title: 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.
authors.affiliation: 1 Mesenchymal Stem Cell Laboratory, Faculty of Health Sciences, School of Medicine, The University of Adelaide , Adelaide, Australia .|2 Cancer Theme, South Australian Health and Medical Research Institute , Adelaide, Australia .
authors.forename: Sarah
authors.initials: S
authors.lastname: Hemming
authors.affiliation: 1 Mesenchymal Stem Cell Laboratory, Faculty of Health Sciences, School of Medicine, The University of Adelaide , Adelaide, Australia .|2 Cancer Theme, South Australian Health and Medical Research Institute , Adelaide, Australia .
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Cakouros
authors.affiliation: 2 Cancer Theme, South Australian Health and Medical Research Institute , Adelaide, Australia .|3 Myeloma Research Laboratory, Faculty of Health Sciences, School of Medicine, The University of Adelaide , Adelaide, Australia .|4 SA Pathology , Adelaide, Australia .
authors.forename: Kate
authors.initials: K
authors.lastname: Vandyke
authors.affiliation: 5 Division of Bioinformatics, Walter and Eliza Hall Institute for Medical Research , Melbourne, Australia .
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Davis
authors.affiliation: 2 Cancer Theme, South Australian Health and Medical Research Institute , Adelaide, Australia .|3 Myeloma Research Laboratory, Faculty of Health Sciences, School of Medicine, The University of Adelaide , Adelaide, Australia .
authors.forename: Andrew C W
authors.initials: AC
authors.lastname: Zannettino
authors.affiliation: 1 Mesenchymal Stem Cell Laboratory, Faculty of Health Sciences, School of Medicine, The University of Adelaide , Adelaide, Australia .|2 Cancer Theme, South Australian Health and Medical Research Institute , Adelaide, Australia .
authors.forename: Stan
authors.initials: S
authors.lastname: Gronthos
mesh_terms: D000328:Adult; D002454:Cell Differentiation / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D000071221:Enhancer of Zeste Homolog 2 Protein / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006657:Histones / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D060588:LIM Domain Proteins / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D008745:Methylation; D009124:Muscle Proteins / Q000378:metabolism; D064547:Myxovirus Resistance Proteins / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D010012:Osteogenesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012097:Repressor Proteins / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics
chemical_list: C101149:FHL1 protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; C579071:MX1 protein, human; D009124:Muscle Proteins; D064547:Myxovirus Resistance Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D000071221:Enhancer of Zeste Homolog 2 Protein; D008239:Lysine