Distinct Trends of DNA Methylation Patterning in the Innate and Adaptive Immune Systems

Abstract

DNA methylation and the localization and post-translational modification of nucleosomes are interdependent factors that contribute to the generation of distinct phenotypes from genetically identical cells. With 112 whole-genome bisulfite sequencing datasets from the BLUEPRINT Epigenome Project, we analyzed the global development of DNA methylation patterns during lineage commitment and maturation of a range of immune system effector cells and the cancers that arise from them. We show clear trends in methylation patterns that are distinct in the innate and adaptive arms of the human immune system, both globally and in relation to consistently positioned nucleosomes. Most notable are a progressive loss of methylation in developing lymphocytes and the consistent occurrence of non-CG methylation in specific cell types. Cancer samples from the two lineages are further polarized, suggesting the involvement of distinct lineage-specific epigenetic mechanisms. We anticipate broad utility for this resource as a basis for further comparative epigenetic analyses.

Keywords: BLUEPRINT; CTCF; DNA methylation; epigenetics; hematopoiesis; nucleosomes; whole-genome bisulfite sequencing.

Figure 1. Genome-wide DNA Methylation Trends in Cells of the Lymphoid and Myeloid Lineages

(A) Methylation in CH and CG contexts in lymphoid (triangles) and myeloid cells (circles). Long-lived lymphoid cells have lower CG methylation. Naive T cells have higher levels of non-CG methylation. Most myeloid-derived cancers gain non-CG methylation. (B) CG methylation. Developing lymphocytes lose methylation (left). Cells of the myeloid lineage are relatively constant or show small gains in methylation (right). (C) Fraction of non-CG cytosines methylated. Fraction methylated is computed as the number of significantly methylated non-CG cytosines divided by the total number of non-CG cytosines read with adequate coverage to make a methylation call, as described in Experimental Procedures. Stem cell: HUES64 cell line; progenitor: CD34⁺, including stem cells, multipotent progenitors, and common lymphoid progenitors. Numbers indicate developmental order relative to the indicated cell type. Blue, female; red, male.

Figure 2. Non-CG Methylation

(A) Example of mCH exclusion from lamina-associated domains (shaded areas) in a naive T cell, chr10:100,600,000–124,600,000. x axis, genomic location; y axis, non-CG methylation level (negative values, minus strand; positive values, plus strand). CpG islands (vertical blue bars) and CTCF-binding sites (red triangles) are shown. (B) Information content in the sequence context of mCH in naive T cells. The methylated cytosine is at position 6. (C) Conversion rate and mCH. After exclusion of under-converted outliers (red, top plot), the fraction of methylated non-CG cytosines is not influenced by conversion rate (bottom expanded plot), as determined using unmethylated spiked-in bacteriophage DNA. mCH is computed as the fraction of non-converted non-CG cytosines.

Figure 3. Exon-Specific CH Non-conversion

Two plots at the same genomic location (x axis) at different resolutions. (Top plot) Spikes rise above background mCH levels. (Bottom plot) Spikes are specific to exons (blue boxes). y axis, fraction of non-converted reads at non-CG cytosines. Negative values indicate cytosines on the minus strand. Only positions with six or more reads informative for methylation status are shown. See also Figure S1.

Figure 4. DNA Methylation Levels at Successive Stages of B Lymphocyte Development Aggregated across Subsets of CTCF-Binding Sites

Development occurs in the order indicated in the legend, with UHP being the least differentiated cell type. (A) Methylation surrounding 23,710 constitutively occupied CTCF-binding sites shows oscillations that increase in amplitude as lymphocytes develop. (B–D) CTCF-binding sites specifically occupied in (B) lymphoblastoid cell line, (C) stem cell line (unoccupied in B cells), and (D) skin cell line (unoccupied in B cells). GC, germinal center B cell; PC, plasma cell; UHP, uncommitted hematopoietic progenitor. See also Figures S2 and S3.

Figure 5. mCH in T Lymphocytes

mCH occurs between nucleosomes (black) in naïve T lymphocytes (red), but not memory T lymphocytes (green and blue). Positive and negative methylation values correspond to cytosines on the plus and minus strand, respectively. Values are aggregated across 23,710 constitutively occupied CTCF sites.

Figure 6. Nucleosome Influence and DNA Methylation in Lymphoid and Myeloid Development

Nucleosome influence (y axis) is defined as the difference in methylation between nucleosome-occupied DNA and the adjacent linker DNA at cCTCF. Data are aggregated across 23,710 constitutively occupied CTCF sites. (A) Normal development in two cell types from each lineage. Two replicates of B lymphocyte development: 0, UHP; 1, pre-B; 2, naive B cell; 3, germinal center B cell; 4, memory B cell; 5, plasma cell. T lymphocytes: CD4⁺ (gray) and CD8⁺ (black). 1, naive T cell; 2, central memory T cell; 3, effector memory T cell. Neutrophil development: 1, promyelocyte; 2, metamyelocyte, 3, banded neutrophil; 4, segmented neutrophil; 5, resting neutrophil; 6, GTX-activated neutrophil. Monocyte/macrophage development: 1, monocyte; 2, resting macrophage (M0); 3, activated macrophages (red:M1; orange:M2). (B) Neoplasms derived from lymphoid and myeloid lineages. (C) Comparison across cell types. Numbers indicate developmental order as in (A). See also Figure S5.