Comprehensive methylome map of lineage commitment from haematopoietic progenitors

Abstract

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.

Figure 1. Examples of known lineage-related genes showing differential DNA methylation between lymphoid and myeloid progenitors

a, Hematopoietic progenitors included in this study. Dashed-arrow indicates existence of intermediate progenitors. DMR in b, Lck and c, Mpo. Upper panels: top half: CpG methylation (p); lower half: CpG dinucleotides (black tick marks), CpG density (curve), CpG islands (orange lines) and the gene annotation (see online Methods). Middle panels: methylation of individual CpGs (in the red boxes), mean values connected by lines. Bottom panels: mRNA expression levels, normalized to the highest expression among the populations (mean ± s.d., n=3; 5 for MPP^FL− for microarrays).

Figure 2. Gene expression correlates strongly with DMRs at shores

DMRs within 2kb of gene TSSs (black circles) were divided into two groups: Island (inside, cover, or overlap more than 50% of a CpG island), and Shores (up to 2000bp away from a CpG island). After RMA preprocessing, the log₂ ratios of the gene expression differences (from leftto right) were plotted against Δp (left group minus right group). Black pluses represent random DMR-gene pairs more than 2kb apart. Wilcoxon rank-sum tests were performed to test the null hypothesis. a, MPP^FL− vs. DN3_DMRs. b, MPP^FL− vs. GMP_DMRs.

Figure 3. CHARM identified genes with previously unknown functions in lymphoid/myeloid lineage commitment and pluripotency maintenance

a and b, Examples of DMRs with methylation changes in lymphoid/myeloid progenitors. a, the DMR in Arl4c. b, the DMR in Jdp2. C, the DMR in Meis1. D, the DMR in Hdac7a. The CHARM plots, pyrosequencing, Affymetrix GeneChip, and RT-PCR data are organized and displayed as in Fig. 1b.