Genome-wide analysis of histone methylation reveals chromatin state-based regulation of gene transcription and function of memory CD8+ T cells

Abstract

Memory lymphocytes are characterized by their ability to exhibit a rapid response to the recall antigen, in which differential transcription is important, yet the underlying mechanism is not understood. We report here a genome-wide analysis of histone methylation on two histone H3 lysine residues (H3K4me3 and H3K27me3) and gene expression profiles in naive and memory CD8(+) T cells. We found that specific correlation exists between gene expression and the amounts of H3K4me3 (positive correlation) and H3K27me3 (negative correlation) across the gene body. These correlations displayed four distinct modes (repressive, active, poised, and bivalent), reflecting different functions of these genes in CD8(+) T cells. Furthermore, a permissive chromatin state of each gene was established by a combination of different histone modifications. Our findings reveal a complex regulation by histone methylation in differential gene expression and suggest that histone methylation may be responsible for memory CD8(+) T cell function.

Figure 1. Scheme for genome-wide parallel analysis of histone methylation by ChIP-Seq and gene expression by microaray in CD8 T cell subsets

Naïve and memory CD8 T cell subsets were isolated by cell sorting. ChIP-seq experiments for H3K4me3 and H3K27me3 was carried out from two individuals and subsequent confirmations of the findings by PCR were done in 3 additional individuals. Microarray gene expression analyses used RNA from 9 individuals in 3 pools and subsequent confirmations by RT-PCR were done with 3 additional individuals.

Figure 2. Global correlation between gene expression and histone methylation in CD8 T cell subsets

(A) The levels of gene expression were positively correlated with the levels of H3K4me3 in naïve and memory CD8 T cells subsets at rest. (B) The levels of gene expressions were negatively correlated with the levels of H3K27me3 in naïve and memory CD8 T cells subsets at rest. Each dot in the plots (A and B) represents the mean value of 100 genes that were grouped based on their rank of mRNA level from the highest to the lowest. (C) The numbers of the genes with high levels of H3K4me3 or H3K27me3 in naïve and memory CD8 T cell subsets are shown.

Figure 3. H3K27me3domains in naïve and memory CD8 T cells

(A) Enriched H3K27me3 regions in naïve CD8 T cells. The chromosomal locations, residing gene loci, and their transcriptional orientations are shown (the scanning method for the selection is discussed in the Experimental Procedures). (B) One representative H3K27me3 island in naïve CD8 T cells. (C) Enriched H3K27me3 regions in memory CD8 (T_EM) T cells. The chromosomal locations, residing gene loci, and their transcriptional orientations are shown. (D) One representative H3K27me3 island in CD8 T_EM cells.

Figure 4. Repressed, active and poised genes in memory CD8 T cell subsets

(A) Comparison of H3K4me3 levels in repressed gene loci between naïve and memory CD8 T cell subsets. The mean H3K4me3 of 105 common repressed genes in memory CD8 T cell subsets and naïve CD8 T cells was compared. (B) Two representative common repressed genes (SH3BGRL2, SYT3) in memory CD8 T cells are presented, indicating their gene loci, the levels of H3K4me3 and H3K27me3 in resting CD8 T cells, and mRNA expression at resting and after 16 hours anti-CD3/28 stimulation in naïve and memory CD8 T cell subsets (a complete list of genes can be found in Table S2). (C) Comparison of H3K4me3 levels in active gene loci between naïve and memory CD8 T cell subsets. The mean H3K4me3 of 150 shared active genes in memory CD8 T cell subsets and naïve CD8 T cells was compared. (D) Two representative shared active genes (PRDM1, KLRG1) in memory CD8 T cells are presented, indicating their gene loci, the levels of H3K4me3 and H3K27me3 in resting CD8 T cells, and mRNA expression at resting and after 16 hours anti-CD3/28 stimulation in naïve and memory CD8 T cell subsets (a complete list of genes can be found in Table S3). (E) Comparison of H3K4me3 levels in poised gene loci between naïve and memory CD8 T cell subsets. The mean H3K4me3 in poised gene loci between naïve and T_CM (N=46) or between naïve and T_EM (N=6) is shown. (F) Two representative poised genes (ID2, PLAGL1) in CD8 T_CM or T_EM, respectively, are presented, indicating their gene loci, the levels of H3K4me3 and H3K27me3 in resting CD8 T cells, and mRNA expression at resting and after 16 hours anti-CD3/28 stimulation in naïve and memory CD8 T cell subsets (a complete list of genes can be found in Table S4). P value is indicated by asterisks (*P<0.01, **P<0.05) used in all figures.

Figure 5. Genes with bivalent chromatins in memory CD8 T cell subsets

(A) Genes with bivalent chromatins of T_CM and/or T_EM CD8 T cells are categorized by cellular function (the criteria for the bivalent genes were described in the Results and Methods. A complete list of bivalent genes can be found in Table S5). (B) One representative common bivalent gene (MLK4) is shown with its gene locus and the levels of H3K4me3 and H3K27me3 at resting in naïve and memory CD8 T cell subsets. (C) Changes in the levels of H3K4me3 and mRNA expression in the MLK4 gene at 0 and 72 hours after anti-CD3/28 stimulation in CD8 T_CM and T_EM are shown. The mean values and SEM are presented from 5 independent donors.

Figure 6. Differentially expressed genes are correlated with histone methylation in CD8 T_CM and T_EM

(A) Differential gene expression profiles in T_CM and T_EM were analyzed by Agilent human whole genome chip. Selected genes that differed between T_CM and T_EM by >3-fold are shown (a complete list of genes can be found in Table S6). (B) Differential gene expressions were positively correlated with the levels of H3K4me3 in T_CM and T_EM. The significantly differentially expressed genes between T_CM and T_EM were selected as described above and in the Experimental Procedures. The ratios of mRNA levels between T_CM and T_EM were compared to the ratios of H3K4me3 levels between T_CM and T_EM in their corresponding genes (N=137, R²=0.39). The Log₁₀ values were used in the plot.

Figure 7. A HAT inhibitor modulates chromatin structure and gene transcription in memory CD8 T cells

(A) High levels of gene expression were not associated with H3K4me3 but with H3K9ac in the TNFRSF9 gene in memory CD8 T cell subsets. (B) High levels of gene expression were associated with both H3K4me3 and H3K9ac in the SLAMF1 gene in memory CD8 T cell subsets. (C) Complete reductions in mRNA levels were associated with reductions in H3K9ac levels alone in the TNFRSF9 gene with curcumin treatment in memory CD8 T cell subsets. Partial reductions in mRNA levels were associated with reductions in H3K9ac levels but not H3K4me3 levels in the SLAMF1 gene with curcumin treatment in memory CD8 T cell subsets. The ACTB gene is shown as a negative control. The mean values and SEM are presented from 4 independent donors.