The methyltransferase, Enhancer of Zeste homology 2 (EZH2), trimethylates histone 3 lysine 27 (H3K27me3) on chromatin and this repressive mark is removed by lysine demethylase 6A (KDM6A). Loss of these epigenetic modifiers results in developmental defects. We demonstrate that Ezh2 and Kdm6a transcript levels change during differentiation of multipotential human bone marrow-derived mesenchymal stem cells (MSC). Enforced expression of Ezh2 in MSC promoted adipogenic in vitro and inhibited osteogenic differentiation potential in vitro and in vivo, whereas Kdm6a inhibited adipogenesis in vitro and promoted osteogenic differentiation in vitro and in vivo. Inhibition of EZH2 activity and knockdown of Ezh2 gene expression in human MSC resulted in decreased adipogenesis and increased osteogenesis. Conversely, knockdown of Kdm6a gene expression in MSC leads to increased adipogenesis and decreased osteogenesis. Both Ezh2 and Kdm6a were shown to affect expression of master regulatory genes involved in adipogenesis and osteogenesis and H3K27me3 on the promoters of master regulatory genes. These findings demonstrate an important epigenetic switch centered on H3K27me3 which dictates MSC lineage determination. STEM CELLS 2014;32:802-815

BackgroundThe 5 hydroxymethylation (5hmC) mark and TET DNA dioxygenases play a pivotal role in embryonic stem cell differentiation and animal development. However, very little is known about TET enzymes in lineage determination of human bone marrow-derived mesenchymal stem/stromal cells (BMSC). We examined the function of all three TET DNA dioxygenases, responsible for DNA hydroxymethylation, in human BMSC cell osteogenic and adipogenic differentiation.ResultsWe used siRNA knockdown and retroviral mediated enforced expression of TET molecules and discovered TET1 to be a repressor of both osteogenesis and adipogenesis. TET1 was found to recruit the co-repressor proteins, SIN3A and the histone lysine methyltransferase, EZH2 to osteogenic genes. Conversely, TET2 was found to be a promoter of both osteogenesis and adipogenesis. The data showed that TET2 was directly responsible for 5hmC levels on osteogenic and adipogenic lineage-associated genes, whereas TET1 also played a role in this process. Interestingly, TET3 showed no functional effect in BMSC osteo-/adipogenic differentiation. Finally, in a mouse model of ovariectomy-induced osteoporosis, the numbers of clonogenic BMSC were dramatically diminished corresponding to lower trabecular bone volume and reduced levels of TET1, TET2 and 5hmC.ConclusionThe present study has discovered an epigenetic mechanism mediated through changes in DNA hydroxymethylation status regulating the activation of key genes involved in the lineage determination of skeletal stem cells, which may have implications in BMSC function during normal bone regulation. Targeting TET molecules or their downstream targets may offer new therapeutic strategies to help prevent bone loss and repair following trauma or disease.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0247-4) contains supplementary material, which is available to authorized users.

Histone three lysine 27 (H3K27) methyltransferase enhancer of zeste homolog 2 (EZH2) is a critical epigenetic modifier, which regulates gene transcription through the trimethylation of the H3K27 residue leading to chromatin compaction and gene repression. EZH2 has previously been identified to regulate human bone marrow-derived mesenchymal stem cells (MSC) lineage specification. MSC lineage specification is regulated by the presence of EZH2 and its H3K27me3 modification or the removal of the H3K27 modification by lysine demethylases 6A and 6B (KDM6A and KDM6B). This study used a bioinformatics approach to identify novel genes regulated by EZH2 during MSC osteogenic differentiation. In this study, we identified the EZH2 targets, ZBTB16, MX1, and FHL1, which were expressed at low levels in MSC. EZH2 and H3K27me3 were found to be present along the transcription start site of their respective promoters. During osteogenesis, these genes become actively expressed coinciding with the disappearance of EZH2 and H3K27me3 on the transcription start site of these genes and the enrichment of the active H3K4me3 modification. Overexpression of EZH2 downregulated the transcript levels of ZBTB16, MX1, and FHL1 during osteogenesis. Small interfering RNA targeting of MX1 and FHL1 was associated with a downregulation of the key osteogenic transcription factor, RUNX2, and its downstream targets osteopontin and osteocalcin. These findings highlight that EZH2 not only acts through the direct regulation of signaling modules and lineage-specific transcription factors but also targets many novel genes important for mediating MSC osteogenic differentiation.

In this study, we examined the functional importance of EZH2 during skeletal development and homeostasis using the conditional deletion of ( ) in early mesenchyme with the use of a Prrx-1-cre driver mouse (). Heterozygous ) newborn and 4-wk-old mice exhibited increased skeletal size, growth plate size, and weight when compared to the wild-type control (), whereas homozygous deletion of () resulted in skeletal deformities and reduced skeletal size, growth plate size, and weight in newborn and 4-wk-old mice. mice exhibited enhanced trabecular patterning. Osteogenic cortical and trabecular bone formation was enhanced in and animals. and mice displayed thinner cortical bone and decreased mechanical strength compared to the wild-type control. Differences in cortical bone thickness were attributed to an increased number of osteoclasts, corresponding with elevated levels of the bone turnover markers cross-linked C-telopeptide-1 and tartrate-resistant acid phosphatase, detected within serum. Moreover, mice displayed increased osteoclastogenic potential coinciding with an upregulation of and expression by mesenchymal stem cells (MSCs). MSCs isolated from mice also exhibited increased trilineage potential compared with wild-type bone marrow stromal/stem cells (BMSCs). Gene expression studies confirmed the upregulation of known target genes in bone tissue, many of which are involved in Wnt/BMP signaling as promoters of osteogenesis and inhibitors of adipogenesis. In summary, EZH2 appears to be an important orchestrator of skeletal development, postnatal bone remodelling and BMSC fate determination and -Hemming, S., Cakouros, D., Codrington, J., Vandyke, K., Arthur, A., Zannettino, A., Gronthos, S. EZH2 deletion in early mesenchyme compromises postnatal bone microarchitecture and structural integrity and accelerates remodeling.

Basic helix-loop-helix (bHLH) transcription factors are pivotal regulators of cellular differentiation and development. The bHLH factor, Twist-1 has previously been found to control bone marrow stromal/stem cells (BMSC) self-renewal, life span, and differentiation, however not much is known about its mechanism of action. In this study, we have discovered a novel Twist-1 regulated bHLH gene, Hes4, expressed in humans, but not in mice. Its closest homologue in both humans and mice is Hes1. Overexpression and knockdown studies demonstrated that Hes4 promotes osteogenesis resulting in an increase in Runx2, osteocalcin, osteopontin, and bone sialoprotein expression. Conversely, Hes4 was found to inhibit adipogenesis accompanied by a decrease in PPARγ2, adiponectin, and adipsin expression. In vitro studies indicate that Hes4 employs a mechanism to counteract the negative function of Twist-1 on osteogenesis by binding to Twist-1 and inhibiting the ability of Twist-1 to bind and inhibit Runx2. In vivo chromatin immunoprecipitation and in vitro reporter assays illustrated that Runx2 recruitment to the osterix promoter, was found to be enhanced in the presence of Hes4 and inhibited in the presence of Twist-1. Therefore, Hes4 antagonizes the function of Twist-1 to regulate lineage commitment of BMSC. These studies highlight the potential differences in molecular mechanisms that regulate BMSC osteogenic differentiation between human and mouse.

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BackgroundDuchenne muscular dystrophy (DMD) is a lethal X-linked muscle wasting disorder caused by the absence of dystrophin, a large cytoskeletal muscle protein. Increasing the levels of the dystrophin-related-protein utrophin is a highly promising therapy for DMD and has been shown to improve pathology in dystrophin-deficient mice. One contributing factor to muscle wasting in DMD is mitochondrial pathology that contributes to oxidative stress and propagates muscle damage. The purpose of this study was to assess whether utrophin could attenuate mitochondria pathology and oxidative stress.MethodsSkeletal muscles from wildtype C57BL/10, dystrophin-deficient mdx, dystrophin/utrophin double knockout (dko) and dystrophin-deficient mdx/utrophin over-expressing mdx-Fiona transgenic mice were assessed for markers of mitochondrial damage.ResultsUsing transmission electron microscopy, we show that high levels of utrophin ameliorate the aberrant structure and localisation of mitochondria in mdx mice whereas absence of utrophin worsened these features in dko mice. Elevated utrophin also reverts markers of protein oxidation and oxidative stress, elevated in mdx and dko mice, to wildtype levels. These changes were observed independently of a shift in oxidative phenotype.ConclusionThese findings show that utrophin levels influence mitochondrial pathology and oxidative stress. While utrophin deficiency worsens the pathology, utrophin over-expression in dystrophic muscle benefits mitochondria and attenuates the downstream pathology associated with aberrant mitochondrial function.Electronic supplementary materialThe online version of this article (10.1186/s13395-017-0139-5) contains supplementary material, which is available to authorized users.

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