BackgroundMicroRNAs (miRNAs) are a family of small, non-coding single-stranded RNA molecules involved in post-transcriptional regulation of gene expression. As such, they are believed to play a role in regulating the step-wise changes in gene expression patterns that occur during cell fate specification of multipotent stem cells. Here, we have studied whether terminal differentiation of C2C12 myoblasts is indeed controlled by lineage-specific changes in miRNA expression.ResultsUsing a previously generated RNA polymerase II (Pol-II) ChIP-on-chip dataset, we show differential Pol-II occupancy at the promoter regions of six miRNAs during C2C12 myogenic versus BMP2-induced osteogenic differentiation. Overexpression of one of these miRNAs, miR-378, enhances Alp activity, calcium deposition and mRNA expression of osteogenic marker genes in the presence of BMP2.ConclusionsOur results demonstrate a previously unknown role for miR-378 in promoting BMP2-induced osteogenic differentiation.

Mesenchymal progenitor cells can be differentiated in vitro into myotubes that exhibit many characteristic features of primary mammalian skeletal muscle fibers. However, in general, they do not show the functional excitation-contraction coupling or the striated sarcomere arrangement typical of mature myofibers. Epigenetic modifications have been shown to play a key role in regulating the progressional changes in transcription necessary for muscle differentiation. In this study, we demonstrate that treatment of murine C2C12 mesenchymal progenitor cells with 10 μM of the DNA methylation inhibitor 5-azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the up-regulation of muscle genes at the myoblast stage, while at later stages nearly 50% of the 5AC-treated myotubes displayed a mature, well-defined sarcomere organization, as well as spontaneous contractions that coincided with action potentials and intracellular calcium transients. Both the percentage of striated myotubes and their contractile activity could be inhibited by 20 nM TTX, 10 μM ryanodine, and 100 μM nifedipine, suggesting that action potential-induced calcium transients are responsible for these characteristics. Our data suggest that genomic demethylation induced by 5AC overcomes an epigenetic barrier that prevents untreated C2C12 myotubes from reaching full maturity.

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.

DNA methylation is an important epigenetic regulator of gene expression. Abnormalities in DNA methylation patterns have been associated with various developmental and proliferative diseases, particularly cancer. Targeting DNA methyltransferases (DNMTs) represents a promising strategy for the treatment of such diseases. Current DNMT inhibitors suffer important drawbacks with respect to their efficacy, specificity, and toxicity. In this study, we have set up a robust in vitro bacterial M.SssI DNMT activity assay to systematically screen a collection of 26 240 compounds that were predicted to compete with the S-adenosyl-L-methionine (SAM) substrate of DNMT. This resulted in the identification of a novel set of structurally distinct inhibitors of M.SssI DNMT activity. Although molecular docking studies using an M.SssI homology model suggest that these compounds might compete with SAM binding, mode of activity (MoA) assays are still needed to confirm this hypothesis. Our set of novel M.SssI DNMT inhibitors, once confirmed in an orthogonal DNMT assay, may thus serve as a starting point to identify and characterize suitable lead candidates for further drug optimization.