Promoter Methylation Profiling of Mesenchymal Progenitor Cells
We addressed the epigenetic relationship, at the DNA methylation level, between progenitor cells isolated from human adipose tissue, bone marrow, and skeletal muscle by MeDIP-chip mapping of promoter DNA methylation profiles in ASCs, BMMSCs, MPCs, and HPCs (A). Immunocaptured DNA fragments enriched in 5-methylcytosine were hybridized on promoter arrays tiling −2 kb to +0.5 kb relative to the transcription start site (TSS) of ~27,000 human promoters at 100-base pair resolution. Correlation analysis of log2 MeDIP/Input ratios for each cell type revealed high reproducibility between replicates (Supplemental Figure S1A).
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 (more ...)
Validation of the MeDIP approach was done at several levels. MeDIP-chip data were corroborated by bisulfite sequencing of randomly chosen promoters (, B and C), by published bisulfite sequencing data for all cell types examined here (Noer et al., 2006
; Sørensen et al., 2009
) and by MeDIP-PCR single-gene analysis (, D and E). MeDIP-PCR data were in addition verified for additional ASC, BMMSC, and MPC donors (Supplemental Figure S2). MeDIP-chip further corroborated published MeDIP-PCR data for methylated and unmethylated promoters in human fibroblasts (Weber et al., 2007
) (Supplemental Figure S1, B and C). MeDIP-PCR also confirmed hypomethylation of the housekeeping UBE2B
promoter and methylation of the H19
imprinting control region (H19ICR
) reported previously in fibroblasts (Weber et al., 2007
) (E). Lastly, the proportions of methylated genes detected by MeDIP-chip in ASCs and BMMSCs (19% of 17,790 RefSeq genes in both cell types) were similar to those detected earlier by combined bisulfite restriction analysis (17 and 16%, respectively) among ~170 genes (Dahl et al., 2008
), and methylation patterns reported for those genes, validated by bisulfite sequencing (Dahl et al., 2008
), were corroborated by MeDIP-chip data.
Adipose Tissue, Bone Marrow, and Muscle Progenitors Share a Large Set of Hypermethylated Genes
We identified with high significance (K-S test p value ≤0.01 for detection of methylation “peaks”) >3300 promoters hypermethylated relative to genome-average methylation in ASCs and BMMSCs, 2630 in MPCs, and 3902 in HPCs (A). These made up 15–22% of all RefSeq promoters represented on the array (A). Hybridization patterns (B) and calculated MaxTen values of methylation intensity for all promoters (C) revealed high similarity and overlap between ASCs, BMMSCs, and MPCs. Intersect analysis of promoters with at least one hypermethylation peak showed that ASCs and BMMSCs shared 2486 hypermethylated genes (74% of all hypermethylated genes in these cell types; , C and D). ASCs and BMMSCs, respectively, shared 1944 (57%) and 2053 (61%) hypermethylated genes with MPCs (, C and D). We also identified a core of 1755 hypermethylated genes common to ASCs, BMMSCs, and MPCs, representing 52–66% of all hypermethylated genes in these cell types (D). Another 20–30% was methylated in two of three cell types, whereas 15–20% was methylated only in one cell type (E). These data indicate a high similarity of promoter DNA methylation patterns in progenitor cells from adipose tissue, bone marrow and skeletal muscle.
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) (more ...)
To determine whether the hypermethylated gene core was specific to mesenchymal progenitors, we also examined BM-derived CD34+ HPCs. We found that 91% of the 1755 core hypermethylated genes also were hypermethylated in HPCs, whereas HPCs contained 2302 hypermethylated genes that distinguished them from mesenchymal progenitors considered as a whole (A). Moreover, 30–50% of genes found to be hypermethylated in ASCs, BMMSCs, or MPCs only (D, crescents) were also hypermethylated in HPCs (Supplemental Figure S3). Lists of these genes hypermethylated in ASCs, BMMSCs, and MPCs are provided in Supplemental Table S2.
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 (more ...)
These results collectively indicate that promoter methylation profiles are similar but not identical among ASCs, BMMSCs, and MPCs, highlighting an intrinsic epigenetic identity between these mesenchymal progenitors. The majority of these genes are also hypermethylated in HPCs, which also contain an additional large set of hypermethylated genes.
Early Developmentally Regulated Genes Are Hypermethylated in Mesenchymal and Nonmesenchymal Progenitors
To address the biological significance of the hypermethylated genes revealed by MeDIP-chip, we identified GO terms enriched among these genes (B and Supplemental Table S3). Interestingly, genes hypermethylated in MSCs as a whole were enriched in signaling and developmental functions pertaining to early fetal development. Genes hypermethylated in HPCs were enriched in signaling functions linked to sensory perception, whereas genes hypermethylated in both MSCs and HPCs were associated with reproduction processes in addition to signaling, transcription regulation, and metabolic functions (B). This finding corroborated the differential epigenetic programming of the germline and the soma shown previously by MeDIP-chip using similar promoter arrays (Weber et al., 2007
). GO analysis therefore suggests that hypermethylation targets developmental functions disabled at the progenitor stage examined here, as well as late differentiation-associated functions.
A randomly chosen subset of early developmental genes identified above was shown to also be hypermethylated in NPCs and KPCs (C), indicating that hypermethylation of these genes can occur in precursors of both mesodermal and ectodermal origin. Nonetheless, among the genes examined some (TBX3
, and PAX5
) were hypomethylated in NPCs and/or KPCs (yet were as expected from our MeDIP-chip data hypermethylated in ASCs and BMMSCs) (C), a pattern that may be linked to their role in neurogenesis and keratinocyte function (Asbreuk et al., 2002
; Norhany et al., 2006
; Pillai et al., 2007
Differentiation Partly Resolves Promoter Methylation Patterns Common to Mesenchymal Progenitors
MeDIP-chip and bisulfite sequencing data have shown that in vitro differentiation of mouse ES cells into neuronal progenitors and subsequently into neurons is accompanied by remarkably few methylation changes, most of which occur during the first step of differentiation (Meissner et al., 2008
; Mohn et al., 2008
). This predicts that at least in this in vitro model, methylation patterns of differentiated cells would be established at the progenitor stage. To address this issue in primary progenitors, ASCs were differentiated in vitro into adipocytes and MPCs were differentiated into multinucleated myocytes. Differentiation was assessed by formation of Oil Red-O–positive lipid inclusions in adipocytes (A), formation of multinucleated myocytes (A), and up-regulation of lineage-specific genes in microarray expression analyses (Supplemental Table S4).
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 (more ...)
Promoter methylation changes after differentiation distinguished adipocytes from ASCs and myocytes from MPCs (B). Nonetheless, most (~80%) hypermethylated promoters in undifferentiated cells remained hypermethylated (C), suggesting that methylation states in differentiated cells are largely established at the progenitor stage. In addition, 29% of all methylated promoters identified in adipocytes were hypermethylated after ASC differentiation, whereas 15% of the methylated promoters in ASCs were hypomethylated (, C and D). Similar observations were made after MPC differentiation (, C and D). Thus, ASC and MPC differentiation is accompanied by methylation changes leading to an increase in the number of hypermethylated promoters in differentiated cells (p < 10−4; chi-square test with Yates' correction).
These data are consistent with enhanced transcriptional restrictions by DNA methylation as cells differentiate. To address the lineage specificity of these methylation changes, we cross-examined genes methylated in adipocytes and myocytes. Twenty percent of genes hypermethylated after differentiation were common to both cell types (E). A subset of these genes was involved in stimulation-dependent changes in metabolism, consistent with differentiation induction (Supplemental Figure S4). Eighty percent of the hypermethylated genes, however, were cell type specific (E). GO term enrichment analysis indicates that these were involved in the regulation of nuclear assembly, nuclear-cytoplasmic transport, and G-protein signaling in adipocytes (consistent with the completion of nuclear reorganization taking place during the formation of mature adipocytes), and in cell–cell interaction and exocytotic and sensory perception functions in myocytes (Supplemental Figure S4 and Supplemental Table S5). The reduced overlap of hypermethylated genes between adipocytes and myocytes, compared with ASCs and MPCs, reflects a greater epigenetic divergence between the two differentiated cell types than between their respective undifferentiated counterparts.
Relationship between Promoter Methylation and Gene Expression upon Differentiation
We next determined the extent to which differentiation-induced changes in promoter methylation reflected transcriptional changes. We first assessed the proportion of expressed genes in ASCs, BMMSCs, and MPCs by using Illumina expression arrays by defining present (expressed), marginal (weakly expressed), and absent (not expressed) cells. In each cell type, 54–57% of the hypermethylated genes were detected as expressed or weakly expressed. These percentages were similar to the proportion of expressed RefSeq genes detected in these cell types irrespective of methylation state (Supplemental Figure S5). Thus, promoter methylation is compatible with transcriptional activity (also see Weber et al., 2007
We next determined transcriptional states associated with promoter hypo- or hypermethylation resulting from differentiation. After adipogenic differentiation, we found 702 genes overexpressed or induced (Supplemental Table S4), 645 of which were included on the Nimblegen platform. Of these, 102 (16%) were hypermethylated in undifferentiated ASCs. Among those methylated genes, 15 became demethylated, whereas 87 retained their methylation state. After myogenic differentiation of MPCs, 444 genes were overexpressed or induced (Supplemental Table S4), 417 of which were covered on the Nimblegen platform. Among those, 49 (12%) were hypermethylated in undifferentiated MPCs. Among those methylated genes, 13 became demethylated, whereas 36 retained their methylation state. These results indicate that the majority of genes up-regulated after MSC differentiation are DNA hypomethylated in undifferentiated cells. Moreover, among hypermethylated genes, only a quarter or less undergo methylation change.
Promoter Methylation Enrichment Profiles Distinguish Promoters of Expressed versus Nonexpressed Genes
We next addressed whether methylation occurred in distinct regions relative to the TSS in expressed versus nonexpressed genes in ASCs, BMMSCs, and MPCs. To this end, we determined average methylation by computing metagene profiles for all hypermethylated promoters. These profiles were distinct for transcriptionally active and inactive promoters (A and Supplemental Figure S6). In all cell types, the amplitude of methylation enrichment was greater on promoters of expressed genes than nonexpressed genes (p values from Welsh two-sample t
tests for methylation intensity amplitude in ASCs: p < 2.2 × 10−16
; BMMSCs: p = 1.34 × 10−14
; and MPCs: p = 3.04 × 10−3
): enrichment was stronger on active promoters but sharply decreased to genome-average or below immediately 5′ of the TSS. In contrast, on inactive promoters, maximum enrichment was lower but was more widely spread by an additional 500-1500 base pairs to include the TSS, as determined by extension of the width at half-maximal enrichment (, A and B, and Supplemental Figure S6). These data indicate that the profile of methylation coverage distinguishes promoters of expressed and nonexpressed genes. Nevertheless, the density of methylated CpGs was lower at the TSS than upstream in both expressed and repressed genes, corroborating recent genome-scale bisulfite sequencing data (Lister et al., 2009
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 (more ...)
Methylation Preferentially Targets Intermediate and Low CpG Content Promoters
The relationship between promoter DNA methylation and gene activity has been shown to depend on CpG content (Weber et al., 2007
). Thus, we asked whether methylation enrichment detected in the tiled regions in progenitor cells was influenced by promoter CpG content. Previous classification of human RefSeq promoters based on CpG density revealed a bimodal distribution from observed/expected CpG ratios, identifying high (HCP), intermediate (ICP), and low (LCP) CpG promoters (Weber et al., 2007
). We applied the algorithm of Weber et al. (2007)
to the tiled regions (−2.5 to +0.5 kb relative to the TSS) of all RefSeq promoters represented on the array, and we identified 11511 HCPs, 3173 ICPs, and 3246 LCPs; these numbers were comparable with those of Weber et al. (2007)
In all cell types examined, CpG methylation targeted a higher proportion of ICPs relative to the proportion of ICPs in the genome (A; p < 10−4
; chi-square test with Yates' correction), at the expense of HCPs whose proportion was reduced among methylated promoters (p < 10−3
). Methylation did not preferentially target LCPs except in hematopoietic progenitors where methylated LCPs were enriched (p = 0.0005). Thus, CpG methylation targets a higher proportion of intermediate to low CpG promoters compared with their proportions in the genome, in consistency with the enhanced protection of CpG islands against methylation (Weber et al., 2007
; Irizarry et al., 2009
; Straussman et al., 2009
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. (more ...)
Differentiation-induced Methylation Changes Distinctively Affect High- and Low-CpG Content Promoters
Having established that methylation differentially affects promoters with distinct CpG contents, we determined whether the nature of methylation changes (hypo- or hypermethylation) after adipogenic or myogenic differentiation differed between promoter classes. To this end, methylation changes identified in D were reanalyzed for HCPs, ICPs, and LCPs. B shows that in ASCs, hypomethylated genes were enriched in HCPs (p = 0.0003; Fisher's exact test) relative to the total number of hypomethylated genes, at the expense of ICPs (p < 10−4) and LCPs (p = 0.033). Furthermore, there was an enrichment of hypermethylated genes in LCPs (p = 0.0004), whereas HCPs and ICPs were not affected (p > 0.5). In MPCs, we also detected a trend in enrichment of hypomethylated genes in HCPs (p = 0.077) and an enrichment of hypermethylated genes in LCPs (p = 0.05) without significantly affecting HCPs and ICPs (B). We concluded that differentiation-elicited hypomethylation predominantly affected methylated HCPs, whereas hypermethylation preferentially concerned LCPs.
Methylation State of Lineage-specific Promoters Is Not a Determinant of Differentiation Capacity
Our previous bisulfite sequencing results suggested no predictability of MSC differentiation capacity based on the methylation state of a few lineage-specific promoters (Sørensen et al., 2009
). Using our MeDIP-chip data, we extended our analysis of ASCs, BMMSCs, MPCs, and HPCs to 200 lineage-priming genes (including 50 HCPs and 150 non-HCPs) linked to differentiation into mesodermal, endodermal, and ectodermal lineages (Supplemental Table S6). These included 107 lineage-priming genes recently reported to be expressed at least at some level in BMMSCs (Delorme et al., 2009
). We detected 57 hypermethylated promoters (28.5%) in at least one cell type, of which 8 (4%) were hypermethylated in all cell types. Methylation of these promoters did not occur in any particular developmental lineage for a given cell type, and we did not observe any significant difference in the proportion of hypermethylated promoters between cells, including HPCs. In fact, most promoters specifying mesodermal (adipogenic, osteogenic, chondrogenic, myogenic, and vascular), endodermal (pancreatic, hepatic) and ectodermal (neurogenic, skin) differentiation were not hypermethylated (Supplemental Figure S7 and Supplemental Table S6). These findings confirm the absence of relationship between methylation state and differentiation capacity of MSCs.
DNA-methylated Promoters in ASCs Are in Majority Not Trimethylated on H3K4, H3K9, or H3K27
The lack of straightforward relationship between promoter DNA methylation and MSC differentiation capacity prompted the interrogation of additional epigenetic states on promoters. We examined by ChIP-on-chip in ASCs and in relation to DNA methylation the promoter enrichment profiles for trimethylated lysine 4 of histone H3 (H3K4me3), a transcriptionally permissive modification; H3K27me3, a Polycomb-mediated transcriptionally repressive mark; and H3K9me3, a mark of heterochromatin associated with repressed promoters (Kouzarides, 2007
We identified 3362 promoters enriched in H3K4me3 and 2321 enriched in H3K27me3 (A). GO term enrichment analysis showed that H3K4me3-marked genes were associated with transcription regulation, macromolecule synthesis, and metabolic processes, whereas H3K27me3-marked genes were distinctively enriched in developmental, differentiation and signaling functions (B and Supplemental Table S7). Moreover, 25% of H3K4me3 promoters were coenriched in H3K27me3 (, A and C) and displayed largely overlapping average enrichment profiles for these modifications, in contrast to promoters exclusively harboring either mark (D). Although we do not have formal proof that these modifications co-occupy individual promoters, the metagene profiles together with previous sequential ChIP results (Noer et al., 2009
) suggest that they might. GO terms enriched among H3K4/K27me3-enriched genes pertained to transcription regulation, development, differentiation, and cell adhesion (B and Supplemental Table S7). These functional groups were similar to those of H3K4/K27me3 “bivalent” genes in ESCs (Bernstein et al., 2006
; Pan et al., 2007
; Zhao et al., 2007
), in hematopoietic progenitors (Cui et al., 2009
) and in embryos (Dahl et al., 2010
). These findings extend the concept that H3K4/K27me3 coenrichment marks developmentally important promoters in stem and progenitor cells.
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 (more ...)
We next examined histone modifications associated with DNA methylated promoters (exemplified in E). We found that of DNA methylated promoters, 22% were enriched in H3K4me3, 17% were enriched in H3K27me3, and <7% were trimethylated on H3K9 (, A and C). These proportions were notably lower than those of H3K4me3-, H3K27me3-, and H3K9me3-enriched promoters among all modified RefSeq promoters (respectively, 37%, 24 and 17%; data not shown; p < 0.001; chi-square test with Yates' correction). Thus, DNA methylation and H3K4, K9, or K27 trimethylation seem to be largely exclusive at least in the promoter regions examined. H3K4/K27me3 coenrichment occurs mainly on weakly or unmethylated promoters (C), a configuration reminiscent of the DNA hypomethylated state of developmentally regulated bivalent promoters in ES cells (Fouse et al., 2008
; Mohn et al., 2008
Nonetheless, a nonnegligible proportion of DNA methylated promoters was found to be enriched in H3K4me3 or H3K27me3 (, A and C). These genes were enriched linked to transcription regulation, metabolic and synthetic processes (H3K4me3), early development, and differentiation (H3K27me3), or transcription and differentiation (H3K4/K27me3). These functional categories were similar to those defined by H3K4 or H3K27 methylation alone (Supplemental Table S7) and were not altered by DNA methylation states. Moreover, we found that the majority (80 to >90%) of H3K27me3- or H3K9me3-enriched genes were not expressed, whereas 60% of H3K4me3 genes were expressed (data not shown). These percentages were similar among DNA methylated genes and among all RefSeq genes bearing these marks (data not shown); thus, DNA methylation does not confer additional repressive effect on promoters harboring any of these histone modifications.
Trimethylated H3K4 and H4K27 Delineate Distinct Epigenetic Markings on a Subset of DNA-methylated Transcriptionally Active and Inactive Promoters
Our earlier data outlined distinct average DNA methylation enrichment profiles on the promoters of expressed versus nonexpressed genes (A). To start addressing the biological significance of this observation, we examined histone modifications patterns on these promoters (Supplemental Figure S8). We first noted that only 23 and 28% of DNA methylated expressed and nonexpressed promoters, respectively, were enriched above genome-average level in any of the histone marks examined (see above). Second, of the active DNA-methylated promoters coenriched in trimethylated H3K4, K9, or K27, 85% were enriched in H3K4me3 alone (74%) or together with H3K27me3 (11%) (A, left). A minority harbored H3K9me3 (2%) or H3K27me3 (13%) only, as expected. Inactive DNA-methylated promoters, in contrast, were predominantly enriched in H3K27me3 only (43%) or together with H3K4me3 (25%), or in H3K4me3 only (25%) (A, right). H3K9me3 enrichment accounted again for only a minor proportion of these DNA-methylated repressed promoters (7%) at least within the tiled region (A, right).
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. (more ...)
We next determined whether these proportions were different from those among all expressed or repressed RefSeq promoters that are also modified. B shows that histone modification enrichment patterns on these promoters were very similar to those of DNA-methylated expressed or repressed promoters. Thus, differential histone modification marking of the promoter of expressed or repressed genes is independent of their DNA methylation state in the genomic regions examined. Lastly, we showed, however, that these histone modification patterns were clearly distinct form those of all RefSeq promoters, regardless of transcriptional status (C). Therefore, in addition to the profile of DNA methylation, epigenetic markings such as trimethylated H3K4 and K27, and to a lesser extent K9, delineate distinct chromatin states on a subset of transcriptionally active versus inactive promoters (A). Of these modifications, however, only H3K9me3 seems to be differentially enriched on DNA hypermethylated versus hypomethylated repressed promoters (p < 0.01; chi-square test; , A and B).
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) (more ...)