indicated in red. (d) Venn diagram showing cell-type specificity of SEs identified by H3K27ac. (e) Heatmap showing gene expression of genes within 50 kb of SE identified only in T_MEM and T_EX, respectively. Selected DEGs are highlighted. (f) Genome tracks showing RNA-seq and H3K27ac data. Putative SE regions identified using H3K27ac data are highlighted in black box. Individual enhancers within SE regions as highlighted in grey. Promoter regions (± 2,500 bp of TSS) are indicated in red.

Fig. 4

Key T_MEM and T_EX genes are poised in T_N and activated upon differentiation. (a) Alluvial plot showing how hPTMs at promoters poised in T_N change in T_MEM and T_EX. Active promoters were defined as either significantly gaining H3K4me3 or losing H3K27me3 or both; repressed promoters were defined as either significantly losing H3K4me3 or gaining H3K27me3 or both. Statistical cutoff of FC > 1.5. Number represents total number of T_N poised protomers; inset table shows number of poised-to-active and poised-to-repressed promoters in T_MEM and T_EX. (b) RNA expression heatmap for DEGs with poised promoters which were activated in either T_MEM (triangle) or T_EX (star). Selected DEGs are highlighted in bold. (c-d) Genome tracks showing poised promoters in T_N that switched to active (c) only in T_MEM or (d) only in T_EX. Promoters were excluded from analysis and are highlighted in grey bars. (e) Bubble plot showing changes in predicted TF binding site accessibility for poised-to-active T_MEM or T_EX genes, with select TFs highlighted.

Fig. 5

T_EX-enriched atypical H3K9me3 peaks cover CTCF sites and are associated with gene expression. (a) Signal intensity heatmap of T_MEM-enriched and T_EX-enriched H3K9me3 regions. (b) Genome track showing broad H3K9me3 regions enriched in T_MEM. (c) Genome track showing narrow H3K9me3 regions enriched in T_EX. (d) Peak size distribution of T_MEM- and T_EX-enriched H3K9me3 regions. (e) Percentage of H3K9me3 peaks that are broad versus narrow. Peaks > = 15 kb are defined as broad, peaks < 15 kb are defined as narrow. N.s. = not significantly different between T_MEM and T_EX. (f) Heatmap of Z-scored repeat element class coverage of T_MEM-enriched, non-significant (n.s.) and T_EX-enriched H3K9me3 narrow and broad peaks. (g) Predicted TF binding motifs in T_MEM- and T_EX-enriched narrow and broad peaks compared to n.s. H3K9me3 peaks. Top 5 motifs shown per comparison. (h) Venn diagram showing overlap between locations of CTCF binding sites and T_EX-enriched narrow H3K9me3 peaks. P value represents a hypergeometric test. (i) Pie chart showing change in CTCF binding at sites within T_EX-enriched narrow H3K9me3 peaks. (j) Violin plot of log2 fold change in RNA expression between T_MEM and T_EX near differentially modified regions for each hPTM (H3K27ac, H3K4me3, H3K27me3 and H3K9me3). (k) Pie chart showing changes in RNA expression between T_MEM and T_EX of genes near T_EX-enriched narrow H3K9me3 peaks. (l) Heatmap of RNA expression for genes from Fig. 5k. (m) Genome tracks showing DEGs near to narrow T_EX-enriched H3K9me3 regions.