Review
Epigenetic approaches to regeneration of bone and cartilage from stem cells 1.
Introduction
2.
Epigenetic gene regulation and chromatin structure
3.
Epigenetics of stem cell
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differentiation 4.
Epigenetics in osteogenic differentiation of MSCs
5.
Epigenetics in chondrogenic differentiation of MSC
6.
Expert opinion
Gun-Il Im† & Kyung-Ju Shin Dongguk University Ilsan Hospital, Department of Orthopaedics, Goyang, Korea
Introduction: Embryonic stem cells (ESCs) or adult stem cells, especially mesenchymal stem cells (MSCs), have been intensively studied for skeletal tissue regeneration including bone and cartilage. Epigenetic mechanisms play essential roles in stem cell maintenance and differentiation. However, little is known about the epigenetic regulation of osteogenesis and chondrogenesis of stem cells. Areas covered: In this review, features of ESCs and adult stem cells, epigenetics and chromatin structure, as well as epigenetic mechanisms, such as chromatin remodeling, DNA methylation and histone modifications, polycomb group (PcG) proteins and microRNAs are described. Epigenetic researches of stem cell are introduced. Expert opinion: Epigenetic alterations of stem cell during the in vitro differentiation can be controlled for clinical applications. MSCs are effective resources for skeletal tissue regeneration in both undifferentiated and differentiated states. Understanding epigenetic signatures of MSC is crucial to maintain the stemness. In addition, investigation of epigenetic changes in the differentiation of MSCs is very important to develop methods or chemicals to promote efficient differentiation of MSCs. Inhibition of PcG protein enhancer of zeste (Ezh2) a chromatin modifier, could be a promising candidate to improve MSC differentiation by decreasing Ezh2-mediated H3K27me3. Keywords: chondrogenesis, epigenetics, osteogenesis, skeletal tissue regeneration, stem cells Expert Opin. Biol. Ther. [Early Online]
1.
Introduction
Regenerative medicine in the musculoskeletal system aims to trigger the intrinsic regenerative ability of damaged tissues. It can provide novel cell-based therapies for the tissues that lack an intrinsic repair process such as articular cartilage or for large tissue defects such as found in massive bone loss [1]. Cell-based therapies have been intensively studied using both embryonic stem cells (ESCs) and adult stem cells, in addition to somatic cells [2-4]. ESCs that are derived from the cells of the inner cell mass of the mammalian blastocysts have an unlimited potential for self-renewal and pluripotency, which is defined as the ability to generate stem cells and differentiate into cells of all three germ layers, ectoderm, mesoderm and endoderm [5]. This pluripotency makes it possible to apply ESCs for cell-replacement therapies for degenerative diseases, including type 1 diabetes, and Parkinson disease [6]. ESCs can be differentiated into osteogenic and chondrogenic lineages, to be used for skeletal regeneration [7,8]. However, the use of ESCs is associated with ethical problems because embryos are destroyed in ESC induction. Safety issues are also concerns as ESCs form teratoma [9,10]. Adult stem cells also have a potential to self-renew and to differentiate, but their differentiation capacity is limited to specific types of cells (multi-lineage 10.1517/14712598.2015.960838 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted
1
G. -I. Im & K. -J. Shin
Article highlights. .
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Connecticut on 10/11/14 For personal use only.
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.
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Embryonic stem cells and mesenchymal stem cells (MSCs) have been extensively studied in the regenerative medicine of musculoskeletal system. Epigenetic mechanisms that play an essential role in differentiation and development are described in detail. Transcriptional gene expression changes during the differentiation of stem cells are regulated by epigenetic mechanisms, such as chromatin remodeling including Polycomb protein chromatin modifiers, DNA methylation and histone modifications. In addition, microRNAs that regulates the osteogenic and chondrogenic differentiation of MSCs are introduced. Epigenetic studies in osteogenesis and chondrogenesis of MSCs are reviewed. DNA methylation of osteocalcin (OC) decreases and its gene expression increases during in vitro osteogenic differentiation of MSCs. Histone acetylation is also mediated with developmental induction of OC in the bone. Histone acetylation of Sox9 through p300 activates Sox9, the master chondrogenic transcription factor, during chondrogenesis. Understanding epigenetic signatures of MSCs is crucial for maintaining the stemness. Investigation of epigenetic changes of MSC differentiation in vitro is also important to promote efficient differentiation of MSCs.
This box summarizes key points contained in the article.
differentiation). In the body, adult stem cells function to preserve normal tissue homeostasis and repair potential injuries in the various tissues [11]. Mesenchymal stem cells (MSCs) are typical adult stem cells that self-renew and differentiate into tissues, including bone, cartilage and adipose [1,12]. MSCs possess a good potential for skeletal tissue regeneration due to their convenient isolation and immunomodulatory capability, as well as their ability to trans-differentiate and to create a tissue microenvironment (stem cell niche) that is favorable for tissue repair [13]. The modulation of osteogenic and chondrogenic potential of MSC is crucial in the clinical application for skeletal regeneration [14]. However, when MSCs undergo chondro-induction, the quality of newly formed cartilaginous tissue is usually inferior to that formed from chondrocytes and has an unstable chondrocytic phenotype [3,15]. One of the underlying causes seems to be epigenetic differences. Epigenetic mechanisms including DNA methylation, histone modifications and chromatin remodeling have essential roles in the maintenance and differentiation of stem cells [11,16,17]. Recently, the discovery of noncoding RNAs that cause changes in the levels of the coded posttranscriptional proteins has expanded the area covered by the term ‘epigenetics’ [4]. During the differentiation of stem cells, genes involved in a specific cell type are up-regulated and genes associated with the stemness are repressed. The cell fate determination requires elaborate balanced control of genetic and epigenetic 2
programs. Epigenetic mechanisms regulate timely expression of lineage-control genes by modifications in chromatin structure, controlling the accessibility of genes [13]. The epigenetic status of adult stem cells that determine their multipotentlineage differentiation is distinct from both ESCs and fully differentiated somatic cells [6,18]. Therefore, understanding the epigenetic status of stem cells would provide a cue in improving stem cell therapy for skeletal tissue regeneration. In this review, an overview of epigenetic features and epigenetic regulations of osteogenic and chondrogenic differentiation derived from stem cells is introduced.
Epigenetic gene regulation and chromatin structure
2.
In eukaryotes, gene expression is regulated at the chromatin level by mechanisms called ‘epigenetics,’ which means heritable changes in gene expression without changes of the underlying DNA nucleotide sequence [19]. Epigenetic alteration is heritable and stably maintained although potentially reversible [20]. Epigenetic gene regulations at the chromatin are involved in diverse biological processes such as cellular differentiation, development and regeneration of tissues (Table 1) [21-23]. The basic molecular repeating unit of chromatin is a nucleosome, in which 145 -- 147 bp of DNA is wrapped nearly twice around a histone octamer, containing two molecules of each histone H2A, H2B, H3 and H4 [24-26]. The linker histone H1 that separates adjacent nucleosomes is required for folding to the higher order chromatin structure. The core histones, especially N-terminal tails of histones H3 and H4, are well conserved from yeast to human, whereas the linker histone H1 is less conserved [27-29]. The chromatin structure is dynamic. Loosely packed chromatin (euchromatin) is more accessible to the transcriptional apparatus leading to active gene expression. On the other hand, tightly packed chromatin (heterochromatin) physically restricts access of the transcriptional machinery to DNA, leading to transcriptional inactivity [30]. There are two distinct heterochromatin genomic regions: facultative and constitutive heterochromatin. Facultative heterochromatin regions contain genes that are differentially expressed and then become silenced in differentiation and development, whereas constitutive heterochromatin regions such as centromeres and telomeres contain permanently silenced genes (Figure 1) [31]. Conversion between the euchromatin and heterochromatin states is controlled by two basic epigenetic processes. The first is the covalent modifications of histone proteins and DNA, which include DNA methylation and histone modification. The second is chromatin remodeling, which includes nucleosome sliding to a new position on the genomic DNA, conformational changes in histone--DNA interactions, and dynamic loss and gain of histones through nucleosome disassembly and reassembly [32-35]. Chromatin remodelers, polycomb group (PcG) proteins and trithorax group (TrxG) proteins are
Expert Opin. Biol. Ther. (2014) 15(2)
Epigenetic approaches to regeneration of bone and cartilage from stem cells
Table 1. Epigenetic studies of musculoskeletal regeneration. Differentiation Osteogenesis
Cell type
Epigenetic mechanism
hMSC
DNA hypermethylation
mMSC
DNA hypomethylation (by mechanical stimulus) DNA hypomethylation
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Rat ROS17/2.8 osteosarcoma cell Rat ROS17/2.8 hMSC
Histone acetylation H3K9Ac/H3K9me2 (ChIP-on-chip)
mC3H10T1/2 mNIH 3T3 rat ROS17/2.8 & primary osteoblast hMSC
Chondrogenesis
Gene
Histone acetylation (HOXA10-mediated)
PcG protein, CDK1-dependent phosphorylation of EZH2 DNA methylation
Human synovium-derived MSC hMSC hMSC h chondrocyte h chondrosarcoma cell line (SW1353)
DNA demethylation Histone acetylation Co-activator CBP/p300
Ref.
Brachyury, LIN28 Trip10 Osteopontin
[65] [66]
Osteocalcin (OC)
[68]
OC OCT4 and NANOG (deacetylation/dimethylation) OPG and ALPL (acetylation/ de-dimethylation) Runx2, alkaline phosphatase, OC, and bone sialoprotein
[69] [70]
Runx2 and Osteopontin
[72]
SOX9, RUNX2, CHM1, CHAD, FGFR3, GEM1, GPR39, and SDF1 COL10A1 Sox9 Col2a1
[90]
[67]
[71]
[91] [92,94]
CBP: CREB-binding protein; CDK1: Cyclin-dependent kinase 1; MSC: Mesenchymal stem cell; PcG: Polycomb group.
HAT HAT
HAT TF
Histone acetylation Euchromatin
DME
DNMT
HAT
HDAC
MBD MBD
HDAC
HDAC
DNA methylation Heterochroamtin
Figure 1. There is euchromatin and heterochromatin structures in the genome. DNA methylation in the heterochromatin serves as a binding site for MBD proteins that can stably associate with HDACs, leading to condense chromatin structure. DME: Demethylase; DNMT: DNA methyltransferase; HAT: Histone acetyltransferase; HDAC: Histone deacetylase; MBD: Methyl binding domain; TF: Transcription factor.
Expert Opin. Biol. Ther. (2014) 15(2)
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G. -I. Im & K. -J. Shin
N-SGRGKQGCKARAKSK.... H2A .. KTESHH......-C 5 9 13 15 119
N-ARTKQTARKSTGGKAPRKQLATKAARKSA...K... 4 9 14 18 23 27 36
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N-SGRGKGGKGLGKGGAKRHRKVLRDNIQ...... 5 8 12 16 20 20
H3
H4
Acetylation Methylation Phosphorylation Ubiquitination
N-PEPAKSAPAPKKGSKKA... 5 12 15
H2B
...KAVT......-C 120
Figure 2. Typical histone tail sites for modification. Representative types of post-translational histone modification are acetylation, methylation, phosphorylation and ubiquitination.
essential for stem cell differentiation and early development [36-38]. These epigenetic mechanisms are described in detail below. DNA methylation DNA methylation is a major mechanism of epigenetics and essential for normal development, imprinting of specific genes, X chromosome inactivation and cell-type-specific gene expression [39,40]. In mammals, CpG dinucleotides are prevalent sites for DNA methylation. CpG islands are the clusters of CpGs associated with the promoters of ~30% of genes. CpG islands are unmethylated in the germline, but DNA methylation occurs in the particular gene promoters during development and tissue differentiation. The addition of a methyl group to cytosine of DNA nucleotides is mediated by DNA methyltransferases, DNMT1, DNMT3A, and DNMT3B [41-45]. 2.1
Histone modifications Histone modifications affect the chromatin structure and play fundamental roles in most biological processes, including DNA replication, transcription and DNA repair [31]. N-terminal tails of histones in the nucleosome protrude from globular histone proteins. Lysines and arginines in the unstructured amino-terminal tails are targets for various types of post-translational modifications, such as acetylation, methylation, phosphorylation, ubiquitination, ADP-ribosylation, sumoylation and deamination (Figure 2) [24,25]. Histone acetylation is one of the most abundant and dynamic modifications. The actual level of histone acetylation is dependent on regulated interplay between histone acetyltransferases (HATs) and histone deacetylases (HDACs) [46]. The 2.2
4
nomenclature for histone modification includes histone, residue and position of that residue in the protein, followed by type and number of modifications to that residue. For example, trimethylation of histone H3 on lysine 4 would be designated H3K4me3 [47]. H3K4me3 are found in the promoter regions of active genes within euchromatin, but not in silent heterochromatic regions [48,49]. Methylation of histones H3K9 and H3K27 are present in the promoters of inactive genes in heterochromatin regions [50]. Unlike histone acetylation, histone methylation does not change the charge of histone protein to weaken the interaction with DNAs. Instead, it recruits specific binding proteins such as PcG proteins and heterochromatin proteins 1 (HP1), which affect the chromatin structure. HP1 is recruited to H3K9 methylation in the promoter to repress genes [20,25]. Chromosome remodeling by PcG proteins PcG and TrxG proteins function to maintain specific cellular gene expression patterns and keep the identity of every cell type during stem cell differentiation. During development, PcG proteins maintain a repressed state of gene expression, whereas TrxG proteins counteract the silencing and maintain active states [38]. PcG proteins form multi-protein complexes, termed polycomb-repressive complexes (PRCs). There are two distinct PRC2 and PRC1 complexes. PRC2 contains enhancer of zeste (Ezh2), embryonic development protein (Eed), and suppressor of zeste12 (Suz12). PRC1 contains ring finger protein1 (Ring1), B lymphoma Mo-MLV insertion region1 (Bmi1), polycomb group ring finger2, and chromobox family proteins. Ring1 possesses an E3 ubiquitin ligase activity, which monoubiquitinates histone H2A at lysine 119 (H2AK119ub1). PcG proteins work together with 2.3
Expert Opin. Biol. Ther. (2014) 15(2)
Epigenetic approaches to regeneration of bone and cartilage from stem cells
TrxG RNA Pol II
Active chromatin
PcG
TrxG
PcG PcG PcG
Repressive chromatin
Bivalent chromatin
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PcG H3K4me3 H3K27me3
TrxG RNA Pol II
Poised chromatin
Figure 3. Bivalent chromatin structure in stem cells is regulated through chromatin remodeling by polycomb repressive complexes and trithorax G proteins. H3K4me3 and H3K27me3 are associated with the bivalent chromatin structure. PcG: Polycomb group; TrxG: Trithorax group.
histone modifications to regulate the chromatin structure and dynamically change gene expression during the differentiation. Ezh2 has an enzyme activity for H3K27me3, which serves as a signal for the binding of PRC1 proteins [36]. Genome-wide mapping of histone methylation profiles of ESCs revealed unique chromatin patterns called ‘bivalent domains,’ which hold both an active gene mark H3K4me3 in smaller regions and a repressive H3K27me3 in large regions in the genome. The function of bivalent domains is to silence developmental genes in ESCs and to keep them poised for activation upon differentiation at the same time (Figure 3) [51,52]. In mouse ESCs, multiple lineage-control essential transcription factor genes, Sox1, Nkx2-2, Msx1, Irx3, and Pax3, are bivalent and possess active marks including H3K9ac and H3K4me3 along with repressive mark H3K27me3 for activation during differentiation [53]. PRCs are essential to control epigenetic modifications in the differentiation to determine cell fate. PRC1 and PRC2 occupy the bivalent domains to regulate differentiation [36]. Eed (a PRC2 component)-deficient ESCs aberrantly induces the expression of lineage-control genes [51]. microRNAs microRNAs (miRNAs) are small 20 -- 23 bp cytoplasmic RNAs that control post-transcriptional gene expression by binding to target mRNAs. miRNAs interact with target mRNA through complementary base pairing between the miRNA and the 3¢-untranslated region of the mRNA, causing degradation of the mRNA target or translational suppression (Figure 4) [52]. Dicer, one of the indispensable components for miRNA synthesis, is essential for normal skeletal development [53]. Dicer-null mice have skeletal growth defects and die prematurely [53]. With the absence of processed miRNAs by removal of the Dicer and Drosha enzymes, osteogenic 2.4
differentiation and adipogenic differentiation of human mesenchymal stem cells (hMSCs) were inhibited [53]. Several miRNAs are known to affect DNA methyltransferases, thus changing the epigenetic status of genes. miRNA-29 targets DNMT 3A and 3B in lung cancer [54]. miRNA-152 targets DNMT 1 in hepatocellular carcinoma [55]. miRNA450a and miRNA-143 affect DNMT 3A in hepatocellular carcinoma and colorectal cancer, respectively [56,57]. Although these miRNAs were mostly reported from the study of oncogenesis, their possible role in osteogenesis and chondrogenesis should be a subject of future investigation.
3.
Epigenetics of stem cell differentiation
Stem cell maintenance and differentiation are associated with the modulation of epigenetic mechanisms, including chromatin remodeling, DNA methylation and histone modifications. ESC differentiation is accompanied by global gene expression changes. Genes regulating pluripotency and self-renewal become silenced, whereas genes associated with specific celltype become active. These global gene expression changes are controlled by epigenetic mechanisms that preferentially target transcription factors [11,16]. Epigenetic alterations occur gene-specifically and globally during ESC differentiation. Many studies have reported that DNA methylation and histone modification were associated with transcriptional changes of ESC differentiation. Heterochromatin mark, H3K9me3, was increased and euchromatin marks, H3 and H4 acetylation, were decreased in the global levels. Treatment of trichostatin A as HDAC inhibitor prevented ESC differentiation due to aberrant global histone acetylations. Global DNA methylation was also slightly increased upon differentiation [58,59].
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G. -I. Im & K. -J. Shin
miRNA gene
Nucleus
Pri-miRNA
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Drosha
Exportin5
Pri-miRNA
Cytoplasm
dsRNA Dicer miRNA: miRNA* duplex
siRNA duplex
mRNA cleavage
Unwind RISC
RISC
Target mRNA
RISC Ribosome RISC ORF Translational repression
Figure 4. Working mechanism of microRNA. dsRNA: Double strand RNA; miRNA: MicroRNA; ORF: Open reading frame; RISC: RNA induced silencing complex; siRNA: Small interfering RNA.
Histone modification states of multipotent adult stem cells such as MSCs, neural progenitor cells (NPCs), and hematopoietic stem cells (HSCs) are different from pluripotent ESCs due to their differentiation capabilities (Figure 5). Nonetheless, epigenetic changes of DNA methylation and histone modifications are essential for the differentiation of adult stem cells. Many studies have shown that DNA methylation played a role in regulating neural stem cell and HSC differentiation. DNMT1 was expressed at high level in the central nerve system such as neurons and glial cells during embryogenesis. Mice lacking this enzyme in neural precursor cells had problems with neural function and died shortly after birth [60]. HSCs give rise to all types of blood including lymphoid and myeloid lineages. The expression of transcription factors involved in HSC differentiation was regulated by promoter methylation. Sp1 was expressed in HSCs and in B cell but not in T cells due to hypermethylation of Sp1 promoter in T cells [61]. De novo methyltransferases (DNMT3a and DNMT3b)-deficient HSCs had no capability for long6
term reconstitution of the hematopoietic system in bonemarrow transplantation assays [62]. These researches showed that DNA methylation functioned in both HSC differentiation and self-renewal. The chromatin of stem cells has bivalent domains controlled by PcG and TrxG proteins and these bivalent domains play a crucial role in developmental gene regulation [36]. Ezh2 that has an enzyme activity for H3K27me3 was highly expressed in adult stem cells such as NPCs and skin stem cells, but down-regulated during differentiation. The down-regulation of PcG protein expression can contribute to the differentiation of adult stem cells by de-repression of differentiation associated genes [63]. The injury signals have been shown to displace PcG proteins from poised differentiation genes to the muscle satellite cell marker Pax7 and to activate bivalent genes [64]. Regeneration signals can affect the balance of Ezh2 and demethylases of PcG proteins at the bivalent domain of adult stem cells. Reduction of repression by PcG proteins induces differentiation by regeneration signals.
Expert Opin. Biol. Ther. (2014) 15(2)
Epigenetic approaches to regeneration of bone and cartilage from stem cells
Differentiation
Neuronal cell NPC Osteocyte
Differentiation
Muscle cell ESC
MSC
Chondrocyte Differentiation
Differentiation
Blood cells Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Connecticut on 10/11/14 For personal use only.
HSC Embryonic stem cell Pluripotent (Oct4, Sox2, Nanog, cMyc, H3K4me3 > H3K27me3 H3K4me3/H3K27me3)
Adult stem cell Multipotent (lineage regulators, H3K4me3/H3K27me3 H3K4me3 < H3K27me3)
Somatic cell Differentiated cell (tissue-specific transcription factors H3K4me3 or H3K27me3)
Figure 5. Histone modification status of ESCs, ASCs, and differentiated cells. ESCs and ASCs possess a bivalent chromatin structure that contains both H3K4me3 and H3K27me3 (H3K4me3/H3K27me3). ASCs: Adult stem cells; ESCs: Embryonic stem cells; HSC: Hematopoietic stem cell; MSC: Mesenchymal stem cell; NPC: Neural progenitor cell.
Epigenetics in osteogenic differentiation of MSCs
4.
Epigenetic control of MSC differentiation into bone and cartilage has been investigated over the past decade. Several epigenetic regulations in osteogenic differentiation of MSCs have been reported [14]. Involvement of DNA methylation in osteogenic differentiation of MSCs has been reported. Osteogenic differentiation of MSCs was accompanied by reduced stemness-related genes Brachyury and LIN28 through hypermethylation of promoters of these genes [65]. The epigenetic modulation can improve osteogenic differentiation from MSCs. The endogenous (Trip10) expression was decreased with progressive accumulation by transfection of exogenous methylated Trip10. Transfection of in vitro methylated thyroid hormone receptor interactor (Trip10) promoter DNA into MSCs resulted in progressive accumulation of cytosine methylation at the endogenous Trip10 promoter, and reduced Trip10 expression. Reduced expression of Trip10 helped to accelerate MSC differentiation into osteocytes [66]. Also, mechanical stimulus was used to improve osteogenic differentiation from MSCs at the epigenetic level. Mechanical signals have been known to regulate the differentiation of stem cells. Mechanical stimulation of MSCs reduced DNA methylation on the promoters of osteogenic candidate genes, leading to increased expressions of these genes [67]. This study first demonstrated that the mechanical microenvironment could induce epigenetic changes that control osteogenic cell fate. Osteocalcin (OC), a bone-specific gene, was regulated by DNA methylation in osteogenic differentiation. Although this gene locus was significantly hypermethylated in undifferentiated cells, DNA methylation of OC decreased and its gene
expression increased during in vitro osteogenic differentiation from MSCs. This finding suggests a compelling example in the context of how epigenetic machinery can be targeted to benefit lives [68]. Histone modification of OC gene was also investigated during osteogenic differentiation. Both the promoter and coding regions of OC contained low level of acetylation of histone H3 and H4 during the proliferative period of osteogenic differentiation when this gene was inactive. However, OC was active in the mature osteoblasts and correlated with enriched H4 acetylation. Histone acetylation was mediated with developmental induction of OC in the bone [69]. ChIP-on-chip was performed to investigate the role of histone modification in the osteogenic differentiation from MSCs. H3K9Ac correlated with activating genes and H3K9me2 was associated with silencing genes. Many vitamin D receptor (VDR) elements were found at the gene promoters in both H3K9Acdecreased and H3K9me2-increased groups, suggesting that VDR might be a potential regulator for mediating deacetylation and dimethylation of H3K9 at specific gene promoters in the osteogenic differentiation from MSCs [70]. Histone methylation has a role in osteogenic differentiation from MSCs. The HoxA10 gene is necessary for embryonic patterning of skeletal elements. HoxA10 contributed to osteogenic lineage determination through total chromatin acetylation and enriched H3K4me3 of the genes. Increased HoxA10 activated Runx2, alkaline phosphatase and OC during osteogenic differentiation [71]. The function of PcG proteins in osteogenic differentiation of MSC has also been investigated. Activation of cyclindependent kinase 1 (CDK1) promoted osteogenic differentiation through disruption of the PRC2 complex. CDK1 phosphorylated Ezh2 and phosphorylated Ezh2 decreased
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G. -I. Im & K. -J. Shin
the interaction with Suz12 and Eed on the promoter of Runx2, one of Ezh2 target genes, which is an important transcription factor for osteogenic differentiation. Decreased H3K27me3 led to activate Runx2 expression during osteogenic differentiation. Genome-wide investigation of Ezh2 target genes was also performed in MSCs, before and after osteogenic differentiation by ChIP-on-chip. Over 4000 genes had Ezh2 binding before differentiation, but
Epigenetic approaches to regeneration of bone and cartilage from stem cells 1.
Introduction
2.
Epigenetic gene regulation and chromatin structure
3.
Epigenetics of stem cell
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Connecticut on 10/11/14 For personal use only.
differentiation 4.
Epigenetics in osteogenic differentiation of MSCs
5.
Epigenetics in chondrogenic differentiation of MSC
6.
Expert opinion
Gun-Il Im† & Kyung-Ju Shin Dongguk University Ilsan Hospital, Department of Orthopaedics, Goyang, Korea
Introduction: Embryonic stem cells (ESCs) or adult stem cells, especially mesenchymal stem cells (MSCs), have been intensively studied for skeletal tissue regeneration including bone and cartilage. Epigenetic mechanisms play essential roles in stem cell maintenance and differentiation. However, little is known about the epigenetic regulation of osteogenesis and chondrogenesis of stem cells. Areas covered: In this review, features of ESCs and adult stem cells, epigenetics and chromatin structure, as well as epigenetic mechanisms, such as chromatin remodeling, DNA methylation and histone modifications, polycomb group (PcG) proteins and microRNAs are described. Epigenetic researches of stem cell are introduced. Expert opinion: Epigenetic alterations of stem cell during the in vitro differentiation can be controlled for clinical applications. MSCs are effective resources for skeletal tissue regeneration in both undifferentiated and differentiated states. Understanding epigenetic signatures of MSC is crucial to maintain the stemness. In addition, investigation of epigenetic changes in the differentiation of MSCs is very important to develop methods or chemicals to promote efficient differentiation of MSCs. Inhibition of PcG protein enhancer of zeste (Ezh2) a chromatin modifier, could be a promising candidate to improve MSC differentiation by decreasing Ezh2-mediated H3K27me3. Keywords: chondrogenesis, epigenetics, osteogenesis, skeletal tissue regeneration, stem cells Expert Opin. Biol. Ther. [Early Online]
1.
Introduction
Regenerative medicine in the musculoskeletal system aims to trigger the intrinsic regenerative ability of damaged tissues. It can provide novel cell-based therapies for the tissues that lack an intrinsic repair process such as articular cartilage or for large tissue defects such as found in massive bone loss [1]. Cell-based therapies have been intensively studied using both embryonic stem cells (ESCs) and adult stem cells, in addition to somatic cells [2-4]. ESCs that are derived from the cells of the inner cell mass of the mammalian blastocysts have an unlimited potential for self-renewal and pluripotency, which is defined as the ability to generate stem cells and differentiate into cells of all three germ layers, ectoderm, mesoderm and endoderm [5]. This pluripotency makes it possible to apply ESCs for cell-replacement therapies for degenerative diseases, including type 1 diabetes, and Parkinson disease [6]. ESCs can be differentiated into osteogenic and chondrogenic lineages, to be used for skeletal regeneration [7,8]. However, the use of ESCs is associated with ethical problems because embryos are destroyed in ESC induction. Safety issues are also concerns as ESCs form teratoma [9,10]. Adult stem cells also have a potential to self-renew and to differentiate, but their differentiation capacity is limited to specific types of cells (multi-lineage 10.1517/14712598.2015.960838 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted
1
G. -I. Im & K. -J. Shin
Article highlights. .
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Connecticut on 10/11/14 For personal use only.
.
.
.
.
Embryonic stem cells and mesenchymal stem cells (MSCs) have been extensively studied in the regenerative medicine of musculoskeletal system. Epigenetic mechanisms that play an essential role in differentiation and development are described in detail. Transcriptional gene expression changes during the differentiation of stem cells are regulated by epigenetic mechanisms, such as chromatin remodeling including Polycomb protein chromatin modifiers, DNA methylation and histone modifications. In addition, microRNAs that regulates the osteogenic and chondrogenic differentiation of MSCs are introduced. Epigenetic studies in osteogenesis and chondrogenesis of MSCs are reviewed. DNA methylation of osteocalcin (OC) decreases and its gene expression increases during in vitro osteogenic differentiation of MSCs. Histone acetylation is also mediated with developmental induction of OC in the bone. Histone acetylation of Sox9 through p300 activates Sox9, the master chondrogenic transcription factor, during chondrogenesis. Understanding epigenetic signatures of MSCs is crucial for maintaining the stemness. Investigation of epigenetic changes of MSC differentiation in vitro is also important to promote efficient differentiation of MSCs.
This box summarizes key points contained in the article.
differentiation). In the body, adult stem cells function to preserve normal tissue homeostasis and repair potential injuries in the various tissues [11]. Mesenchymal stem cells (MSCs) are typical adult stem cells that self-renew and differentiate into tissues, including bone, cartilage and adipose [1,12]. MSCs possess a good potential for skeletal tissue regeneration due to their convenient isolation and immunomodulatory capability, as well as their ability to trans-differentiate and to create a tissue microenvironment (stem cell niche) that is favorable for tissue repair [13]. The modulation of osteogenic and chondrogenic potential of MSC is crucial in the clinical application for skeletal regeneration [14]. However, when MSCs undergo chondro-induction, the quality of newly formed cartilaginous tissue is usually inferior to that formed from chondrocytes and has an unstable chondrocytic phenotype [3,15]. One of the underlying causes seems to be epigenetic differences. Epigenetic mechanisms including DNA methylation, histone modifications and chromatin remodeling have essential roles in the maintenance and differentiation of stem cells [11,16,17]. Recently, the discovery of noncoding RNAs that cause changes in the levels of the coded posttranscriptional proteins has expanded the area covered by the term ‘epigenetics’ [4]. During the differentiation of stem cells, genes involved in a specific cell type are up-regulated and genes associated with the stemness are repressed. The cell fate determination requires elaborate balanced control of genetic and epigenetic 2
programs. Epigenetic mechanisms regulate timely expression of lineage-control genes by modifications in chromatin structure, controlling the accessibility of genes [13]. The epigenetic status of adult stem cells that determine their multipotentlineage differentiation is distinct from both ESCs and fully differentiated somatic cells [6,18]. Therefore, understanding the epigenetic status of stem cells would provide a cue in improving stem cell therapy for skeletal tissue regeneration. In this review, an overview of epigenetic features and epigenetic regulations of osteogenic and chondrogenic differentiation derived from stem cells is introduced.
Epigenetic gene regulation and chromatin structure
2.
In eukaryotes, gene expression is regulated at the chromatin level by mechanisms called ‘epigenetics,’ which means heritable changes in gene expression without changes of the underlying DNA nucleotide sequence [19]. Epigenetic alteration is heritable and stably maintained although potentially reversible [20]. Epigenetic gene regulations at the chromatin are involved in diverse biological processes such as cellular differentiation, development and regeneration of tissues (Table 1) [21-23]. The basic molecular repeating unit of chromatin is a nucleosome, in which 145 -- 147 bp of DNA is wrapped nearly twice around a histone octamer, containing two molecules of each histone H2A, H2B, H3 and H4 [24-26]. The linker histone H1 that separates adjacent nucleosomes is required for folding to the higher order chromatin structure. The core histones, especially N-terminal tails of histones H3 and H4, are well conserved from yeast to human, whereas the linker histone H1 is less conserved [27-29]. The chromatin structure is dynamic. Loosely packed chromatin (euchromatin) is more accessible to the transcriptional apparatus leading to active gene expression. On the other hand, tightly packed chromatin (heterochromatin) physically restricts access of the transcriptional machinery to DNA, leading to transcriptional inactivity [30]. There are two distinct heterochromatin genomic regions: facultative and constitutive heterochromatin. Facultative heterochromatin regions contain genes that are differentially expressed and then become silenced in differentiation and development, whereas constitutive heterochromatin regions such as centromeres and telomeres contain permanently silenced genes (Figure 1) [31]. Conversion between the euchromatin and heterochromatin states is controlled by two basic epigenetic processes. The first is the covalent modifications of histone proteins and DNA, which include DNA methylation and histone modification. The second is chromatin remodeling, which includes nucleosome sliding to a new position on the genomic DNA, conformational changes in histone--DNA interactions, and dynamic loss and gain of histones through nucleosome disassembly and reassembly [32-35]. Chromatin remodelers, polycomb group (PcG) proteins and trithorax group (TrxG) proteins are
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Epigenetic approaches to regeneration of bone and cartilage from stem cells
Table 1. Epigenetic studies of musculoskeletal regeneration. Differentiation Osteogenesis
Cell type
Epigenetic mechanism
hMSC
DNA hypermethylation
mMSC
DNA hypomethylation (by mechanical stimulus) DNA hypomethylation
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Rat ROS17/2.8 osteosarcoma cell Rat ROS17/2.8 hMSC
Histone acetylation H3K9Ac/H3K9me2 (ChIP-on-chip)
mC3H10T1/2 mNIH 3T3 rat ROS17/2.8 & primary osteoblast hMSC
Chondrogenesis
Gene
Histone acetylation (HOXA10-mediated)
PcG protein, CDK1-dependent phosphorylation of EZH2 DNA methylation
Human synovium-derived MSC hMSC hMSC h chondrocyte h chondrosarcoma cell line (SW1353)
DNA demethylation Histone acetylation Co-activator CBP/p300
Ref.
Brachyury, LIN28 Trip10 Osteopontin
[65] [66]
Osteocalcin (OC)
[68]
OC OCT4 and NANOG (deacetylation/dimethylation) OPG and ALPL (acetylation/ de-dimethylation) Runx2, alkaline phosphatase, OC, and bone sialoprotein
[69] [70]
Runx2 and Osteopontin
[72]
SOX9, RUNX2, CHM1, CHAD, FGFR3, GEM1, GPR39, and SDF1 COL10A1 Sox9 Col2a1
[90]
[67]
[71]
[91] [92,94]
CBP: CREB-binding protein; CDK1: Cyclin-dependent kinase 1; MSC: Mesenchymal stem cell; PcG: Polycomb group.
HAT HAT
HAT TF
Histone acetylation Euchromatin
DME
DNMT
HAT
HDAC
MBD MBD
HDAC
HDAC
DNA methylation Heterochroamtin
Figure 1. There is euchromatin and heterochromatin structures in the genome. DNA methylation in the heterochromatin serves as a binding site for MBD proteins that can stably associate with HDACs, leading to condense chromatin structure. DME: Demethylase; DNMT: DNA methyltransferase; HAT: Histone acetyltransferase; HDAC: Histone deacetylase; MBD: Methyl binding domain; TF: Transcription factor.
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N-SGRGKQGCKARAKSK.... H2A .. KTESHH......-C 5 9 13 15 119
N-ARTKQTARKSTGGKAPRKQLATKAARKSA...K... 4 9 14 18 23 27 36
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N-SGRGKGGKGLGKGGAKRHRKVLRDNIQ...... 5 8 12 16 20 20
H3
H4
Acetylation Methylation Phosphorylation Ubiquitination
N-PEPAKSAPAPKKGSKKA... 5 12 15
H2B
...KAVT......-C 120
Figure 2. Typical histone tail sites for modification. Representative types of post-translational histone modification are acetylation, methylation, phosphorylation and ubiquitination.
essential for stem cell differentiation and early development [36-38]. These epigenetic mechanisms are described in detail below. DNA methylation DNA methylation is a major mechanism of epigenetics and essential for normal development, imprinting of specific genes, X chromosome inactivation and cell-type-specific gene expression [39,40]. In mammals, CpG dinucleotides are prevalent sites for DNA methylation. CpG islands are the clusters of CpGs associated with the promoters of ~30% of genes. CpG islands are unmethylated in the germline, but DNA methylation occurs in the particular gene promoters during development and tissue differentiation. The addition of a methyl group to cytosine of DNA nucleotides is mediated by DNA methyltransferases, DNMT1, DNMT3A, and DNMT3B [41-45]. 2.1
Histone modifications Histone modifications affect the chromatin structure and play fundamental roles in most biological processes, including DNA replication, transcription and DNA repair [31]. N-terminal tails of histones in the nucleosome protrude from globular histone proteins. Lysines and arginines in the unstructured amino-terminal tails are targets for various types of post-translational modifications, such as acetylation, methylation, phosphorylation, ubiquitination, ADP-ribosylation, sumoylation and deamination (Figure 2) [24,25]. Histone acetylation is one of the most abundant and dynamic modifications. The actual level of histone acetylation is dependent on regulated interplay between histone acetyltransferases (HATs) and histone deacetylases (HDACs) [46]. The 2.2
4
nomenclature for histone modification includes histone, residue and position of that residue in the protein, followed by type and number of modifications to that residue. For example, trimethylation of histone H3 on lysine 4 would be designated H3K4me3 [47]. H3K4me3 are found in the promoter regions of active genes within euchromatin, but not in silent heterochromatic regions [48,49]. Methylation of histones H3K9 and H3K27 are present in the promoters of inactive genes in heterochromatin regions [50]. Unlike histone acetylation, histone methylation does not change the charge of histone protein to weaken the interaction with DNAs. Instead, it recruits specific binding proteins such as PcG proteins and heterochromatin proteins 1 (HP1), which affect the chromatin structure. HP1 is recruited to H3K9 methylation in the promoter to repress genes [20,25]. Chromosome remodeling by PcG proteins PcG and TrxG proteins function to maintain specific cellular gene expression patterns and keep the identity of every cell type during stem cell differentiation. During development, PcG proteins maintain a repressed state of gene expression, whereas TrxG proteins counteract the silencing and maintain active states [38]. PcG proteins form multi-protein complexes, termed polycomb-repressive complexes (PRCs). There are two distinct PRC2 and PRC1 complexes. PRC2 contains enhancer of zeste (Ezh2), embryonic development protein (Eed), and suppressor of zeste12 (Suz12). PRC1 contains ring finger protein1 (Ring1), B lymphoma Mo-MLV insertion region1 (Bmi1), polycomb group ring finger2, and chromobox family proteins. Ring1 possesses an E3 ubiquitin ligase activity, which monoubiquitinates histone H2A at lysine 119 (H2AK119ub1). PcG proteins work together with 2.3
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Epigenetic approaches to regeneration of bone and cartilage from stem cells
TrxG RNA Pol II
Active chromatin
PcG
TrxG
PcG PcG PcG
Repressive chromatin
Bivalent chromatin
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PcG H3K4me3 H3K27me3
TrxG RNA Pol II
Poised chromatin
Figure 3. Bivalent chromatin structure in stem cells is regulated through chromatin remodeling by polycomb repressive complexes and trithorax G proteins. H3K4me3 and H3K27me3 are associated with the bivalent chromatin structure. PcG: Polycomb group; TrxG: Trithorax group.
histone modifications to regulate the chromatin structure and dynamically change gene expression during the differentiation. Ezh2 has an enzyme activity for H3K27me3, which serves as a signal for the binding of PRC1 proteins [36]. Genome-wide mapping of histone methylation profiles of ESCs revealed unique chromatin patterns called ‘bivalent domains,’ which hold both an active gene mark H3K4me3 in smaller regions and a repressive H3K27me3 in large regions in the genome. The function of bivalent domains is to silence developmental genes in ESCs and to keep them poised for activation upon differentiation at the same time (Figure 3) [51,52]. In mouse ESCs, multiple lineage-control essential transcription factor genes, Sox1, Nkx2-2, Msx1, Irx3, and Pax3, are bivalent and possess active marks including H3K9ac and H3K4me3 along with repressive mark H3K27me3 for activation during differentiation [53]. PRCs are essential to control epigenetic modifications in the differentiation to determine cell fate. PRC1 and PRC2 occupy the bivalent domains to regulate differentiation [36]. Eed (a PRC2 component)-deficient ESCs aberrantly induces the expression of lineage-control genes [51]. microRNAs microRNAs (miRNAs) are small 20 -- 23 bp cytoplasmic RNAs that control post-transcriptional gene expression by binding to target mRNAs. miRNAs interact with target mRNA through complementary base pairing between the miRNA and the 3¢-untranslated region of the mRNA, causing degradation of the mRNA target or translational suppression (Figure 4) [52]. Dicer, one of the indispensable components for miRNA synthesis, is essential for normal skeletal development [53]. Dicer-null mice have skeletal growth defects and die prematurely [53]. With the absence of processed miRNAs by removal of the Dicer and Drosha enzymes, osteogenic 2.4
differentiation and adipogenic differentiation of human mesenchymal stem cells (hMSCs) were inhibited [53]. Several miRNAs are known to affect DNA methyltransferases, thus changing the epigenetic status of genes. miRNA-29 targets DNMT 3A and 3B in lung cancer [54]. miRNA-152 targets DNMT 1 in hepatocellular carcinoma [55]. miRNA450a and miRNA-143 affect DNMT 3A in hepatocellular carcinoma and colorectal cancer, respectively [56,57]. Although these miRNAs were mostly reported from the study of oncogenesis, their possible role in osteogenesis and chondrogenesis should be a subject of future investigation.
3.
Epigenetics of stem cell differentiation
Stem cell maintenance and differentiation are associated with the modulation of epigenetic mechanisms, including chromatin remodeling, DNA methylation and histone modifications. ESC differentiation is accompanied by global gene expression changes. Genes regulating pluripotency and self-renewal become silenced, whereas genes associated with specific celltype become active. These global gene expression changes are controlled by epigenetic mechanisms that preferentially target transcription factors [11,16]. Epigenetic alterations occur gene-specifically and globally during ESC differentiation. Many studies have reported that DNA methylation and histone modification were associated with transcriptional changes of ESC differentiation. Heterochromatin mark, H3K9me3, was increased and euchromatin marks, H3 and H4 acetylation, were decreased in the global levels. Treatment of trichostatin A as HDAC inhibitor prevented ESC differentiation due to aberrant global histone acetylations. Global DNA methylation was also slightly increased upon differentiation [58,59].
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miRNA gene
Nucleus
Pri-miRNA
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Drosha
Exportin5
Pri-miRNA
Cytoplasm
dsRNA Dicer miRNA: miRNA* duplex
siRNA duplex
mRNA cleavage
Unwind RISC
RISC
Target mRNA
RISC Ribosome RISC ORF Translational repression
Figure 4. Working mechanism of microRNA. dsRNA: Double strand RNA; miRNA: MicroRNA; ORF: Open reading frame; RISC: RNA induced silencing complex; siRNA: Small interfering RNA.
Histone modification states of multipotent adult stem cells such as MSCs, neural progenitor cells (NPCs), and hematopoietic stem cells (HSCs) are different from pluripotent ESCs due to their differentiation capabilities (Figure 5). Nonetheless, epigenetic changes of DNA methylation and histone modifications are essential for the differentiation of adult stem cells. Many studies have shown that DNA methylation played a role in regulating neural stem cell and HSC differentiation. DNMT1 was expressed at high level in the central nerve system such as neurons and glial cells during embryogenesis. Mice lacking this enzyme in neural precursor cells had problems with neural function and died shortly after birth [60]. HSCs give rise to all types of blood including lymphoid and myeloid lineages. The expression of transcription factors involved in HSC differentiation was regulated by promoter methylation. Sp1 was expressed in HSCs and in B cell but not in T cells due to hypermethylation of Sp1 promoter in T cells [61]. De novo methyltransferases (DNMT3a and DNMT3b)-deficient HSCs had no capability for long6
term reconstitution of the hematopoietic system in bonemarrow transplantation assays [62]. These researches showed that DNA methylation functioned in both HSC differentiation and self-renewal. The chromatin of stem cells has bivalent domains controlled by PcG and TrxG proteins and these bivalent domains play a crucial role in developmental gene regulation [36]. Ezh2 that has an enzyme activity for H3K27me3 was highly expressed in adult stem cells such as NPCs and skin stem cells, but down-regulated during differentiation. The down-regulation of PcG protein expression can contribute to the differentiation of adult stem cells by de-repression of differentiation associated genes [63]. The injury signals have been shown to displace PcG proteins from poised differentiation genes to the muscle satellite cell marker Pax7 and to activate bivalent genes [64]. Regeneration signals can affect the balance of Ezh2 and demethylases of PcG proteins at the bivalent domain of adult stem cells. Reduction of repression by PcG proteins induces differentiation by regeneration signals.
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Epigenetic approaches to regeneration of bone and cartilage from stem cells
Differentiation
Neuronal cell NPC Osteocyte
Differentiation
Muscle cell ESC
MSC
Chondrocyte Differentiation
Differentiation
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HSC Embryonic stem cell Pluripotent (Oct4, Sox2, Nanog, cMyc, H3K4me3 > H3K27me3 H3K4me3/H3K27me3)
Adult stem cell Multipotent (lineage regulators, H3K4me3/H3K27me3 H3K4me3 < H3K27me3)
Somatic cell Differentiated cell (tissue-specific transcription factors H3K4me3 or H3K27me3)
Figure 5. Histone modification status of ESCs, ASCs, and differentiated cells. ESCs and ASCs possess a bivalent chromatin structure that contains both H3K4me3 and H3K27me3 (H3K4me3/H3K27me3). ASCs: Adult stem cells; ESCs: Embryonic stem cells; HSC: Hematopoietic stem cell; MSC: Mesenchymal stem cell; NPC: Neural progenitor cell.
Epigenetics in osteogenic differentiation of MSCs
4.
Epigenetic control of MSC differentiation into bone and cartilage has been investigated over the past decade. Several epigenetic regulations in osteogenic differentiation of MSCs have been reported [14]. Involvement of DNA methylation in osteogenic differentiation of MSCs has been reported. Osteogenic differentiation of MSCs was accompanied by reduced stemness-related genes Brachyury and LIN28 through hypermethylation of promoters of these genes [65]. The epigenetic modulation can improve osteogenic differentiation from MSCs. The endogenous (Trip10) expression was decreased with progressive accumulation by transfection of exogenous methylated Trip10. Transfection of in vitro methylated thyroid hormone receptor interactor (Trip10) promoter DNA into MSCs resulted in progressive accumulation of cytosine methylation at the endogenous Trip10 promoter, and reduced Trip10 expression. Reduced expression of Trip10 helped to accelerate MSC differentiation into osteocytes [66]. Also, mechanical stimulus was used to improve osteogenic differentiation from MSCs at the epigenetic level. Mechanical signals have been known to regulate the differentiation of stem cells. Mechanical stimulation of MSCs reduced DNA methylation on the promoters of osteogenic candidate genes, leading to increased expressions of these genes [67]. This study first demonstrated that the mechanical microenvironment could induce epigenetic changes that control osteogenic cell fate. Osteocalcin (OC), a bone-specific gene, was regulated by DNA methylation in osteogenic differentiation. Although this gene locus was significantly hypermethylated in undifferentiated cells, DNA methylation of OC decreased and its gene
expression increased during in vitro osteogenic differentiation from MSCs. This finding suggests a compelling example in the context of how epigenetic machinery can be targeted to benefit lives [68]. Histone modification of OC gene was also investigated during osteogenic differentiation. Both the promoter and coding regions of OC contained low level of acetylation of histone H3 and H4 during the proliferative period of osteogenic differentiation when this gene was inactive. However, OC was active in the mature osteoblasts and correlated with enriched H4 acetylation. Histone acetylation was mediated with developmental induction of OC in the bone [69]. ChIP-on-chip was performed to investigate the role of histone modification in the osteogenic differentiation from MSCs. H3K9Ac correlated with activating genes and H3K9me2 was associated with silencing genes. Many vitamin D receptor (VDR) elements were found at the gene promoters in both H3K9Acdecreased and H3K9me2-increased groups, suggesting that VDR might be a potential regulator for mediating deacetylation and dimethylation of H3K9 at specific gene promoters in the osteogenic differentiation from MSCs [70]. Histone methylation has a role in osteogenic differentiation from MSCs. The HoxA10 gene is necessary for embryonic patterning of skeletal elements. HoxA10 contributed to osteogenic lineage determination through total chromatin acetylation and enriched H3K4me3 of the genes. Increased HoxA10 activated Runx2, alkaline phosphatase and OC during osteogenic differentiation [71]. The function of PcG proteins in osteogenic differentiation of MSC has also been investigated. Activation of cyclindependent kinase 1 (CDK1) promoted osteogenic differentiation through disruption of the PRC2 complex. CDK1 phosphorylated Ezh2 and phosphorylated Ezh2 decreased
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the interaction with Suz12 and Eed on the promoter of Runx2, one of Ezh2 target genes, which is an important transcription factor for osteogenic differentiation. Decreased H3K27me3 led to activate Runx2 expression during osteogenic differentiation. Genome-wide investigation of Ezh2 target genes was also performed in MSCs, before and after osteogenic differentiation by ChIP-on-chip. Over 4000 genes had Ezh2 binding before differentiation, but
