DNA methylation restricts spontaneous multi-lineage differentiation of mesenchymal progenitor cells, but is stable during growth factor-induced terminal differentiation

Abstract

The progressive restriction of differentiation potential from pluripotent embryonic stem cells, via multipotent progenitor cells to terminally differentiated, mature somatic cells, involves step-wise changes in transcription patterns that are tightly controlled by the coordinated action of key transcription factors and changes in epigenetic modifications. While previous studies have demonstrated tissue-specific differences in DNA methylation patterns that might function in lineage restriction, it is unclear at what exact developmental stage these differences arise. Here, we have studied whether terminal, multi-lineage differentiation of C2C12 myoblasts is accompanied by lineage-specific changes in DNA methylation patterns. Using bisulfite sequencing and genome-wide methylated DNA- and chromatin immunoprecipitation-on-chip techniques we show that in these cells, in general, myogenic genes are enriched for RNA polymerase II and hypomethylated, whereas osteogenic genes show lower polymerase occupancy and are hypermethylated. Removal of DNA methylation marks by 5-azacytidine (5AC) treatment alters the myogenic lineage commitment of these cells and induces spontaneous osteogenic and adipogenic differentiation. This is accompanied by upregulation of key lineage-specific transcription factors. We subsequently analyzed genome-wide changes in DNA methylation and polymerase II occupancy during BMP2-induced osteogenesis. Our data indicate that BMP2 is able to induce the transcriptional program underlying osteogenesis without changing the methylation status of the genome. We conclude that DNA methylation primes C2C12 cells for myogenesis and prevents spontaneous osteogenesis, but still permits induction of the osteogenic transcriptional program upon BMP2 stimulation. Based on these results, we propose that cell type-specific DNA methylation patterns are established prior to terminal differentiation of adult progenitor cells. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

PDGFRβ Expression and Function in Fibroblasts Derived from Pluripotent Cells is Linked to DNA Demethylation

“…For example, gene promoters related to mesenchymal cell fate determination, such as LEP and PPARG2, demonstrate demethylation upon differentiation of mesenchymal cells but not other cell types or lineages (Noer et al, 2006). In addition, it has been shown that DNA demethylation in myoblasts can activate mesenchymal gene expression and induce trans-differentiation to osteogenic and adipogenic fates (Hupkes et al, 2011). Interestingly, it has recently been shown that differences exist in methylation at lineage-specific genes between adult MSCs and putative MSCs derived from ESCs that might account for differences in differentiation potential of MSC populations (Sørensen et al, 2010).…”

Impact of Tissue-Specific Stem Cells on Lineage-Specific Differentiation: A Focus on the Musculoskeletal System

“…Hupkes et al . postulated that DNA methylation preprogramming could underlie the default differentiation of C2C12 cells toward the myogenic lineage (81). Collas described 400–700 hypermethylated genes specific to ADSCs, BMSCs, and muscle progenitor cells (MPCs) and commented that these methylation patterns might be determined by the tissue-specific stem cell niche (82).…”

“…While CpG methylation is a well-studied epigenetic modification to DNA, research indicates poor correlation between gene expression and promoter methylation, suggesting that other epigenetic mechanisms may also be important determinants for lineage preference (77, 81, 83). Additionally, many studies have discovered general hypomethylation of lineage-specific promoter regions in mesenchymal and non-mesenchymal ASCs, regardless of origin (61, 84, 85).…”

DNA methylation in stem cell renewal and multipotency

“…Characterization of DNA methylation profiles of all human RefSeq promoters in mesenchymal adult stem cells from various origins, including adipose, hematopoietic, and neural progenitors and muscle tissue, shows that the majority of the lineage-specific genes are hypomethylated even if the progenitor is not able to differentiate into this specific lineage [10]. There are some examples of epigenetic silencing associated with restriction to differentiation: endothelial markers such as CD31 and CD144 are strongly methylated in ASCs that show very limited capacity for endothelial differentiation [36] or osteogenic and adipogenic restriction of C2C12 myoblast cell line differentiation [37]. Furthermore, the restriction for differentiation in specific programs imposed by means of DNA methylation is established early in development, in the progenitor state, and persists after differentiation, as most of the hypermethylated promoters in undifferentiated cells remain hypermethylated in somatic cells [10,37].…”