Epigenetic Control of Osteogenesis: Pathways Toward Improved Bone Regeneration

Abstract

Purpose of Review

In this review, we summarize our evolving understanding of the epigenetic mechanisms directing the osteogenic differentiation of skeletal progenitor cells.

Recent Findings

Advances in genome-wide approaches used to profile chromatin accessibility and histone modifications in skeletal progenitors have uncovered chromatin remodeling associated with progression of osteoblast differentiation and the key regulatory nodes driving this process. Utilization of cell culture systems and genetic mouse models highlight the key enzymes regulating histone posttranslational modifications and DNA methylation that promote the transition of cells from progenitor to mature osteoblast stage. Herein, the described studies provide emerging insights gained from pharmacologic targeting of chromatin modifiers promoting osteogenic differentiation of skeletal progenitors.

Summary

While our fundamental understanding of chromatin modifiers and factors regulating chromatin accessibility and transcriptional activity in skeletal progenitors continues to develop, future research may inform new therapeutic approaches to promote osteoblast differentiation and enhance mineralization to augment fracture repair.

Introduction

Fractures are among the most common trauma-induced injuries seen in the clinic. In 2019, the global burden of fractures was estimated to be approximately 178 million new cases [1]. Bone is unique in its capacity for regeneration. Nevertheless, insufficient or delayed bone healing affects up to 10% of bone fracture patients and remains a significant clinical challenge [2, 3]. Complications associated with fractures may lower life expectancy, with an estimated 1-7 year life loss. Comorbidities such as age-related bone loss, osteoporosis-induced bone loss, and inflammatory conditions like diabetes further complicate bone repair strategies [5, 6]. This public health concern is further exacerbated by a rapid increase in the aging population [7].

The development of effective clinical strategies that stimulate native bone-forming processes remains a significant challenge. The process of bone regeneration following bone fracture proceeds through one of two well-established pathways: endochondral and intramembranous ossification [8]. Endochondral ossification involves the initial formation of a cartilaginous callus that is remodeled and eventually replaced by bone. Healing via this pathway occurs primarily in long bone fractures treated with the intent of relative stability. Conversely, the intramembranous ossification pathway involves the direct differentiation of skeletal stem and progenitors (SSPC) into bone-depositing osteoblasts [8]. Bone healing through intramembranous ossification is commonly observed in stable and rigidly fixed fractures. Both types of healing require intricate spatial, temporal and epigenetic control of SSPC proliferation, differentiation, and extracellular matrix deposition.

Bone-forming osteoblasts are the key cells involved in reparative processes and originate from SSPCs [9]. The quantity and activity of SSPCs within the bone marrow and periosteum are key determinants of effective bone regeneration following injury. The molecular markers used to define bona fide SSPCs however, vary depending on the developmental stage (i.e., embryonic, adolescence, adult), anatomical site, and local environment; therefore, the term “skeletal stem and progenitor cells” encompasses all immature progenitor-like cells with the intrinsic capacity to differentiate into osteoblasts [10]. Differentiation of SSPCs toward the osteogenic lineage can be divided into four stages, including lineage commitment, osteoblast proliferation, matrix maturation, and mineralization [10–12]. Osteogenic lineage commitment requires the activity of bone morphogenetic protein 2 (Bmp-2) to activate the expression of the runt-related transcription factor 2 (Runx2), which in turn induces the expression of other downstream signaling molecules and transcription factors [13]. Runx2 activation promotes the transcription of several key signaling molecules, including components of the Indian Hedgehog pathway (such as Ihh, Gli1, and Ptch1), fibroblast growth factor receptors (Fgfr1, Fgfr2, and Fgfr3), Wnt signaling members (including Tcf7, Wnt1, and Wnt10b), and elements of the parathyroid hormone-related signaling axis (such as Pthlh-Pthr1) [14, 15]. Additionally, Runx2 influences the expression of genes associated with the production of collagenous and non-collagenous components of the bone matrix, including collagen type I alpha 1 (Col1a1), osteocalcin (Ocn), osteopontin (Opn), bone sialoprotein (Bsp), and alkaline phosphatase (Alp) [14].

The activity and fate of SSPCs are highly responsive to external cues such as growth factors, including BMPs, FGFs, and WNT ligands. These signals guide their proliferation and differentiation into osteoblasts via activation of intracellular pathways that ultimately converge on chromatin to alter gene expression profiles. Epigenetic mechanisms—including histone posttranslational modifications, and DNA hydroxymethylation regulate the accessibility of bone-related transcription factors on chromatin. For instance, signaling through the BMP pathway recruits histone modifiers that open chromatin at key regulatory regions of osteoblast-specific genes, while Wnt signaling may lead to depletion of repressive marks like H3K27me3, facilitating SSPC commitment to the osteoblast lineage [16–19]. This review will focus on select recent studies that have advanced our understanding of the epigenetic mechanisms governing osteogenesis.

Epigenetic Mechanisms

Epigenetic modifications can facilitate gene expression or suppression via changes in chromatin configuration. Generally, “activating” histone modifications, including acetylation (ac), contribute to an open chromatin or euchromatin state that facilitates binding of transcription factors to promote gene expression. Conversely, “repressive” modifications, including methylation (me) at specific lysine residues in histone 3 and DNA methylation, contribute to a condensed chromatin configuration called heterochromatin. While heterochromatin is generally less accessible and associated with gene silencing, it can still be accessible to specific proteins.

The regulation of gene expression by DNA and histone-modifying enzymes is critical for normal skeletal development, stem cell maintenance, lineage specification, and bone repair [10]. Specific post-translational modifications of lysine residues in histone 3, such as H3K4me3, H3K27me3, H3K9me2 H3K36me3, H3K9ac and H3K27ac have been implicated in controlling osteogenic and chondrogenic gene programs during fracture healing [20, 21]. Studies using cell culture systems, mouse models with tissue-specific deletion of chromatin modifying enzymes, and small molecules that selectively inhibit the catalytic activity of chromatin modifiers have shed key insights into the functional relevance of epigenetic regulation of osteogenesis during development and bone healing (Fig. 1).

Fig. 1. Representative histone modifiers, model systems, and technologies used to study epigenetic regulation of osteogenesis.

Schematic illustration of a subset of epigenetic regulators—focusing on histone arginine and lysine post-translational modifications—involved in modulating osteogenic differentiation and bone regeneration. Histone arginine methylation by protein arginine methyltransferases (PRMTs) can have context-dependent effects on osteogenesis, while histone lysine acetylation and methylation are governed by a range of enzymes with either promotive or suppressive roles depending on the specific context and chromatin landscape. Listed epidrugs represent small-molecule inhibitors used experimentally to interrogate the functional role of these enzymes. Multi-omics approaches and in vivo / in vitro model systems have enabled the dissection of these pathways in both developmental and injury-induced bone formation settings. This is not an exhaustive list, but rather a curated selection of modifiers and tools discussed in this review that highlight key regulatory nodes in the epigenetic control of osteogenesis

Interrogation of Chromatin Landscapes in Osteogenic Differentiation of Progenitor Cells

The integration of multiple high-throughput sequencing approaches reveals the dynamic chromatin changes occurring at key genomic loci over the course of osteogenic differentiation. Recently, Chen et al. combined RNA sequencing (RNA-seq) and transposase-accessible chromatin sequencing (ATAC-seq) to reveal associations of chromatin accessibility with gene expression in mouse bone marrow-derived mesenchymal stem cells (MSCs) over the stages of osteoblast differentiation (proliferative phase, matrix maturation, mineralization) [22]. Their data revealed a progressive decline in global chromatin accessibility, particularly over the proliferation phase (days 0 to 7). This marked a critical transition from multipotent to osteoblast-committed state. Promoter accessibility peaked early, while intergenic and intronic elements were increasingly utilized at later stages of matrix maturation and mineralization, reflecting shifting regulatory inputs. Overlaying RNA-seq and ATAC-seq datasets allowed the authors to identify genes whose expression was tightly coupled to promoter accessibility, including Col6a3, Serpina3n, Ms4a4d, Lyz2, Phf11b, and Grin3a. Motif enrichment analysis further pinpointed dynamic changes in transcription factor binding accessibility, implicating Mef2A, Prrx1, Shox2, and Hoxb13 as key regulators of osteogenesis. These findings underscore how epigenomic remodeling shapes gene expression during differentiation, highlighting the utility of ATAC-seq and RNA-seq to uncover stage-specific epigenetic mechanisms relevant to bone formation and repair [22].

Recent studies by Weng et al. applied fracture models in mice, coupled with multiple -omics approaches, including ATAC-seq, ChIP-seq, and CUT&Tag, to identify novel transcriptional regulators of the initial stages of bone regeneration by skeletal stem and progenitor cells. Their studies revealed that the transcription factor Zfp260 (Zinc finger protein 260) controls the transition of skeletal stem cells (SSCs) to osteoprogenitors during early bone repair and maintains chromatin accessibility at osteogenic loci, particularly within super-enhancer regions regulating Runx2 and Zfhx4 (Zinc finger homeobox 4). Loss of Zfp260 impaired chromatin accessibility and transcriptional activation of these genes, blocking osteogenic lineage commitment and hindering both intramembranous and endochondral ossification [23]. These findings emphasize the importance of stage-specific chromatin remodeling in skeletal lineage progression and highlight how chromatin profiling technologies can uncover new regulatory nodes critical for bone regeneration [23].

Recent developments towards defining the functional contributions of chromatin modifying enzymes in osteogenesis.

Histone modifications dynamically regulate gene expression programs that are essential for osteoblast lineage progression. Activation or repression of gene expression is modulated, at least in part, by post-translational modifications on lysine or arginine residues in histones within the promoters or gene bodies of key osteogenic genes. The catalytic effects of select chromatin modifying enzymes on either the activation or repression of gene expression in osteogenic differentiation are discussed below and in Table 1.

Table 1.

Epigenetic regulation of osteogenic differentiation by histone lysine methylation

Histone Modifier	Catalytic Site	Recent studies on functional role in osteogenesis cited in this review	Ref
Methyltransferases Ezh1 Ezh2 Kmt2d Nsd2 Setb1 Smyd2	H3K27 H3K27 H3K4 H3K36 H3K9 H3K4, H3K36	Ezh1 overexpression accelerated periosteum-mediated fracture repair through recruitment of Dnmt1 and repression of TFPI2. Repression of osteogenic genes; pharmacologic inhibition relieves inhibition of osteogenic gene expression and promotes mineralization. Expression levels upregulated during hMSC osteogenic differentiation; knockdown in human bone marrow stromal cells attenuated osteogenic differentiation through reduction of p-AKT. In aging bone, melatonin upregulates NSD2 to promote osteogenesis. In osteoporotic bone, NSD2 negatively regulates bone formation. SETDB1 along with cofactor ATF7IP promotes osteoblast proliferation via epigenetic silencing of Macrod2. Smyd2 interacts with Ezh2 to negatively regulate osteoblast proliferation and mineralization.	[24–26, 32–34] [25–31] [35–38] [39, 40] [41, 42] [30]
Demethylases Kdm4b Kdm5a Kdm6b	H3K9, H3K36 H3K4 H3K27	Loss in oral bone-derived MSCs impaired osteogenesis, while increasing adipogenesis and senescence. Impaired osteoporotic fracture healing by attenuating repression of miR-495 and protein SKP2, leading to degradation of Runx2 and reduced osteogenic differentiation capacity. Estrogen receptor 2/Kdm5b regulatory axis modulates osteogenic differentiation in hMSCs. Increased expression of Kdm6b by allyl sulfide restored osteogenesis in oxidative stress environment.	[43] [45–48] [49, 50]

Histone Lysine Methyltransferases

The polycomb repressor complex 2 (PRC2) consists of the core proteins (Suz12, Eed, RbAsp48) and one of two H3K27 methyltransferases, enhancer of zeste homolog 1 or 2 (Ezh1, Ezh2) [24]. Ezh1/2 catalyzes the mono-, di- and tri-methylation of histone 3 at lysine 27 (H3K27) at gene promoters and regulatory elements. H3K27me3, a key gatekeeper of chromatin accessibility, is associated with chromatin condensation and transcriptional repression [25]. Ezh2 has been widely studied for its role in skeletal development and osteogenic differentiation [26]. Human mutations in EZH2 cause a skeletal overgrowth condition known as Weaver Syndrome, characterized by distinct facial features and varying degrees of intellectual disability [25]. The role of Ezh2 in osteogenesis differs depending on the developmental context, timing, and mode of inhibition. Recent studies using functional inhibition of Ezh2 in MC3T3-E1 preosteoblasts with the small molecule Tazemetostat (EPZ 6438) determined that inhibition of Ezh2 significantly increased expression of osteoblast-specific genes via relief of H3K27me3-mediated repression at bivalent domains of key genes required for bone formation, such as Runx2 and markers of mineralization (Alpl, Bglap, Phex, Ibsp, and Phospho1) [25]. Interestingly, the co-treatment with Ezh2 inhibitors and BMP2 further enhanced osteogenic stimulation. Bmp-2 has been used extensively to stimulate bone growth in clinical settings, but when used at high concentrations, can lead to adverse effects [27]. Strategies to reduce Bmp-2 dose through combinatorial drug treatments may provide efficacious alternatives that yield bone anabolic effects without adverse effects. Interestingly, co-stimulation of human BMSCs with GSK126, a small molecule inhibitor or Ezh2 and suboptimal doses of Bmp-2, increased osteogenic differentiation and led to increased vascularization when implanted in vivo [25, 28, 29, 31].

Recent studies showed regulation of Ezh2 by the SET and MYND domain containing-protein 2, Smyd2. The lysine methyltransferase SMYD2/Smyd2 is highly expressed in human bone tissue and mouse osteoblasts (as shown by RNA-seq). Dashti et al., showed Smyd2 acts in parallel to the suppressive effects of Ezh2 in osteoblast proliferation and mineralization [30]. Smyd2 depletion in MC3T3 cells resulted in increased levels of Ezh2 and H3K27me3 protein levels. Combined siRNA-mediated knockdown of Smyd2 and Ezh2 resulted in greater enhancement of matrix mineralization than either gene knockdown alone, supporting a model whereby Smyd2 and Ezh2 operate in parallel, but via distinct pathways, to restrain osteoblast maturation. The findings suggest that targeting Smyd2, particularly in conjunction with Ezh2 inhibition, may represent an effective strategy to enhance osteogenesis through coordinated modulation of proliferation and chromatin repression [30].

Liu et al., demonstrated that expression of Ezh1 within the periosteum promotes osteogenic differentiation of SSPCs and enhances fracture repair by epigenetically repressing tissue factor pathway inhibitor 2 (TFPI2), a negative regulator of osteogenesis [32]. It does so by depositing the repressive histone mark H3K27me3 at the TFPI2 promoter and recruiting DNA methyltransferase 1 (Dnmt1) to induce DNA methylation, leading to stable transcriptional silencing [32]. After fracture, TFPI2 expression increased while Ezh1 levels decreased, and experimental overexpression of Ezh1 restored bone healing by suppressing TFPI2. These findings highlight Ezh1 as a positive regulator of bone regeneration through its repressive epigenetic control over inhibitory genes [32]. Together, these studies show that Ezh1 and Ezh2 both regulate H3K27 methylation but do so via distinct mechanisms. Interestingly, Ezh2 exhibits much stronger histone methyltransferase activity, whereas Ezh1 is more effective at compacting chromatin through methylation-independent means. Although they share some target genes and exhibit partial redundancy, Ezh2 is predominantly active in early, proliferating mesenchymal stem cells, whereas Ezh1 appears more important during later stages of differentiation [26, 33, 34]. These differences in function and expression suggest complementary but distinct roles in mesenchymal and skeletal development.

Histone-lysine N-methyltransferase 2D (Kmt2D) catalyzes the mono-methylation of histone H3 at lysine 4 (H3K4me1), an epigenetic mark that is typically distributed at poised enhancers and promoters [35]. H3K4me1 marks also contribute to gene repression, especially when paired with repressive marks like H3K27me3 at bivalent regulatory regions. Hence, the role of Kmt2d is context-dependent, varying by cell type and chromatin environment [36, 37]. Knockdown of KMT2D in human BMSCs impaired osteogenic differentiation in vitro and interfered with bone formation in vivo as shown by subcutaneous transplantation of BMSCs in nude mice (an athymic mouse model). The inhibitory effects of KMT2D knockdown on human BMSCs osteogenesis may be mediated, at least partially, through suppression of Akt signaling [38]. The enzymatic function of Kmt2d on regulation of Akt signaling - a key pathway for stimulation of osteoblast differentiation– will require further investigation.

Recent studies suggest an epigenetic link to bone marrow stromal cell senescence. Senescent bone marrow stromal cells exhibited diminished expression of NSD2 (Nuclear receptor binding SET domain protein 2), leading to reduced H3K36me2 and increased accumulation of the repressive H3K27me3 modification at the promoters of osteogenic genes Runx2 and bone gamma-carboxyglutamate protein (Bglap) [39]. Interestingly, Xie et al. found that reduced NSD2 levels in senescent BMSCs coincided with reduced melatonin levels. Treatment with melatonin upregulated NSD2 expression, thus restoring the balance between H3K36me2 and H3K27me3, and enhancing chromatin accessibility of osteogenic gene loci [39]. Conversely, a more recent study using ovariectomized (OVX) mouse models revealed that NSD2-mediated H3K36me2 can exacerbate osteoporosis by activating the transcription of Hoxa2, a known inhibitor of osteogenic differentiation [40]. Inhibition of NSD2 in human osteoporotic bone marrow-derived MSCs led to increased expression of osteogenic markers like Runx2 and Bsp, suggesting that NSD2 may suppress osteogenesis under certain pathological conditions [40]. These findings suggest that NSD2’s role in osteogenesis is context dependent. In aging-related bone loss, enhancing NSD2 activity may be beneficial, while in conditions like osteoporosis, where NSD2-mediated Hoxa2 activation suppresses osteogenesis, inhibiting NSD2 could be beneficial.

Setdb1 (SET domain bifurcated 1, also known as ESET), catalyzes the trimethylation of histone H3 at lysine 9 (H3K9me3), a modification associated with transcriptional repression. Recent studies on bone loss induced by mechanical unloading used a small interfering RNA (siRNA) approach to study the role of Setdb1 in bone formation [41]. The results indicated that Setdb1 enhances osteoblast proliferation by repressing negative regulators of bone formation in tibial hindlimb unloading mouse models and in MC3T3-E1 pre-osteoblast cells. Specifically, it represses the expression of Macrod2 (mono-ADP ribosylhydrolase 2), a gene that inhibits osteoblast proliferation, through H3K9me3-mediated chromatin remodeling. This repression is facilitated by the cofactor ATF7IP (ATF7-interacting protein 1), which in conjunction with mechanical loading stimuli assists in the nuclear localization and activity of Setdb1 [41]. Setdb1 expression is downregulated in osteoporotic conditions, leading to impaired osteogenic differentiation. Overexpression of Setdb1 in osteoblasts and OVX mouse models can inhibit the transcription factor orthodenticle homeobox 2 (Otx2) via H3K9me3 modification at its promoter region. This inhibition activates the Bmp-Smad and Wnt/β-catenin signaling pathways, thereby promoting osteogenic differentiation [42].

Histone Lysine Demethylases

Histone lysine methylation must be appropriately counterbalanced by the removal of methyl groups from lysine residues on histones through the activity of histone lysine demethylases (KDMs). Lysine demethylase 4B (Kdm4b) demethylates the repressive mark, H3K9me3 at promoter distal-less homeobox 5 (Dlx5), facilitating its transcription and promoting the differentiation of oral bone-derived mesenchymal stem cells (MSCs) into osteoblasts [43]. Kdm4b expression is upregulated in response to osteogenic stimuli from Bone Morphogenetic Proteins (BMPs) and Nerve Growth Factor (NGF) [44]. Loss of Kdm4b leads to impaired osteogenesis and fat-bone imbalance with increased adipogenesis and cellular senescence [43]. Given its role in maintaining a healthy skeletal stem and progenitor pool, modulation of Kdm4b could be critical in aging-related bone disorders and repair mechanisms.

The removal of activating H3K4me3 modification is achieved by the lysine-specific demethylase 5 A (Kdm5a). Kdm5a serves as a negative regulator of osteoblast differentiation and bone formation. Recent findings on KDM5A’s role in osteogenesis confirm its repressive epigenetic function through H3K4me3 demethylation in human bone marrow mesenchymal stem cells (BMSCs), specifically in the context of steroid-induced osteonecrosis of the femoral head (SONFH) [45]. The authors show that KDM5A is upregulated in BMSCs exposed to dexamethasone and directly binds to the promoters of key osteogenic genes—RUNX2, OCN, and OPN—reducing their H3K4me3 marks and thus suppressing their transcription. Silencing KDM5A increased H3K4me3 levels at these promoters, enhanced gene expression, and promoted osteogenic differentiation. Notably, the study also identifies miR-107 as an upstream negative regulator of KDM5A, providing a novel regulatory axis (miR-107/KDM5A) that enhances osteogenesis [45]. This study confirms prior reports showing that KDM5A inhibition with small interfering RNA (siRNA) targeting KDM5A (si-KMD5A) or a small molecule inhibitor JIB-04 enhances osteoblast differentiation by maintaining H3K4me3 at osteogenic gene loci [46, 47]. Importantly, their findings from human BMSCs under pathological conditions contribute novel insight into microRNA-mediated regulatory mechanisms. Another study builds upon these findings using an OVX model [48]. Kdm5a impaired osteoporotic fracture healing in an OVX mouse by promoting demethylation at H3K4me3, which in turn led to increased expression of miR-495 and Skp2 (S-phase kinase-associated protein 2) protein. Subsequently, this caused the degradation of Runx2 and repressed osteoblast differentiation [48]. Targeting Kdm5a presents a promising therapeutic strategy for enhancing osteogenesis and improving bone repair, especially in osteoporotic conditions. By inhibiting Kdm5a activity, it may be possible to upregulate osteogenic gene expression and promote bone formation.

The lysine demethylase 6B (Kdm6b) is responsible for the removal of repressive methyl groups from histone H3 at lysine 27 (H3K27me3). Kdm6b plays a role in cell differentiation by modulating the expression of components of the Wnt signaling pathway and other transcription factors [49]. In fact, disruption of the demethylase activity of Kdm6b by shRNA prevented Alp expression and hindered mineralization after osteogenic induction. Liu et al. expanded on this finding in dental mesenchymal stem cells, which share core properties of MSCs, to conclude that the catalytic activity of Kdm6b is responsible for activation at osteogenic promoter sites on Bmp-2 and Hoxc. Kdm6b was also upregulated by the estrogen receptor 2, which provides insight into the upstream effects of enhancers and transcription factors on demethylases [49]. Further, Behera et al. Showed that a chemical compound, allyl sulfide enhances Kdm6b expression and reduces H3K27me3 at the Runx2 promoter, restoring osteogenesis in oxidative stress environments [50].

Histone Acetyltransferases and Deacetylases

The acetylation status of gene promoter regions is directly correlated to gene expression and is under tight regulatory control by multiple histone acetyltransferases (HATs) and histone deacetylases (HDACs). Histone acetylation is classified as an activating chromatin modification (stimulates gene expression). The addition of negative charges via acetylation leads to the loosening of histone-DNA interactions and thus increased accessibility of transcriptional factors and RNA polymerases to promote gene expression [51, 52]. Conversely, the removal of acetyl groups via the activity of histone deacetylases (HDAC 1–7) is associated with the suppression of gene expression.

Mutations in the MYST family histone acetyltransferase gene KAT6B underlie several human syndromes and congenital disorders, like Genitopatellar syndrome, the Say-Barber-Biesecker-Young-Simpson variant of Ohdo syndrome, and congenital scoliosis, all characterized by craniofacial and skeletal abnormalities [53, 54]. Global loss of Kat6b in mice causes premature ossification in development, leading to shortened craniofacial elements and tibias with an expansion of pre-hypertrophic chondrocytes [55]. Mechanistically, the premature ossification in Kat6b knockout mice was attributed to up-regulation of Runx2 activators, and enhanced commitment of skeletal progenitor to the osteoblastic lineage [55].

Another target to alleviate osteoporosis and age-related bone loss is histone deacetylase Hdac6. Aged bones show hypoacetylation of H3K9/K14 and H4K12 and accumulation of Hdac6 with increased binding capacity on Runx2 promoters in BMSCs, leading to reduced osteogenesis [56]. A study on aged mice shows that inhibition of Hdac6 with Tubastatin A rescues Runx2 expression and osteogenic differentiation of BMSCs to alleviate age-related bone loss [56].

Beyond cellular and molecular factors, mechanical stimuli have been associated with regulating histone-modifying enzymes and influencing osteogenic outcomes. Mechanical unloading or disuse reduces osteogenic capacity by increasing Hdac1 and repressing Wnt signaling, highlighting how biomechanical forces shape the epigenetic landscape in fracture healing. Zinc finger RNA-binding protein, ZFP36L2, upregulated by shear stress, degrades Hdac1 mRNA, increasing histone acetylation and enhancing Runx2 expression and mineralization [57].

Histone Arginine Methyltransferases

Histone arginine methylation, while far less studied relative to histone lysine methylation or acetylation, also contributes to the regulation of osteogenic differentiation. Protein arginine methyltransferases (PRMTs) deposit methyl groups to arginine residues within histones and other cytoplasmic proteins [58]. As with histone lysine methylation, the consequence of arginine methylation on gene activation or repression depends on the specific arginine moiety within the histone tail or less often globular domains. Previous genome-wide transcriptomic analyses revealed stage-specific expression of PRMTs during the process of MSC differentiation to the osteogenic lineage [58]. Prmt1, Prmt4/Carm1 (coactivator-associated arginine methyltransferase 1), and Prmt5 are examples of highly expressed arginine methyltransferases found in human BMSCs, mouse calvarial bone, and MC3T3 pre-osteoblasts, each with distinct roles in osteoblast differentiation. Prmt5, a type II PRMT that catalyzes symmetric dimethylation at H3 (H3R17me2a), emerged as a positive regulator of osteogenesis: its depletion via siRNA resulted in a marked suppression of osteogenic gene expression (e.g., Alpl, Ibsp, Phospho1) and a reduction in mineralization of MC3T3 cells [58]. In contrast, Prmt1 (type I) appeared to function as a repressor of osteoblast maturation, with its knockdown or inhibition (by small molecule GSK715) enhancing calcium deposition. Prmt4/Carm1 had more nuanced effects, influencing the expression of certain osteogenic genes (e.g., Alpl, Bglap) without significantly altering mineral deposition. Interestingly, treatment with GSK715 selectively reduced the H3R17me2a mark (a product of Carm1 activity) and mineralization [58]. The authors note that the results pertaining to Carm1 may be explained by compensatory mechanisms of other PRMTs in the siRNA knockout or the possibility that GSK715 is targeting another unknown H3R17me2 methyltransferase. In a complementary mechanistic study, Liu et al. demonstrated that Prmt5-mediated histone arginine methylation can also modulate other epigenetic pathways through crosstalk with PRC2 [59]. Specifically, Prmt5-mediated methylation at H3R2 and H3R8 antagonizes PRC2/Ezh2-catalyzed trimethylation at H3K27 (H3K27me3), a repressive mark that silences osteogenic genes. Loss of Prmt5 led to enhanced H3K27me3 at promoter regions of target genes, thereby reducing their expression. Additionally, Prmt1 could limit osteogenesis through methylation of R342 on the repressive Ezh2 [59]. PRMT3, another class I enzyme, was shown to promote osteogenic differentiation of human mesenchymal stem cells (hMSCs) via asymmetric dimethylation of H4R3 (H4R3me2a) [60]. Knockdown (via shRNA) or pharmacologic inhibition of PRMT3 (via SGC707) in human and mouse MSCs impaired alkaline phosphatase activity, calcium deposition, and expression of Runx2 and Ocn in vitro and led to low bone mass in vivo. Mechanistically, Prmt3 upregulated the expression of miR-3648, a microRNA that enhanced osteogenic differentiation and rescued impaired bone formation in Prmt3-deficient mouse BMSCs. Prmt3 directly deposited H4R3me2a at the miR-3648 promoter, revealing an epigenetic axis where arginine methylation regulates non-coding RNAs with downstream osteogenic effects [60]. Collectively, these findings emphasize that histone arginine methylation actively contributes to the epigenetic orchestration of osteoblast differentiation.

Epigenetic Control of Osteogenic Differentiation by Vitamin C

The importance of dietary micronutrients like Vitamin C (ascorbate/L-hexuronic acid) on bone health and healing are well-documented. Recent studies from van Wijnen and colleagues uncovered a novel epigenetic role for Vitamin C in supporting bone formation and osteogenic differentiation. Vitamin C selectively primed both mouse and human bone marrow-derived MSCs towards osteogenesis by inducing a permissive chromatin state that facilitates the expression of bone-related genes. The actions of Vitamin C were required at all stages of osteogenic differentiation (yet dispensable for adipogenesis). The study revealed Vitamin C stimulated the activity of DNA cytosine dioxygenases of the TET family, leading to activating 5-hydroxy-methyl cytosine (5hmC) on DNA in addition to de-repressed H3K9/H3K27 histone marks at proximal and distal regions (superenhancer like elements) in bone-selective genes encompassing transcription factors, collagens, collagen cross-linking enzymes, and non-collagenous ECM protein involved in mineralization. Moreover, genetic deletion of Tet1 and Tet2, the key enzymes responsible for catalyzing the conversion of repressive 5-methyl-cytosine (5mc) on DNA to activating 5-hydroxy-cytosine DNA methylation, caused skeletal defects mimicking Vitamin C deficiency. Interestingly, mechanistic studies in MC3T3-E1 preosteoblasts further revealed that 5hmc generation, rather than loss of repressive 5mc on DNA, is responsible for the effect of Vitamin C on osteoblastic differentiation. Collectively, these studies established an essential role for Vitamin C-mediated regulation of chromatin accessibility and gene activation in osteogenic lineage commitment and progression via modulation of Tet2-dependent 5-hydroxy-cytosine DNA methylation.

Analyses of Chromatin Conformation

While chromatin Immunoprecipitation sequencing (ChIP-seq) has been used extensively to map the distribution of specific histone modifications at genomic loci, newer approaches offer significant advantages. CUT&RUN or CUT&Tag have reduced cell input requirements, improved signal-to-noise, and faster workflow, all important factors when working with SSPC populations harvested directly from bone [64]. Though integrating the transcriptome with analyses of histone post-translational modifications and chromatin accessibility provides a wealth of information, these do not address chromatin looping and long-range interactions. Chromatin loops bring distal elements close to their target promoters to regulate gene transcription further. Hi-C and related chromatin conformation techniques have emerged to address the limitations of linear profiling by capturing 3D chromatin architecture and long-range interactions [65–67]. However, these remain less commonly applied in osteogenic contexts due to resolution constraints and computational complexity [68]. A recent advance, Puzzle-HiC (a software in development), provides improved accuracy in scaffolding chromatin loops but still relies heavily on assumptions about genome structure and may not fully resolve rearranged regions [69]. Despite the power of these techniques, the relatively low abundance and heterogeneity of SSPCs make it difficult to achieve sufficient resolution and reproducibility, particularly in single-cell assays. Studies report that open chromatin signals in stem-like MSC subpopulations do not always correlate with distinct gene expression programs, possibly due to pre-established chromatin landscapes that are not yet transcriptionally active [70].

Challenges and Perspectives

Targeting of the epigenome to augment bone regeneration is attractive due to the reversible nature of epigenetic modifications [25, 29, 56]. Several challenges must be first be overcome to realize the translational potential of epigenomic targeting to enhance osteogenesis in SSPCs. Firstly, few studies have examined chromatin in SSPCs. The mineralized nature of the tissues where SSPCs reside makes it difficult to isolate these cells for mechanistic studies [71]. Culturing SSPCs or heterogenous bone marrow stromal cells (BMSCs) may induce changes that do not reflect in vivo activities. The complex cellular heterogeneity within different bones and bone compartments, and a mixture of cell types within the fracture callus pose a significant challenge towards defining epigenetic signatures. Additionally, the identification of overlapping, as well as distinct molecular markers used to identify SSPCs within different bone compartments and using multiple Cre reporters across different studies complicates lineage tracing for SSPC isolation and data interpretation. The sensitivity and resolution of single-cell sequencing may be limited as SSPCs comprise less than 1% of the total bone marrow population, where datasets are often contaminated with hematopoietic lineage cells that are non-specifically labeled and sorted [15]. While spatial transcriptomics and epigenomics are emerging technologies for the study of bone, these applications remain technically challenging [72].

Ultimately, the challenge of targeted delivery remains a major barrier to the clinical translation of epidrugs in bone, where systemic administration can result in low bioavailability and may elicit undesired effects in other cell types. Moreover, therapeutic applications of epidrugs to such as the Ezh2 inhibitors, EPZ6438 and GSK126 to augment bone regeneration after fracture must balance the bone-promoting effects of inhibitors, while minimizing potential drawbacks on progenitor cell proliferation [24, 25, 28, 29, 31]. Emerging insights from integrative epigenomic approaches and advancing technologies for cell-specific drug delivery during bone repair may position epidrugs as a promising therapeutic approach to enhance bone regeneration.

Conclusions

Epigenetic regulation, especially through post-translational histone modifications, is pivotal in orchestrating fracture repair by controlling gene expression, stem cell fate, and cell–cell communication. New studies highlight the importance of epigenetic regulators and their influence on key osteogenic pathways and fracture outcomes. As new knowledge is generated using cutting-edge, integrative approaches, future research on epigenetic modulation in bone repair must focus on delivery strategies, lineage-specific mechanisms, and the integration of mechanical and inflammatory cues to optimize bone regeneration therapies.

Data Availability

No datasets were generated or analysed during the current study.