Identification of Novel EZH2 Targets Regulating Osteogenic Differentiation in Mesenchymal Stem Cells

Abstract

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.

EZH2 deletion in early mesenchyme compromises postnatal bone microarchitecture and structural integrity and accelerates remodeling

“…Moreover, EZH2 was shown to function as a repressor of osteogenic differentiation through the direct methylation of genes critical for osteogenic lineage specification. More recently, bioinformatic analyses of gene expression and ChIPseq data sets of human MSC osteogenic differentiation revealed several novel Ezh2 target molecules mediating MSC osteogenesis (41). Although some of these genes (summarized in Fig.…”

“…We analyzed the gene expression patterns of known Ezh2 target genes regulated during human MSC osteogenic differentiation (41) in long-bone samples collected from wild-type and newborn Ezh2 2/2 mice. The data showed that most of these genes (with the exception of FHFL1) were significantly upregulated in the long bones of Ezh2-null mice compared with wild-type controls, correlating to an increase in the expression of osteogenesis inductive factors such as Bmp2 and Runx2 (Fig.…”

Epigenetics as a New Frontier in Orthopedic Regenerative Medicine and Oncology

“…Studies on the EZH2 null mutation in chondrocytes (using the COL2a1‐Cre driver) revealed that EZH2 deficiency reduces H3K27me3 levels, yet remarkably had minimal impact on skeletal development or maturation of articular cartilage . Nevertheless, EZH2 target gene analysis in either osteoblasts or chondrocytes revealed that EZH2 enhances expression of osteogenic markers in chondrocytes, including BMP2 and BMP2 responsive transcription factors SP7 and extracellular matrix proteins (e.g., IBSP and BGLAP) as well as a large number other targets that have been identified in MSCs . However, EZH1 partially compensates for loss of EZH2 during skeletal development.…”

Local delivery of tetramethylpyrazine eliminates the senescent phenotype of bone marrow mesenchymal stromal cells and creates an anti‐inflammatory and angiogenic environment in aging mice

“…Of note, several studies have demonstrated that Ezh2 is an essential regulator of bone formation through modulating the osteogenic differentiation of MSCs (Chen et al., 2016; Hemming et al., 2016, 2017). Additionally, Li et al.…”