Promoter DNA methylation patterns of differentiated cells are largely programmed at the progenitor stage

Abstract

Mesenchymal stem cells (MSCs) isolated from various tissues share common phenotypic and functional properties. However, intrinsic molecular evidence supporting these observations has been lacking. Here, we unravel overlapping genome-wide promoter DNA methylation patterns between MSCs from adipose tissue, bone marrow, and skeletal muscle, whereas hematopoietic progenitors are more epigenetically distant from MSCs as a whole. Commonly hypermethylated genes are enriched in signaling, metabolic, and developmental functions, whereas genes hypermethylated only in MSCs are associated with early development functions. We find that most lineage-specification promoters are DNA hypomethylated and harbor a combination of trimethylated H3K4 and H3K27, whereas early developmental genes are DNA hypermethylated with or without H3K27 methylation. Promoter DNA methylation patterns of differentiated cells are largely established at the progenitor stage; yet, differentiation segregates a minor fraction of the commonly hypermethylated promoters, generating greater epigenetic divergence between differentiated cell types than between their undifferentiated counterparts. We also show an effect of promoter CpG content on methylation dynamics upon differentiation and distinct methylation profiles on transcriptionally active and inactive promoters. We infer that methylation state of lineage-specific promoters in MSCs is not a primary determinant of differentiation capacity. Our results support the view of a common origin of mesenchymal progenitors.

Figure 1.

MeDIP in progenitor cells. (A) MeDIP-chip approach used in this study. (B) MeDIP-chip profiles in the tiled regions of indicated promoters in ASCs. Red bar indicates a methylation peak. Lower track shows coding region and TSS (arrow). (C) Bisulfite sequencing analysis of CpG methylation of promoters shown in B. ○, unmethylated CpG; •, methylated CpG. (D) MeDIP-chip profiles of adipogenic (LEP, LPL, FABP4, and PPARG2), myogenic (MYOG), and housekeeping (UBE2B) promoters in ASCs, BMMSCs, MPCs, and HPCs, shown as log₂ IP/input ratios. Transcripts and tiled regions are shown. (E) MeDIP-PCR analysis of promoter methylation for indicated genes. IP, MeDIP; In, input; Ig, precipitation with control nonimmune immunoglobulin.

Figure 2.

MeDIP-chip analysis of promoter DNA hypermethylation in mesenchymal progenitors. (A) Number and percentage of hypermethylated RefSeq promoters in ASCs, BMMSCs, MPCs, and HPCs. (B) Methylation profiles showing methylated (left) and unmethylated (right) promoters on two segments of chromosome 1 (log₂ IP/input). (C) Two-dimensional scatter plots of MaxTen values of methylation intensity in one cell type versus another. Average MaxTen values of both MeDIP replicates are plotted. Data points were colored to indicate classification according to peak calling algorithm to show hypermethylated promoters in one (purple, green) or both (blue) cell types. (D) Venn diagram analysis of hypermethylated promoters in ASCs, BMMSCs, and MPCs. (E) Percentages of hypermethylated promoters unique to each cell type and shared between cell types, identified from D.

Figure 3.

GO term enrichment for genes hypermethylated in MSCs and HPCs. (A) Venn diagram analysis of hypermethylated genes included in the MSC methylation core versus HPCs. (B) Enriched GO terms for genes hypermethylated in MSCs and HPCs. (C) A subset of developmentally regulated promoters is hypermethylated in NPCs and KPCs. MeDIP-PCR analysis of promoter methylation for indicated genes. IP, MeDIP; In, input. UBE2B and H19ICR methylation states in ASCs and BMMSCs are shown in Figure 1E.

Figure 4.

MSC differentiation partially resolves promoter methylation profiles. (A) In vitro differentiation of ASCs into adipocytes (stained with Oil Red-O) and of MPCs into multinucleated myocytes (stained with Hemacolor). Bars, 100 μm. (B) Two-dimensional scatter plots of MaxTen values for methylation intensities in ASCs or MPCs versus their differentiated counterparts (ASCad and MPCmd). Average values of both MeDIP replicates are plotted. Data points were colored to indicate classification according to peak calling to show hypermethylated promoters in differentiated cells (green), undifferentiated cells (purple), and common to both (blue). ASCad, adipogenic-differentiated ASCs; MPCmd, myogenic-differentiated MPCs. (C) Percentage of hypo- and hypermethylated promoters after adipogenic and myogenic differentiation of ASCs and MPCs. Venn diagrams of hypermethylated promoters in undifferentiated versus differentiated ASCs and MPCs (D) and between differentiated ASCs and MPCs (E).

Figure 5.

Distinct DNA methylation enrichment profiles on promoters of expressed versus nonexpressed genes. (A) Metagene analysis of average DNA methylation enrichment on hypermethylated promoters of expressed and repressed genes in ASCs. (B) Base pair coverage of methylation on promoters of expressed and nonexpressed genes, shown as width at half-maximal enrichment intensity calculated from metagene profiles. The difference in width at half-maximal enrichment intensity between expressed and nonexpressed genes for each cell type is also shown (right 3 columns).

Figure 6.

Promoter CpG content differentially affects methylation targeting and methylation response to differentiation induction. (A) Proportion of LCPs, ICPs, and HCPs among hypermethylated genes in ASCs, BMMSCs, MPCs, HCPs, and among all human RefSeq genes. Numbers of genes included in the analysis are shown on top. **p ≤ 0.0005 relative to the RefSeq data set. (B) Evolution of promoter methylation after adipogenic (left) and myogenic (right) differentiation as a function of promoter CpG class. “All” refers to all hypo- or hypermethylated promoters identified in Figure 4D. **p ≤ 0.0003, *p = 0.033, and (*)p = 0.077, relative to the All data set.

Figure 7.

Relationship between promoter DNA methylation and post-translational histone modifications in ASCs. (A) Venn diagram analysis of DNA methylated, H3K4me3-, H3K27me3-, and H3K9me3-enriched promoters. (B) GO term enrichment of genes with a promoter enriched in H3K4me3, H3K27me3, or in both marks. (C) Proportions of promoters coenriched in methylated DNA and/or indicated combinations of modified histones. (D) Metagene analysis of average enrichment profiles for indicated histone modifications. (E) Differential DNA methylation and histone modification enrichment profiles on exemplified loci on chromosome 10.

Figure 8.

Promoters of expressed and nonexpressed genes are enriched in distinct proportions of trimethylated H3K4, K9, and K27 irrespective of DNA methylation. (A) Histone modifications associated with DNA-methylated promoters of expressed or repressed genes. Percentages were calculated from MeDIP-chip and ChIP-on-chip data, and are those of DNA-methylated promoters also enriched in the indicated histone modifications (see Supplemental Figure S8 for Venn diagrams). (B) Histone modifications associated with all expressed and nonexpressed RefSeq promoters, regardless of DNA methylation. (C) Histone modifications associated with all RefSeq promoters enriched in H3K4me3, H3K9me3, H3K27me3, or in H3K4/K27me3.

Figure 9.

Chromatin states in human mesenchymal stem cells. (A) DNA methylation and histone modification patterns are grouped into several combinations on promoters of genes involved in indicated cellular functions. Promoter CpG content is shown on the left. (B) Model of MSC differentiation capacity in relation to DNA methylation of lineage-specific promoters. Hypermethylation is likely to be repressive; hypo- or unmethylation constitutes a permissive configuration, although it is of no predictive value on differentiation potential. (C) Changes in promoter DNA methylation after MSC differentiation: summary drawn from adipogenic ASC differentiation and myogenic MPC differentiation. Thickness of arrows reflects the proportion of promoters undergoing the indicated methylation change, or absence thereof.