DNA methylome in human CD4+ T cells identifies transcriptionally repressive and non-repressive methylation peaks

Abstract

DNA methylation is an epigenetic mark that is critical in determining chromatin accessibility and regulating gene expression. This epigenetic mechanism has an important role in T-cell function. We used genome-wide methylation profiling to characterize the DNA methylome in primary human CD4+ T cells. We found that only 5% of CpG islands are methylated in CD4+ T cells, and that DNA methylation peak density is increased in subtelomeric chromosomal regions. We also found an inverse relationship between methylation peak density and chromosomal length. Our data indicate that DNA methylation in gene promoter regions is not always a repressive epigenetic mark. Indeed, about 27% of methylated genes are actively expressed in CD4+ T cells. We demonstrate that repressive methylation peaks are located closer to the transcription start site (TSS) compared with functionally non-repressive peaks (-893±110 bp versus -1342±218 bp (mean±s.e.m.), P-value <0.05). We also show that both a larger number and an increased CpG island density in promoter sequences predict transcriptional permissiveness of DNA methylation. TSS in the majority of genes with permissive DNA methylation peaks is in DNase I hypersensitive sites, indicating a failure of DNA methylation to induce chromatin inaccessibility in these loci.

Figure 1

Methylation enrichment signals represented as −log₁₀ P-value scores in CD4+ T-cell DNA from five normal healthy participants in (a) the H19/IGF2/KCNQ1 genetic locus on chromosome 11, and (b) the HOXA gene cluster on chromosome 7. Bisulfite DNA sequencing was used to validate the methylation status in methylated regions within the HOXA3 gene and the KCNQ1/TRPM5 promoter region, and hypomethylated regions in the HOXA1 and HOXA13 promoter regions in an independent set of samples.

Figure 1

Methylation enrichment signals represented as −log₁₀ P-value scores in CD4+ T-cell DNA from five normal healthy participants in (a) the H19/IGF2/KCNQ1 genetic locus on chromosome 11, and (b) the HOXA gene cluster on chromosome 7. Bisulfite DNA sequencing was used to validate the methylation status in methylated regions within the HOXA3 gene and the KCNQ1/TRPM5 promoter region, and hypomethylated regions in the HOXA1 and HOXA13 promoter regions in an independent set of samples.

Figure 2

(a) DNA methylation is significantly associated with transcriptional repression in CD4+ T cells (P<0.0001). About 27% of genes with promoter region methylation escape transcriptional repression and are actively expressed. (b) Methylation peak density normalized to tiled regions (peak/Mb) negatively correlates with chromosomal length (r² = 0.34).

Figure 3

Functional network analysis of methylated genes that are expressed in primary human CD4+ T cells identified involvement in basic cell functions, including cellular signaling and interaction, and cellular growth and proliferation (a). Genes that are methylated and non-expressed are functionally associated with antigen presentation, cell-mediated immune response and humoral immune response (b). Gene key: solid lines indicate direct interaction, dotted lines indicate indirect interaction, an arrow from a to b indicates that a acts on b, a line without an arrowhead indicates binding only and a line with a small vertical line at the end from a to b indicates a inhibits b. Gray indicates genes that are methylated, white indicates genes that are not user specified but incorporated into the network through relationships with other genes. Node shapes are: square, cytokine; diamond (vertical), enzyme; diamond (horizontal), peptidase; dotted rectangular (vertical), ion channel; solid rectangular (vertical), G-protein-coupled receptor; triangle, kinase; oval (horizontal), transcription regulator; oval (vertical), transmembrane receptor; trapezoid, transporter; circle, other.

Figure 4

A schematic representation showing distinguishing features between repressive and permissive DNA methylation. (a) Repressive methylation peaks are on average 893±110 bp upstream of the transcription start site of target genes, which are characterized by a relatively lower maximum CpG island density. Chromatin is closed at the transcription start site as indicated by the absence of DNase I HS sites. (b) Permissive methylation peaks are further upstream from transcription start site compared with repressive peaks, and are characterized by higher maximum CpG island densities within promoter sequences of target genes. These methylation peaks fail to maintain a closed chromatin configuration. This results in accessible chromatin at the transcription start sites of target genes as evidenced by the presence of DNase I HS sites and gene expression. It is possible that these methylation peaks fail to efficiently recruit transcriptional repressor complexes, such as the MECP2–SIN3A–HDAC complex. MECP2, methyl-CpG-binding protein 2; SIN3A, SIN3 homolog A; HDAC, histone deacetylase.