Chromatin modifiers and histone modifications in bone formation, regeneration, and therapeutic intervention for bone-related disease

Abstract

Post-translational modifications of chromatin such as DNA methylation and different types of histone acetylation, methylation and phosphorylation are well-appreciated epigenetic mechanisms that confer information to progeny cells during lineage commitment. These distinct epigenetic modifications have defined roles in bone, development, tissue regeneration, cell commitment and differentiation, as well as disease etiologies. In this review, we discuss the role of these chromatin modifications and the enzymes regulating these marks (methyltransferases, demethylases, acetyltransferases, and deacetylases) in progenitor cells, osteoblasts and bone-related cells. In addition, the clinical relevance of deregulated histone modifications and enzymes as well as current and potential therapeutic interventions targeting chromatin modifiers are addressed.

Introduction

The study of epigenetics has been widely defined as any heritable changes in an organism that occur without direct alteration in DNA sequence. This broad definition encompasses a wide range of chemical modifications of proteins and DNA that can alter the structure and accessibility of chromatin in the nucleus leading to altered gene expression. A great deal of research has defined multiple epigenetic mechanisms regulating transcription factor and transcriptional machinery access to chromatin, including nucleosome positioning [1, 2], chromatin-chromatin interactions, lncRNA and miRNA binding [3, 4], and several unique chemical post-translational modifications to DNA and DNA-associated proteins [5]. As these epigenetic mechanisms are too complicated to cover in brief, this review will focus on the two most prominent (and therefore widely studied) epigenetic modifications; DNA methylation and post-translational modifications of histone proteins (i.e. lysine acetylation and methylation, and serine phosphorylation) as they relate to bone development, regeneration and disease.

DNA methylation

Methylation of cytosine bases at the fifth carbon (5-methylcytosine or 5mC) is a highly conserved DNA modification found in all mammals [6]. In somatic cells, cytosine methylation is essentially limited to palindromic CpG dinucleotides, however non-CpG methylation has been observed in some cells including; oocytes, pluripotent embryonic stem cells (ESCs), and mature neurons [7, 8]. DNA methylation is dynamically regulated during development and contributes to developmental stage- and cell-type specific epigenetic signals that profoundly impact gene expression [9]. Nearly 70% of annotated gene promoter regions and a large number of transcriptional start sites (TSS) are associated with short CpG-dense sequences [10]. This would suggest that a large number of genes are susceptible to methylation at these CpG islands (CGIs), however CGIs are frequently non-methylated, regardless of the transcriptional state of the corresponding genes [10]. There are several examples of CGI methylation during normal development resulting in stable silencing of gene transcription, however it would appear that CGI methylation may not be an initiating event in gene silencing, but rather acting to permanently maintain gene inactivation [10, 11]. Therefore at most genomic sites methylation patterns are static amongst specific tissues and change in cell-specific context as specialized cellular processes are activated or inactivated. Mechanistically, CpG methylation is thought to mediate gene silencing through direct inhibition of transcription factor binding due to DNA methylation or by recruiting methyl-binding domain proteins that in turn, recruit chromatin-modifying enzymes and complexes to methylated DNA.

Biological regulation of DNA methylation

Most CpG methylations are deposited in a coordinated effort by three conserved enzymes, DNA methyltransferase 1 (DNMT1), DNMT3A, and DNMT3B. During mitosis, DNMT1/ UHRF1 recognizes methylated CpGs on the template strand and correspondingly deposits a methyl group on the nascent DNA [12]. Although maintenance of DNA methylation at mitosis ensures epigenetic inheritance at most genomic regions, there are many instances in which methylation must be actively targeted and others in which methylation must be inhibited or indirectly removed by exclusion of DNA methyltransferases. For instance, many CpG island promoters are unmethylated, but a subset of promoters is methylated during development (i.e. de novo methylation) or lineage commitment leading to gene inactivation. In theses cases, DNMT3A and DNMT3B target promoters in complex with other epigenetic repressors, including histone deacetylases (HDACs) and methyltransferases, to remove H3K9 and/or H3K27 methylation and co-operatively repress gene activity [12]. The methylation status of CpG islands also allows for binding of regulatory complexes through specific protein domains. The SRA domain in UHRF1 is able to recognize hemimethylated (mCG:GC) and methyl-CpG-binding domains (MBD) in proteins such as MECP2, MBD1, MBD2, and MBD4 are able to recognize symmetrically methylated (mCG:GCm) CpG dyads [13]. Several zinc finger-CxxC domain proteins including KDM2a (Fbxl11), KDM2b (Fbxl10), and MLL1 specifically recognize non-methylated DNA (CG:GC) and recruit chromatin-modifying activities to CpG islands [13]. Active DNA demethylation of CpG residues is initiated by the TET family of DNA dioxygenases. TET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine (5hmc) and subsequently 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) leading to nucleotide or base excision repair [9].

DNA methylation in bone

The involvement of DNA methylation in musculoskeletal diseases has been most studied in the context of osteoarthritis. Studies have linked increased methylation at the MMP13 and iNOS promoters in human articular chondrocytes with the progression of arthritic disease [14-16]. Several studies have examined DNA methylation at specific gene promoters as a functional consequence or potential driver of osteoblast differentiation. Differentiation of human mesenchymal stem/stromal cells (MSCs) into the osteoblast lineage resulted in the progressive methylation of promoters associated with pluripotency/ stem cell genes [17] or genes involved in ERα signaling [18]. In cells undergoing osteogenic differentiation changes in the methylation status of the osteoblast-related gene Bglap2 (osteocalcin) gene were observed as a process of mechanical stimulation or differentiation [19, 20]. Treatment of MC-3T3-E1 osteoblasts with homocysteine in addition to IL-6 increased levels of DNMT1 and subsequent methylation at specific gene promoters [21]. In the same cell model system, osteoblasts plated on a collagen matrix or treated with DMSO demonstrated increased expression of DNMT1 and TET and an increase in global DNA methylation and methylation of the promoter of the apoptotic gene Fas [22, 23]. In a study involving and osteosarcoma model, a DNMT inhibitor reduced DNA methylation and increased expression of osteoblast-related genes such as IL-6, IL6ST, BMP7, ATP6B1, IGF1, WNT1, TNFs and ALPL [24].

Differentiation of osteoblasts from MSCs is an integral part of bone development and homeostasis and as such, the role of DNMTs in maintaining MSC quiescence, senescence and self-renewal has been studied. DNMT1 knockdown in early-passage MSCs induces senescence and reduces differentiation potential through a pRB-mediated pathway, whereas DNMT1 overexpression in late-passage MSCs prevents senescence [25]. Other studies demonstrated that the inhibition of DNMT1 in MSCs prevents differentiation to osteoblasts, adipocytes or chondrocytes by inducing replicative senescence mediated by p16 and p21 expression [26].

Histone post-translational modifications

Eukaryotic chromatin assembled into nucleosomes, which are complexes of multiple histone subunits and DNA that act both as physical barrier to DNA access and a source of epigenetic information. Post-translational modifications of histones proteins are a key component of epigenetic regulation influencing lineage commitment and gene expression. Reversible covalent modifications of histones occur at chemically labile residues (i.e. lysine, arginine, serine, threonine, tyrosine and histidine) on accessible N-and C-terminal tail regions and in the less accessible histone fold or globular domains in the nucleosome core [27, 28]. Each specific modified residue on the histone protein may relay a specific piece of information. For example, methylation of lysine 4 of histone 3 (H3K4me3), which is a well-studied histone modification, generally indicates that a gene is transcriptionally-poised or active. This information is conferred through multiple mechanisms including altered histone protein-protein or histone protein-DNA interactions or recruitment of binding factors that affect histone modifying and remodeling activities [29]. The most commonly modified histone methylation sites include lysines; H3K4, H3K9, H3K27, H3K36, H3K79 and H4K20 and arginines (R); H3R2, H3R8, H3R17, H3R26 and H4R3. However, several other residues throughout histone H1 and the core histone proteins H2A, H2B, H3 and H4 are modified in various contexts [30]. Although a specific histone modification can act individually and provide information on the expression state of a particular gene, the total of all modifications in a cell represent a comprehensive histone code relating the epigenetic state of a particular cell. This code becomes particularly complex as particular modifications may act synergistically or antagonistically and the extent or type of modification may vary at individual nucleosomes or individual cells in a population [5]. For example, many promoters in embryonic stem cells are marked by both an activating H3K4me3 mark and a repressive H3K27me3 mark which would appear conflicting with respect to simple models in which these marks are linked to, respectively, active and repressed transcriptional states [31]. A large number of histone-modifying enzymes are found in multi-subunit complexes and nearby, modified residues can create requisite binding sites for the components of the complex helping to anchor an enzyme to a nucleosome. For example, the COMPASS-like histone-modifying complexes can recognize and demethylate H3K27me3 (through the actions of UTX) while simultaneously methylating H3K4 (through the actions of MLL3/4) and acetylating H3K9 and H4K16 (through WDR5) [32].

Regulation of histone methylation and acetylation

Histone marks are regulated by proteins (or protein complexes) that incorporate (write) or remove (erase) specific covalent modifications and can be recognized by proteins that bind (read) specific modifications leading to altered gene expression (Figure 1). These proteins can be loosely characterized on their enzyme activity (e.g. histone/lysine deacetylase (HDAC/KDAC), histone/lysine acetyltransferase (HAT/KAT), lysine demethylase (KDM), or binding activity (e.g. bromodomain: proteins that bind acetylated lysines or chromodomain: proteins that bind methylated lysines). Three main families of enzymes have been identified thus far that catalyze the addition of methyl groups to lysine residues of histones (KMTs): the SET-domain-containing proteins, DOT1-like proteins, and protein arginine N-methyltransferases (PRMT) [30]. Enzymes that reverse the methylation of lysine residues (demethylases/KDMs) can be grouped into two basic families: amine oxidases and jumonji C (JmjC)-domain-containing, iron-dependent dioxygenases [30]. These enzymes are highly conserved from yeast to humans and are able to demethylate histone and non-histone substrates.

Figure 1. Regulation of gene expression by chromatin modifiers.

Schematic overview of activity of chromatin modifiers regulating gene expression. Opposing actions of lysine methyltransferases (KMTs) and lysine demethylases (KDMs) serve to add or remove methyl groups to histone proteins of chromatin, respectively. DNA methyltransferase (DNMT) activity results in transfer to methyl groups to cytosine on chromatin-associated DNA. Removal or addition of acetyl residues is facilitated by histone deacetylases (HDACs) or lysine acetyltransferases (KATs), respectively.

Acetylation of lysine residues entails the addition of an acetyl group on histone residues by lysine acetyltransferases (KATs (sometimes referred to as histone acetyltransferases (HATs)) and their removal by histone deacetylases (HDACs). There are two major classes of KATs based on homology to enzymes discovered in other model systems: the GNAT (Gcn5) family and the MYST (Moz/Ybf2/Sas2/Tip60) family. Other enzymes with less clearly conserved catalytic domains (such as CBP/p300 (KAT3A/KAT3B)) are functionally important in higher eukaryotes [33]. There are four classes of HDAC: classes I and II are closely related to yeast scRpd3 and scHda1, respectively, class III (referred to as sirtuins) are NAD⁺-dependent analogs to the yeast Sir2 protein, while class IV has only a single member (HDAC11) [34]. In addition to histone proteins many KATs and HDACs can act on a wide range of proteins both in the cytosol and the nucleus, extending their functional significance not only to epigenetic events but also cellular processes [35].

Histone modifications in bone

The histone code or epigenetic events leading to bone formation are relatively uncharacterized, however two recent studies have evaluated global histone modifications in the context of Runx2 binding in human MSCs and MC-3T3-E1 preosteoblasts during osteogenesis [36, 37]. In the study by Håkelien and colleagues, dynamic changes of several different histone methylations and acetylations were evaluated at two different osteogenic time points [36].

While the basic epigenetic modifications leading to bone formation are somewhat under studied, a significant amount of research has defined the regulatory roles of several histone modifiers in relation to bone formation (Table 1). The functions of several methyltransferases and demethylases have been directly evaluated in osteoblast or MSC models. Ito and colleagues determined through a complementary deletion/transgenic genetic approach, that the arginine methyltransferase PRMT4 (CARM1) is involved in endochondral ossification and chondrocyte proliferation through methylation of Sox9 [38]. However, chromatin targets were not tested in this study. The WHSC1 gene was originally identified due to its role in the human disease Wolf-Hirschhorn syndrome, a syndrome associated with bone abnormalities including craniofacial defects and growth abnormalities [39]. WHSC1 (also known as NSD2) encodes for a KMT that methylates H3K36 and deletion of this gene has severe effects on bone formation in mouse models through interactions with Runx2 and p300 [40]. In addition the related protein NSD1 also a H3K36-specific methyltransferase is associated with Soto syndrome, a condition characterized by macrocephaly and advanced bone ageing [41]. Several reports have suggested that the PRC2-associated H3K27 methyltransferase Ezh2 (KMT6) directly inhibits osteoblatogenesis either by promoting adipogenesis or blocking expression of phenotypic bone genes [42-44]. However it would appear that Ezh2 is required for osteochondrogenic progenitor establishment during differentiation from neural-crest cells [45]. Stimulation of MSCs with Wnt5a can induce the phosphorylation of histone methyltransferase, SETDB1 (KMT1E) leading to histone H3K9 methylation repressing PPAR-gamma expression and adipogenesis thereby inducing osteogenesis [46].

Table 1.

Chromatin modifying enzymes involved in bone formation or regulation of bone genes

Enzyme	Alias	Histone residue targets	Role in bone formation	Reference
Methyltransferases
CARM1	PRMT4	H3R2; H3R17; H3R26	Endochondral bone formation	[38]
WHSC1	NSD2	H3K36	Regulation of bone genes; developmental bone formation	[40, 93]
NSD1	KMT3B	H3K36	Regulation of bone genes; Advanced bone age; Soto syndrome	[41]
MLL1	KMT2A	H3K4	Advanced bone age	[94]
SETD7	SET7/9, KMT7	H3K4	Regulation of chondrocyte-related genes in chondrocytes	[95]
EZH2	KMT6	H3K27	Required for progenitor formation; inhibits osteogenesis	[42-45]
SETDB1	KMT1E	H3K9	Suppression of adipogenesis in progenitors	[46]
Demethylases
KDM1A	LSD1, AOF2	H3K4; H3K9	Regulation of Nfat1-mediated chondrogenesis	[96]
KDM4B	JMJD2B	H3K9; H3K36	Promotes osteogenic differentiation	[48]
KDM6A	UTX	H3K27	Inhibits osteogenesis	[44]
KDM6B	JMJD3	H3K27	Promotes osteogenic differentiation	[48]
C14orf169	NO66	H3K4; H3K36	Inhibits osteogenesis	[49, 50]
Acetyltransferases
KAT2B	PCAF	H2B: H3	Regulation of bone-related genes	[60, 64]
CREBBP
	CBP, KAT3A	H2A; H2B: H3	Co-regulation of bone genes; Rubenstein-Taybi syndrome	[51-57]
EP300	KAT3B	H3	Co-regulation of bone-related genes	[58-61]
KAT6A	MOZ, MYST3, RUNXBP2	H3; H4	Bone formation in Genitopatellar syndrome; Interaction with Runx2	[65, 66]
KAT6B	MORF, MYST4	H3; H4	Interaction with Runx2	[66]
WDR5	BIG-3, SWD3	H3K4; H4K8	Developmental bone formation	[63, 97]
Deacetylases
HDAC1	Histone Deacetylase 1; KDAC1	H2A; H2B: H3; H4	Transcriptional co-regulator; Regulation of osteoblast differentiation	[70, 98]
HDAC2	Histone Deacetylase 2	H2A; H2B: H3; H4	Regulation of osteoblast differentiation	[71]
HDAC3	Histone Deacetylase 3	H2A; H2B: H3; H3K27; H4	Craniofacial development; Maintenance of bone mass during aging; Negative regulator of osteoblast gene expression	[72, 99, 100]
HDAC4	Histone Deacetylase 4	H2A; H2B: H3; H4	Transcriptional co-regulator; Regulator of chondrocyte hypertrophy and skeletogenesis: Modulation of Runx2 activity	[73, 101-103]
HDAC5	Histone Deacetylase 5	H2A; H2B: H3; H4	Negative regulation of osteoblast gene expression; Modulation of Runx2 activity	[104, 105]
HDAC6	Histone Deacetylase 6; Tdac	H2A; H2B: H3; H4	Modulation of Runx2 activity; Regulation of gene expression	[106]
HDAC7	Histone Deacetylase 7	H2A; H2B: H3; H4	Regulation of chondrocyte hypertrophy; Negative regulation of osteoblast gene expression; Modulation of transcriptional activators	[68, 77, 107]
HDAC8	Histone Deacetylase 8	H2A; H2B: H3; H3K9; H4	Negative regulation of osteoblast differentiation; Cranialfacial development	[69, 108]
SIRT1	SIR2L1
	H3; H4	MSC renewal; Regulation of cartilage and bone formation; Regulation of osteoblast differentiation	[109-112]
SIRT6	SIR2L6
	H3; H4; H3K9; H3K56
	Osteoporosis and reduced bone mineral density in null-animals; Regulation of chondrocyte proliferation and hypertrophy	[113, 114]

Several studies have evaluated the role of demethylases in osteoblast commitment and differentiation. The PRC2-associated H3K27 methyltransferase Ezh2 (KMT6) directly inhibits osteoblastogenesis either by promoting adipogenesis or blocking expression of phenotypic bone genes [42-44]. In contrast, expression of the H3K27 demethylase UTX (KDM6A) during mesenchymal commitment promotes osteogenesis over adipogenesis [44]. Decreased expression of Jmjd3 (KDM6B) or JMJD2B (KDM4B) resulted in increases H3K27 or H3K9 methylation (respectively) thereby suppressing osteoblast differentiation and reducing expression of several bone-related genes [47, 48]. The histone demethylase NO66 regulates the expression of several bone-related genes including Osx through active demethylation of H3K4 and H3K36, resulting in impaired osteogenesis [49, 50]. Bone-related signaling molecules can also induce epigenetic modifications. Stimulation of MSCs with Wnt5a increases the phosphorylation of histone methyltransferase, SETDB1 (KMT1E), leading to histone H3K9 methylation and repression of PPAR-gamma expression, thereby suppressing adipogenesis and inducing osteogenesis [46].

Several acetyltransferases have been demonstrated to be important to osteogenesis or bone formation. Mutations in the gene encoding CREB binding protein (CBP/KAT3A), a non-specific histone acetyltransferase are associated with Rubinstein-Taybi syndrome, an autosomal dominant disorder characterized by face and limb anomalies [51-53]. CBP has been also implicated as a direct- or co- regulator of several bone-related genes through acetylation of histone residues [54-57]. A frequent co-factor of CBP, p300 also functions as a general histone acetyltransferase and like CBP has been implicated in the acetylation and activation of bone-related gene promoters during osteogenesis [58-61]. WDR5 (WD repeat domain 5) is a component of the multi-subunit SET1 complex comprised of several histone-modifying proteins. WDR5 itself has both H3K4-specific methyltransferase and H4K8-specific acetyltransferase activity [62]. Transgenic overexpression of Wdr5 using a 2.3-kb α(1)I-collagen promoter induced accelerated osteoblast and chondrocyte differentiation resulting in substantially increased endochondral bone growth suggesting [63]. Several other acetyltransferases have been implicated in epigenetic regulation of bone formation, osteoblast-related gene expression or deregulated in osteoarthritis including PCAF (KAT2B) [60, 64], MOZ/MORF (KAT6A/KAT6B) [65, 66], and the circadian-rhythm specific acetyltransferase: Clock (KAT13D) [67].

Amongst the histone-modifying enzymes, the most widely researched in the context of bone are the HDACs. Several studies have defined roles for several specific HDACs in regulating bone-related genes, in turn affecting osteoblast differentiation [68-73] (and reviewed in [74]). In addition, modulation of HDAC function can modulate extracellular signaling pathways, thereby affecting osteogenic commitment and differentiation in a context-dependent manner [75, 76]. A complicating factor with analysis of HDAC function is that like many acetyltransferases, HDACs can act on acetylated lysines of non-histone proteins. Many HDACs can deacetylate multiple cellular proteins, including the bone-related transcription factor Runx2, regulating their function through removal of acetylation or direct physical interaction [72, 77, 78]. As most HDAC proteins have no intrinsic chromatin binding ability, they are frequently associated with chromatin as transcriptional co-repressors. Several bone-related transcriptional regulators have been shown to be complexed with specific HDACs to control osteoblast-related gene expression in a context dependent manner including; Runx2, NFATc1, Zfp521 and Pbx1 [58, 78-80]. In addition to their direct involvement in osteoblastogenesis, HDACs have also been demonstrated to contribute to osteoarthritis [81, 82]

Clinical relevance/mouse models of deregulated histone modifications

Drugs that alter the epigenome are entering the clinic an unprecedented rate [83]. While hematopoietic and solid tumors are often the subject of clinical trials with these drugs, other conditions, including various forms of arthritis, diabetes, epilepsy, HIV infection, and neurodegeneration, are also being treated with epigenome-targeted therapies [84]. The first class of epigenetic drugs inhibited DNA methylation. Subsequently histone post-translational modifications were targeted with inhibitors of HDACs, KATs, KMTs, and KDMs. Histone deacetylase inhibitors (HDIs) are the most studied at this time, but drugs targeting histone methyltransferases (DotL1 and Ezh2) are now entered into clinical trials for cancer treatment. The mechanisms by which these drugs act are multifaceted and complex with both histone and non-histone proteins affected. However, it is broadly understood that changes in chromatin structure will alter gene expression and thus influence disease progression. Chromatin relaxation may also make cancerous cells more susceptible to DNA damage from radiation or chemotherapies.

The ubiquitous expression of histone modifiers raises the issue of specificity of the epigenetic drugs for diseased tissues and the risk of adverse events, especially for chronic diseases. Surprisingly, HDIs are fairly well tolerated, likely because they have short half-lives and their effects are reversible. The FDA-approved HDI, vorinostat, causes some generally mild problems, including thrombocytopenia, neutropenia, fatigue, diarrhea and dehydration, but all are temporary and cease after therapy ends [85]. A common feature of platelets, neutrophils and gastric epithelial cells is that they undergo continuous cellular regeneration. Bone is another tissue that is constantly remodeled and regenerated at both the tissue and cellular level. Considerable evidence suggests that long-term HDI use increases fracture risk and is teratogenic for fetuses [86, 87]. The assessment of animals treated with HDIs has produced a better understanding of how deregulated histone modifications affect the skeleton.

Several studies examined the effects of HDIs on bone tumor growth and bone density in animal models. Pratap and colleagues tested the ability of vorinostat to treat bone metastases of human breast and prostate cancer [88]. While daily HDI treatment (100 mg/kg, ip) for 4 weeks was effective at reducing tumor growth and osteolysis, it also caused significant cancellous bone loss in the contralateral non-tumor injected limbs. Because these studies required immunocompromised mice and micrometastases to the contralateral limbs could not be excluded, a parallel study was performed in tumor-free male C57Bl/6 mice. Using an identical treatment schedule, McGee-Lawrence and colleagues also observed severe cancellous bone loss in vorinostat-treated animals [89]. Cortical bone density was unaffected, as was bone resorption. Paradoxically, while osteoblast numbers were reduced in vorinostat treated bones, measures of osteoblast activity such as mineral apposition rate and bone formation rate were elevated. In vitro studies showed that immature osteoblasts were highly susceptible to DNA damage and cell cycle arrest when cultured in the presence of vorinostat [89, 90].

Although bone resorption was unaffected in the above study, there is a growing body of evidence that HDIs inhibit osteoclasts in animal models of chronic inflammatory diseases, including rheumatoid arthritis and periodontal disease [91]. A variety of HDIs suppress NF-κB or NFAT signaling in osteoclasts and reduce expression of inflammatory cytokines, thereby slowly disease progression. Administration of HDIs via topical ointments or creams was effective in some studies and avoids systemic damage to the skeleton.

The observation that broad-acting HDIs can increase osteogenic activity of committed osteoblasts and other cells of mesenchymal origin has been independently reproduced in nearly a dozen laboratories around the world [92]. Thus, HDIs and perhaps other epigenetic drugs could be effectively used to engineer bone in vitro. However, the direct effects of the drugs on precursor cells remain a concern for in vivo bone regeneration [75]. Although some studies suggest that systemic administration of vorinostat at lower doses or reduced frequency may prevent the adverse skeletal effects of HDI therapy [90], finding an effective dose of a broad acting histone modifying drug that has no effects on a renewing tissue like bone, blood, or gastric epithelium may be impossible, just as it is for many cancer therapies, including chemotherapy and hormone-deprivation, and anti-inflammatory medications (e.g., corticosteroids). One way to address this problem is to gain a better understanding of which epigenetic modulators (readers, writers, and erasers) are expressed in bone cells so that more specific inhibitors can be designed for specific diseases.