Role and mechanisms of histone methylation in osteogenic/odontogenic differentiation of dental mesenchymal stem cells

ABSTRACT Dental mesenchymal stem cells (DMSCs) are pivotal for tooth development and periodontal tissue health and play an important role in tissue engineering and regenerative medicine

because of their multidirectional differentiation potential and self-renewal ability. The cellular microenvironment regulates the fate of stem cells and can be modified using various

optimization techniques. These methods can influence the cellular microenvironment, activate disparate signaling pathways, and induce different biological effects. “Epigenetic regulation”

refers to the process of influencing gene expression and regulating cell fate without altering DNA sequences, such as histone methylation. Histone methylation modifications regulate pivotal

transcription factors governing DMSCs differentiation into osteo-/odontogenic lineages. The most important sites of histone methylation in tooth organization were found to be H3K4, H3K9, and

H3K27. Histone methylation affects gene expression and regulates stem cell differentiation by maintaining a delicate balance between major trimethylation sites, generating distinct

chromatin structures associated with specific downstream transcriptional states. Several crucial signaling pathways associated with osteogenic differentiation are susceptible to modulation

via histone methylation modifications. A deeper understanding of the regulatory mechanisms governing histone methylation modifications in osteo-/odontogenic differentiation and

immune-inflammatory responses of DMSCs will facilitate further investigation of the epigenetic regulation of histone methylation in DMSC-mediated tissue regeneration and inflammation. Here

is a concise overview of the pivotal functions of epigenetic histone methylation at H3K4, H3K9, and H3K27 in the regulation of osteo-/odontogenic differentiation and renewal of DMSCs in both

non-inflammatory and inflammatory microenvironments. This review summarizes the current research on these processes in the context of tissue regeneration and therapeutic interventions.

SIMILAR CONTENT BEING VIEWED BY OTHERS CELL SIGNALING AND TRANSCRIPTIONAL REGULATION OF OSTEOBLAST LINEAGE COMMITMENT, DIFFERENTIATION, BONE FORMATION, AND HOMEOSTASIS Article Open access 02

July 2024 HSA_CIRC_0001599 PROMOTES ODONTOGENIC DIFFERENTIATION OF HUMAN DENTAL PULP STEM CELLS BY INCREASING ITGA2 EXPRESSION AND STABILITY Article Open access 17 January 2025 DIRECT

REPROGRAMMING OF EPITHELIAL CELL RESTS OF MALASSEZ INTO MESENCHYMAL-LIKE CELLS BY EPIGENETIC AGENTS Article Open access 20 January 2021 Bones and teeth are integral components of the

craniofacial skeleton, providing indispensable support for the overlying soft tissues and allowing for complex and intricate movements.1 Missing teeth, due to congenital or acquired

diseases, and trauma, is by far the most common oral health problem.2 Maxillofacial tumors can invade the normal tissue structures of the jaw, teeth, muscles, and skin, severely affecting

maxillofacial form and function. The use of autologous or allogeneic bone grafts to repair maxillofacial bone defects caused by tumors or traumatic injuries is common, but problems such as

poor osseointegration stability, immune rejection, and interference with normal physiological functions remain. In elderly individuals or those with severe periodontitis, severe damage to

the dental tissue can result in malnutrition due to difficulty in eating and chewing, which may subsequently affect an individual’s self-esteem and lead to severe psychosocial and mental

health issues.2 Prevailing techniques for the restoration of damaged and missing teeth rely on the use of synthetic materials to repair structural defects. However, these techniques do not

facilitate the promotion of biological functions such as supplying blood and nerves. Mesenchymal stem cells (MSCs) are a population of stem cells that function as self-renewing and

differentiating progenitor cells. The International Society for Cell & Gene Therapy (ISCT) Mesenchymal Stromal Cell committee defines them as readily adherent, expressing CD105, CD73,

and CD90 (but not CD11b, CD14, CD19, CD34, CD45, CD79a, HLA-DR). In addition, these cells are capable of differentiating into osteoblasts, chondroblasts, and adipocyte lineages in vitro.3,4

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are a category of seed cells currently employed with considerable frequency in the field of organizational engineering.5,6 They are

commonly used to repair bone defects owing to their ability to rapidly adapt to the body’s bone microenvironment and accelerate angiogenesis when implanted after mixing with scaffolding

materials in vitro. DMSCs have been identified as an optimal cell source for regenerative therapies owing to their excellent proliferative capacity and multidirectional differentiation

potential.7 Compared to BM-MSCs, DMSCs demonstrate augmented proliferative, neural differentiation, and odontogenic capabilities,8 which are highly promising for bone and neural tissue

regeneration.9 The cellular microenvironment exerts a significant influence on the fate of stem cells and can be modified using various optimization techniques. These include hormone-stem

cell combination therapy, gene modification to regulate stem cells, epigenetic regulation, cell membrane sheets, scaffolding materials, and hydrogels.10 The transplantation of PDLSCs has

been shown to facilitate the regeneration of the cementum, periodontium, and alveolar bone in in vivo experiments conducted on rats, beagles, minipigs, and humans.9 In clinical trials,

autologous PDLSC cell membrane sheets were used to treat three patients with periodontitis, and it was found that the formation of neoplastic dentin and periodontium was visible around the

periodontal tissue.11 Dental pulp stem cells (DPSCs) can be combined with collagen sponge scaffolds to form complexes for the repair of human mandibular defects12 and the reconstruction of

the pulp-dentin complex, thereby generating functional biological tooth roots.13 However, current in vitro organoid constructs lack the requisite functional robustness and physiological

relevance, particularly in the context of tooth regeneration, which is inherently challenging because of their high structural complexity. Therefore, it is imperative that a potentially

effective tissue engineering approach is developed to regenerate teeth that are functional, biocompatible, sustainable, and reproducible, with the ultimate goal of clinical application.14 In

1942, Waddington initially proposed that the intricate developmental process from genotype to phenotype could be described by the concept of epigenetics. Epigenetic regulation can alter the

chromatin state without modifying the DNA sequence, which, in turn, affects gene expression, protein function, and RNA processing.15 Epigenetic modifications can be categorised into three

main classifications: These are DNA methylation, histone modifications and chromatin remodelling. DNA methylation is primarily associated with gene regulation and can maintain chromosome

integrity. Histone modification is a key process in various biological mechanisms, such as transcriptional regulation, DNA repair, DNA replication, alternative splicing, and chromosome

aggregation.16 Epigenetic regulation is implicated in a range of biological processes such as embryonic development,17 disease onset and progression,18 bone homeostasis,19 and stem cell fate

determination.20 A reduction in MSC functionality has been identified as a significant underlying cause of numerous pathological conditions. Epigenetic regulation is of critical importance

for the establishment and maintenance of the homeostasis equilibrium of MSCs within the body. Previous studies have demonstrated that osteo-/odontogenic differentiation of DMSCs is regulated

by epigenetic modifications.21,22 Histone modifications affect osteogenic differentiation by regulating the expression of genes associated with this process. For example, the histone

acetyltransferase PCAF has been demonstrated to affect the BMP pathway by elevating histone H3K9 acetylation in the promoter regions of BMP2/4, BMPR1B, and Runx2.23 Additionally, histone

methylation represents another histone modification that is closely associated with the regulation of osteogenic-related gene expression, either through activation or repression. This paper

presents a systematic and comprehensive review of the current research on BM-MSCs and DMSCs in the field of regenerative medicine. We further elucidate the role of epigenetic regulation in

tooth regeneration and focus on the role and mechanism of histone methylation in the regulation of osteogenesis-related genes and tooth-derived stem cell functions. OSTEOGENIC/ODONTOGENIC

DIFFERENTIATION-RELATED GENES Bone formation primarily depends on osteoblast differentiation, which is strictly controlled by key transcriptional regulators. These regulators are activated

in response to specific external signals and signaling pathways that are regulated during development. The transcription factors (TFs) RUNX2 and SP7 (or Osterix, OSX) are essential for the

differentiation of mesenchymal precursor cells into the osteoblast lineage.24 Furthermore, the knockout of either RUNX2 or OSX in vivo impedes the normal formation of bone tissue. RUNX2

RUNX2 is a member of the RUNX family of TFs that forms a heterodimer with core-binding factor b (CBFb). This heterodimer enhances the ability of RUNX2 to bind to DNA and maintains protein

stability.25 Runx2 is an indispensable regulator of osteoblast and odontoblast differentiation, controlling the expression of specific genes involved in these processes. Runx2 has the

capacity to exert a variety of functions by regulating several key signaling pathways (such as FGF, Hedgehog, Wnt, and Pthlh) and certain TFs (such as OSX and Dlx5).26 Wnt signaling has been

demonstrated to regulate the function of Runx1 and Runx2.27 The canonical Wnt/β-catenin signalling pathway has been shown to regulate osteoblast differentiation. The pathway is initiated by

the binding of Wnt to the FZD receptor, which leads to the accumulation of β-catenin. Subsequently, β-catenin moves to the nucleus, leading to the transcription of target genes.27 The

noncanonical Wnt signaling ligand, WNT5A, has been identified as a key lineage-specific gene in the regulation of differentiation of dental mesenchymal cells. FGFs are a family of secreted

peptides controlling a series of secreted peptides in intrachondral and intramembranous ossification.28 The Twist, Msx2, and PLZF genes are primarily situated upstream of the Runx2 gene. The

principal downstream genes were OSX, ATF4, and ZFP521.29 ATF4 has been demonstrated to interact with Runx2, thereby upregulating the expression of osteocalcin (OCN) and Osx.30 BMP signaling

is essential for Runx2-dependent induction of the osteoblast phenotype.31 Upon activation of Smads (Smad1/5/8), BMP2 initiates Runx2 gene transcription through its distal P1 promoter and

proximal P1 promoter. Runx2 is also phosphorylated by the non-canonical BMP signaling pathway (TAK1-MEK-p38 or ERK), which promotes its association with the co-activator CREB binding

protein.32 In addition, BMP also promotes Runx2 acetylation via p300 for Runx2 stabilization.32 OSTERIX (OSX/SP7) OSX/Sp7 is an osteoblast-specific TF expressed in dental embryonic

mesenchymal cells. Novel variants of the OSX/Sp7 gene have been identified as a cause of recessive osteogenesis imperfecta, a disorder characterized by bone fragility and hearing

impairment.33 Sp7/Osx regulates and influences zebrafish larval tooth development and bone mineralization.34 During osteoblast differentiation, the molecular switch OSX/Sp7 plays a crucial

role in the formation of active chromatin states.35 Mice lacking Sp7 are unable to express osteoblast-associated genes such as SPARC(secreted protein, acidic and rich in cysteine),

Spp1(secreted phosphoprotein 1)/osteopontin, BSP. Additionally, they exhibited a significant reduction in Cola1 expression, a major component of the bone matrix. Sp7 knockout in mice

resulted in serious craniofacial malformations, dentin hypoplasia, and root abnormalities. However, initial tooth morphogenesis and dentin formation in the crowns were not impaired. SP7 is

involved in bone and tooth formation through a variety of molecular mechanisms, including FGF and TGF-β/BMP signaling. CHIP-seq data revealed that OSX binds to the BSP promoter, thereby

promoting osteoclastic differentiation and mineralization.36 SP7 has been demonstrated to promote dentinogenic cell differentiation by upregulating DSPP, DMP1, nestin, and ALP. Furthermore,

Sp7 acts as a cofactor of Dlx5 and physically interacts with Dlx5. The Sp7-Dlx5 complex regulates osteoblast differentiation. Other TFs containing homologous structural domains, including

Msx1/2, Satb2, and Alx4, may also interact with Sp7 in the osteogenic genome.34 DENTIN SALIVARY PHOSPHOPROTEIN (DSPP) DSPP represents the primary non-collagenous protein present in dentin,

osteoblasts, and alveolar bone, and is essential for the normal mineralization of dentin and enamel.37 The Runx2 and OSX/Sp7 are essential for the differentiation of osteoblasts and

odontoblasts, whereas DSPP exerts a regulatory effect on the latter process.38 The functions of DMP1 and DSPP in tooth formation and maintenance can be considered to be complementary and/or

synergistic.39 It is hypothesized that Runx2 may affect the development of the tooth embryo before the bell stage, yet not during the subsequent stage. Osx may be consistently expressed in

adult dentin and pulp cells, stimulating the differentiation and expression of DSPP via a non-Runx2-dependent signaling pathway in the later phase of tooth development. The BMP2 signaling

pathway plays a critical role in the differentiation and maturation of odontoblasts. BMP2-deficient teeth display morphological features similar to dentinogenesis imperfecta (DGI), which is

associated with mutations in the DMP1 and DSPP genes. Upregulation of BMP2-driven Dlx3, Dmp1, DSPP, and Sp7 promotes mesenchymal cell differentiation and biomineralization.40 THE ROLE OF

HISTONE METHYLATION REGULATION IN THE REGULATION OF OSTEO-/ODONTOGENIC DIFFERENTIATION OF DMSCS MECHANISMS OF REGULATION OF HISTONE METHYLATION The basic unit of chromatin is the nucleosome,

which is formed by the association of histones (H1, H2A, H2B, H3, and H4) with 147 bp of DNA41,42 (Fig. 1). The DNA is densely wrapped in the nucleus of a eukaryotic cell, forming a tightly

organized and compact structure that presents a significant challenge to transcription factors attempting to access the genes. The degree of chromatin openness or accessibility is a crucial

factor influencing gene expression. This reflects the transcriptional activity of chromatin and represents a pivotal area of investigation in the regulation of gene expression. This field

of study holds considerable significance in the context of epigenetic mapping, cell differentiation and development, and the pathogenesis of various diseases. Histone modifications within

promoter regions are indispensable for regulating chromatin accessibility and gene expression.43 Amino acid residues located at the N-terminus of histones are susceptible to

post-translational modifications, including methylation, acetylation, ribosylation, and ubiquitination, among others.44 The pathways involved in these post-translational modifications are

clinically approved targets for the treatment of human diseases. Currently, acetylation and methylation have been extensively studied,45 and acetylation of histones enhances gene

transcription by loosening histone-DNA complexes to allow transcription factor binding,46 whereas histone methylation can stimulate or inhibit gene transcription.47 Histone methylation has

long been implicated in heterochromatin formation and X chromosome inactivation.48,49 Dynamic regulation of histone methylation by site-specific histone methyltransferases (HMTs) and

demethylases (HDMs) is essential for establishing and maintaining epigenetic modifications.50 These enzymes are involved in a variety of essential cellular processes such as cell

proliferation and differentiation, DNA damage response and senescence, individual development, and cancer. Histone methylation is primarily observed at arginine (S) and lysine (K) residues

within the tails of H3/H4. This is mediated by lysine methyltransferases (PKMTs) and arginine methyltransferases (PRMTs).50 Additionally, Lys residues of histones can undergo mono-,

dimethyl-, and trimethylation.51 The study of lysine methylation sites in mammalian cells has been a prominent area of research, with six sites–K4, K9, K27, K36, and K79 on H3 and K20 on

H4–receiving particular attention.49 Of these, H3K4, H3K36, and H3K79 have been identified as active markers that occupy the active regions of transcribed genes within chromatin. Conversely,

H3K9, H3K27, and H4K20 are regarded as repressive markers that are typically related to silencing of gene expression and condensation of chromatin.22 In contrast to H3K9me3, which is a

heterochromatin marker, H3K27me3 is usually found in bivalent structural domains together with H3K4me3, and regulates stem cell differentiation. In mammalian cells, H3K27me3 protects

heterochromatin tissues following H3K9 methylation deletion.52 The most influential histone methylation sites in dental tissues identified in the current study are H3K4, H3K9, and H3K27. For

example, dental follicular progenitor cells and alveolar bone osteoblasts display elevated levels of the H3K4me3 mark, whereas periodontal ligament fibroblasts and odontoblasts exhibit high

levels of repressive H3K9me3 and H3K27me3 marks on the OSX, IBSP, and BGLAP/Ocn promoters.47,53 Specific enzymes are responsible for the methylation of H3K4, H3K9, and H3K27, and their

activities are essential for this process. H3K4 methyltransferases can be divided into two main categories: SET structural domain-containing proteins (including Mixed-Spectrum Leukemia

Proteins, MLLs, and Set1A/B) and unrelated histone methyltransferases (ASH1, SMYD, and PRDM).54 The COMPASS complex, in which mono-, di-, and trimethylates H3K4, comprises MLLs, Set1A/B,

WDR5, RbPB5, DYP30, and ASH2. (Fig. 3a) WDR5 is indispensable for the assembly of the SET1 complex and HMT activity. Furthermore, other subunits specifically interact with different KMT2

complexes, thereby increasing the diversity and functional modifications of these methyltransferases. The histone methyltransferases KMT1, KMT2H, and KMT8A/D are involved in methylation at

H3K9. The trimethylation of H3K27 is catalyzed by the enzyme EZH254. H3K4 demethylases include the LSD/KDM1 and JARID/KDM5 families. LSD1 removed the methyl group from K4me2 or K4me1,

yielding K4me0, whereas KDM5 removed the methyl group from K3me3 or K4me2, producing K4me1.54 The histone demethylase classes JMJD1/KDM3, JMJD2/KDM4, and KDM7 are responsible for H3K9

demethylation, whereas KDM6A/B is involved in H3K27 demethylation (Fig. 2). The impact of histone methylation is determined by the maintenance of a precise equilibrium between H3K4me3,

H3K9me3, and H3K27me3, which serve as major trimethylation sites. These modifications are essential for the formation of distinct chromatin structures, which, in turn, regulate the

expression of downstream genes.47,55,56 ROLE OF H3K4 HISTONE METHYLATION IN OSTEOGENIC/ODONTOGENIC DIFFERENTIATION MLL FAMILY H3K4 methylation is mediated by the SET1/COMPASS complex, which

comprises six catalytic subunits: MLL1-4(KMT2A-D), SETD1A/KMT2F, and SETD1B/KMT2G. The MLL1/2 complex contains the tumor suppressor Menin, which facilitates the recruitment of the MLL1/2

complex to HOX and other sites. The MLL3/4 complex contains NCOA6, PTIP, and PA-1, which contributes to the targeting of the MLL3/4 complex to a distinct set of genes. The MLL3/4 complex

also comprises the H3K27 demethylase UTX57 (Fig. 3a). An enhancer is a cis-transcription element critical for the regulation of gene expression. Upon activation, an enhancer binds to various

transcription factors, transcriptional co-activators, and RNA Pol II, thereby initiating the activation of target genes. In mammals, H3K4me1 is typically observed in enhancer regions and

serves as a hallmark marker of enhancers. Trimethylation of H3K4 (H3K4me3), on the other hand, is predominantly distributed near transcription start sites (TSS) and is associated with

transcriptional activation.58 H3K4 monomethylation is dependent on the recruitment of MLL3/4. The MLL1/2 core complex is responsible for the conversion of H3K4 to H3K4me1 and H3K4me2.

Methyltransferases MLL3(KMT2C) and MLL4 (KMT2D) are indispensable for the activation of enhancers, cellular differentiation, and overall development. They also regulate the early stages of

embryonic development and differentiation of embryonic stem cells,59 and their functions potentially occur independently of their enzymatic activities.60 The MLL2/3-COMPASS complex can bind

to the Runx2 P1 promoter, which enriches H3K4me3 levels and decreases H3K4me1 levels. This process has been observed to promote and maintain RUNX2 expression in osteoblasts.61 It has been

demonstrated that MLL1 is the enzyme responsible for the deposition of H3K4me3 at the promoters of developmental genes. Conversely, the MLL2-COMPASS-like complex has been demonstrated to

deposit H3K4me3 at “bivalent” promoters.60 Previous research has revealed that the catalytic activity of MLL1 is indispensable to skeletal development.60 MLL1 facilitates the deposition of

H3K4me3 at the promoter of the Hox gene, a crucial developmental regulator.62 Additionally, it has been observed to regulate dental mesenchymal cell differentiation by modulating the

formation of H3K4me3 at the WNT5A promoter.63 MLL1 forms a functional complex with nuclear factor I family member NFIB and targets the developmental genes DLX5 and Cebpa to promote

osteogenic differentiation in MSCs.64 SET7 catalyzes H3K4 trimethylation and is significantly expressed in dental papillae at the bell stage, enamel formation stage of developing dental

embryos (E17.5, P0 and P3), suggesting that SET7 plays a role in the odontogenic differentiation of dental follicle precursor cells (DFPCs).65 The phytohormone, Ferutinin, has been shown to

promote the expression of osteocalcin and collagen 1A1 mRNA and protein levels by activating H3K9 acetylation and H3K4 trimethylation markers in the promoter regions of WNT3A and DVL3 and

promote the osteogenic differentiation of DPSCs.66 Histone demethylation is a crucial epigenetic mechanism in the context of tooth-mandibular regeneration.67 The JARID1 family of proteins,

LSD1, and the NO66 protein, all of which contain the JmjC structural domain, have been identified as catalysts for H3K4 demethylation. The JARID1 family of proteins demethylates H3K4me2 and

H3K4me3, while LSD1 demethylates H3K4me1 and H3K4me2. Additionally, NO66 was identified as a catalyst for all three methylated H3K4 states. These proteins have been observed to exert

transcriptional repression through demethylation or recruitment of other inhibitory proteins. KDM5A JARID1B/KDM5A has been demonstrated to remove H3K4me2 and H3K4me3, thereby repressing

transcription of the Runx2 P1 promoter of osteogenesis-related genes. This results in the inhibition of osteogenic differentiation and exerts a negative regulatory role in bone formation in

osteoporotic mice.68,69 KDM5A binds to the promoters of RUNX2, OCN, and OPN, resulting in reduced levels and downregulation of H3K4me3. Several miRNAs have been shown to regulate the

function of KDM5A, which in turn regulates osteogenic differentiation. Examples of such miRNAs include miR-10769 and miR-29b-3p.70 During the odontogenic differentiation of human dental pulp

cells (hDPCs), there was a notable increase in mRNA expression of KDM5A at the initial stage and in a time-dependent manner throughout the induction of tooth formation. Knockdown of KDM5A

resulted in elevated expression of DMP1, DSPP, OSX, and OCN. A notable increase in total H3K4me3 levels was observed, whereas H3K4me2 levels remained unaltered. It has been hypothesized that

KDM5A may inhibit the odontogenic differentiation of hDPCs by removing H3K4me3 from the promoters of specific genes. This suggests that KDM5A-dependent histone demethylation is a crucial

factor in restorative dentinogenesis.71 LSD1 Lysine-specific demethylase 1 (LSD1, also designated as KDM1A) is a member of the flavin adenine dinucleotide (FAD)-dependent amine oxidase

family of demethylases. LSD1 is responsible for removing mono- and bi-methylation modifications of histones H3K4 and H3K9.72 As an initial member of the histone demethylation family, it

plays a crucial role in the differentiation of stem cells. LSD1 occupies the binding site for H3K4me2/3, and its activity is essential for the normal formation of bone tissue.73 The

administration of LSD1 inhibitors have been demonstrated to prevent osteoporosis after ovarietomy (OVX) in mice by increasing the number of osteoblasts.74 KDM1A/LSD1 has been shown to

exhibit dynamic bidirectional effects during osteogenic/odontogenic differentiation of DMSCs. Knockdown of KDM1A increased the expression of BSP, DSPP, DMP1, OSX, Runx2, and DLX in SCAP.

Conversely, in an in vivo transplantation experiment, more mineralized tissue formation was observed in the KDM1Ash group than in the control group, accompanied by upregulated expression of

BSP and DSPP. Further studies demonstrated that KDM1A can form a protein complex with PLOD2, an important gene involved in the osteogenic differentiation of BM-MSCs, and that this complex

can inhibit the osteo-/odontogenic differentiation of SCAP.75 HOXC8 gene has been found to controls the differentiation of spinal motor neurons.76 HOXC8 has been seen to binds directly to

the KDM1A promoter region, thereby regulating KDM1A. This results in a negative regulation of the osteo-/odontogenic differentiation of SCAP and the expression of Runx2 and Osx.77 The JmjC

structural domain-containing NO66 is a histone demethylase that can catalyze the removal of mono-, di-, and trimethyl marks at H3K4, di- and trimethyl marks at H3K36. In mammals, NO66 is

important for bone formation during early development and maintenance of adult bone homeostasis. During osteogenesis induction, NO66 has been observed to interacts with OSX, thereby

inhibiting its transcriptional activity and suppressing osteogenesis. This is achieved by removing labeling from the promoters H3K4me3 and H3K36me3 of OSX target genes, including bone

salivary protein (BSP), which in turn inhibits osteogenesis.35,78 Although NO66 has been shown to inhibit osteoblast differentiation, whether it plays a similar role in the differentiation

of DMSCs into odontoblasts remains unanswered. INFLAMMATORY CONDITION During the process of inflammation, various inflammatory factors (e.g., TNF-a, IL-1, IL-6) have been observed to affect

the epigenetic characteristics of cells, thereby influencing gene expression.79 Moreover, the inflammatory microenvironment has been demonstrated to impact stem cell differentiation and

function. In patients with chronic periodontitis, bacteria can alter histone modifications in periodontal tissue and activate a series of inflammatory and bone metabolism-related signaling

pathways.80,81 Lipopolysaccharide (LPS) has been identified as a pivotal periodontal pathogenic factor, capable of activating NF-κB signaling and promoting the release of inflammatory

cytokines such as IL-6 and IL-8. Research has demonstrated that rat dental capsule stem cells possess the capacity to modulate the expression of ERK1/2 and NF-κB signaling pathway via the

paracrine pathway. This modulation involves the repression of IL-1β, IL-6, and TNF-α gene expression, while concurrently promoting the expression of IL-4 and TGF-β. This regulatory mechanism

contributes to the preservation of dental pulp regeneration in rats afflicted with inflammation.82 In response to LPS stimulation, there was an increase in H3K27me3 enrichment in the

extracellular matrix and osteogenic gene promoters, whereas H3K4me3 enrichment increased in the inflammatory response gene promoters.83 In an LPS-induced periodontitis model, the histone

lysine methyltransferase SETD1 promoted the expression of inflammatory genes (such as IL-1β, IL-6, and MMP2) in PDLSCs through H3K4 trimethylation.80 SETD1 and p65 have been shown to have a

synergistic role in inflammatory regulation. Additionally, LPS increased the intranuclear localization of SETD1, which, in turn, mediated the nuclear translocation of p65 through SETD1 in a

p65-dependent manner. Furthermore, the use of an NF-κB inhibitor has been shown to reduce SETD1B expression and enhance osteogenesis in vivo. The expression of KDM5A and LSD1 was increased

in LPS-treated hPDLSCs.84 KDM5A knockdown enhanced ALP activity, promoted mineralization, and upregulated the expression of Runx2, OCN, and OPN. ChIP analysis confirmed that KDM5A binds to

the miR-495-3p promoter and inhibits the expression of miR-495-3p by demethylating H3K4me3, thereby enhancing HOXC8 transcription and inhibiting the osteogenic differentiation,

proliferation, and migration of hPDLSCs in patients with periodontitis.85 miR-590-3p targets LSD1 transcription, upregulates H3K4me2 methylation, and promotes OSX transcription, which in

turn facilitates the osteogenic differentiation of hPDLSCs in periodontitis.84 Markers of H3K4me3 activity are present in the promoters of TFs of the early osteogenic lineage, including

RUNX2, MSX2, and DLX5. The regulation of histone demethylases/methylases may affect the level of H3K4me2/3 at the promoters of osteo-/odontogenic genes, which in turn affect the

transcription of DMP1, DSPP, OSX, and OCN, thereby affecting the osteo-/odontogenic differentiation of DMSCs. KDM5A and LSD1 are the major demethylases of H3K4 (Table 1). Modification of

these enzymes or the utilization of miRNAs to affect their transcription may represent promising targets for enhancing dental-derived stem cell-mediated tissue regeneration (Fig. 3b). ROLE

OF H3K9 HISTONE METHYLATION IN OSTEOGENIC/ODONTOGENIC DIFFERENTIATION Notably, more than 50% of vertebrate genomes are packaged as condensed and transcriptionally repressed

heterochromatin.86 Epigenetic markers of heterochromatin are H3K9me2 or H3K9me3. H3K9 methylation is a key epigenetic mark, and this modification has been associated with tissue-specific

gene silencing. Theoretically, H3K9 methylation and chromatin densification inhibit transcription in three ways. First, they restrict the entry of RNA polymerase complexes. Second, they can

limit the elongation of the transcript or stability of the mRNA. Third, they can block pathways or functions that activate transcription factors. G9A, GLP, PRDM2, AND SUV39H1 In mammals,

H3K9 methyltransferases with different catalytic activities and target genes are involved in various cellular processes. These enzymes include the SUV39H1/2, dimeric G9a-GLP, and PRDM

families. Synergistic expression of G9a, GLP, PRDM2, and SUV39H1 during mouse dental embryo development is critical for the regulation of tooth development. The differential distribution of

these factors in the mesenchyme may be associated with the epigenetic regulation of signaling molecules engaged in mesenchymal-epithelial interactions. SETDB1 catalyzes the monomethylation

of H3K9 in the periplasmic region. Furthermore, evidence suggests that SETDB1 may inhibit osteogenic differentiation of BM-MSCs, regulate osteogenic differentiation through the

Setdb1/miR-212-3p/Hmgb1 pathway in hFOB cells.87 G9a may serve as a catalyst for mono/dimethylation of H3K9.88 G9a deficiency may result in delayed expression of Shh and BMP2/4 in dental

embryos. In addition, G9a may bind to Runx2 to form a complex, which is recruited to the endogenous Runx2 binding site and is directly involved in regulating tooth development.89 In

pulp-derived MSCs, low levels of G9a reduce inhibitory H3K9me2 production and promote osteogenesis.90 SUV39H1/2 catalyzes the dimethylation and trimethylation of H3K9 in constitutive

heterochromatin. LIM homobox8 (Lhx8) is a highly conserved transcription factor that regulates dentin development and regeneration through the Wnt and TGF-β pathways.91 Elevated Lhx8 levels

facilitate the activation of these two pathways, thereby ensuring the maintenance of mesenchymal development during the initial stages of tooth development. Conversely, the differentiation

of odontoblast cells is inhibited by Wnt and TGF-β in the subsequent phase. Through RNA hybridization, Zhou et al. discovered that the co-expression pattern of Lhx8 and Suv39h1 in the

mesenchyme was consistent with the dynamic expression profiles of the early epithelial signaling molecule FGF8 and the later mesenchymal signaling molecule BMP2.92 In hDPSCs, Suv39h1 forms a

complex with Lhx8, which inhibits the expression of the tooth-forming genes Runx2 and DSPP by controlling the formation of H3K9me2/3. (Fig. 4a) The PRDM family members are also involved in

H3K9 methylation. Although Prdm3 and Prdm16 have been demonstrated to have a significant impact on craniofacial development by maintaining the spatiotemporal expression of genes associated

with cranial neural crest cell development,93 their involvement in DMSCs differentiation has seldom been investigated and the underlying mechanisms remain unclear. JHDM3(JMJD2)/KDM4 The

primary enzymes implicated in the demethylation of H3K9 are members of the JHDM2/KDM3, JHDM3(JMJD2)/KDM4, and PHF8/KDM7B families.94 The JHDM2A-C proteins mediate the demethylation of

H3K9me1/2, thereby regulating hormone-dependent transcriptional activation. In addition, JHDM3 catalyzes the demethylation of H3K9me2 and H3K9me3 in vitro. KDM3A and KDM4C have been shown to

regulate heterodimerization through transcriptional activation of the lectin components NCAPD2 and NCAPG2. Chromatin reorganization inhibits progression of DNA damage and cell senescence.95

KDM4B was identified as an epigenetic coordinator of β-catenin/Smad1-mediated transcription, facilitating the removal of the repressive marker H3K9me3.96 JMJD2/ KDM4B regulates the

expression of H3K9me3 in the Runx2 promoter region. Its deletion inhibits osteogenic differentiation of MSCs isolated from oral bones (OMSCs) and promotes oral and maxillofacial bone

senescence.97 JMJD2B/ KDM4B facilitates the osteogenic differentiation of hBM-MSCs via regulating the methylation level of H3K9me2 on the Runx2 promoter.98 DLX homologous proteins are

involved in the formation of functional dental epithelial tissues and crown morphogenesis, regulating the differentiation of odontoblast cells.99 KDM4B binds to the DLX5 promoter and removes

H3K9me3 from the DLX5 gene promoter, and DLX5 plays a key role in osteogenic differentiation by regulating OSX expression.100 The DLX5 gene is highly expressed in DMSCs. DLX5 and KDM4B

exert positive effects on BMP signaling. These two factors appear to regulate each other through a positive feedback mechanism.101 The induction of Osterix expression by BMP-2 is facilitated

by Dlx5, yet it functions independently of Runx2. This mechanism promotes the osteo-/odontogenic differentiation potential of SCAP by promoting the expression of ALP, DSPP, DMP1, OPN, and

OSX.102 In addition, KDM4B can act in conjunction with c-Jun within the JNK signaling pathway, facilitating its recruitment to the DLX5 promoter region, thereby regulating the NGF-mediated

osteogenic differentiation of DMSCs.101 KDM4D enhanced the migratory and chemotactic capabilities of SCAP. Using protein profiling technology, co-binding proteins of KDM4D and ribosomal

protein S5 (RPS5) were successfully identified. The KDM4D-RPS5 complex jointly promotes osteo-/odontogenic differentiation potential and migratory capacity of SCAP by increasing the

expression of ALP, DSPP, and DMP1. These findings suggest that KDM4D is a promising candidate for dentin tissue.103 KDM3B/JMJD1B is a demethylase that alters chromatin modifications through

demethylation of H3K9me2. This demethylation process promotes the osteo-/odontogenic differentiation of SCAP by upregulating the expression of specific genes (such as Runx2, Osx, Ocn, and

Dspp)67 (Fig. 4b). INFLAMMATORY CONDITION While the epigenetic mechanism of KDMs plays a pivotal role in numerous biological processes, evidence suggests that KDM3C exerts an

anti-inflammatory effect on oral bacterial infections in periapical and periodontal tissues through the inhibition of NF-κB signaling and osteoclast (OC) production.104 However, its role in

the inflammatory response to oral bacterial infections remains unclear. H3K9me3 is predominantly associated with transcriptional silencing and occupies the promoter region. KMTs and KDMs

determine histone methylation and are associated with osteogenic differentiation. KMTs are involved in the repression of the histone marker H3K9, which includes Setdb1 and G9a, and have been

demonstrated to inhibit the osteogenic differentiation of DMSCs. However, KDM4B/D and KDM3B have been shown to promote the osteo-/odontogenic differentiation of DMSCs by regulating the

expression of specific genes (Table 2). ROLE OF H3K27 HISTONE METHYLATION IN OSTEO-/ODONTOGENIC DIFFERENTIATION H3K27me3 is associated with gene silencing and predominantly deposited in

CpG-rich promoters. H3K27me3-labeled promoters can still be bound by general transcription factors and by suspended Pol II binding, and are therefore considered parthenogenetic

heterochromatin, which exhibits a mechanistic profile distinct from that of H3K9me3. H3K27 methylation is mediated by the Polycomb Repressive Complex 2 (PRC2), which is composed of four core

subunits: EZH2, SUZ12, EED, and RBAP46/48.49,105,106 (Fig. 5a) EZH2, the core component of PRC2, facilitates the addition of a methyl group to histone H3K27, ultimately contributing to the

maintenance of gene silencing.107 SUZ12 and EED are essential for the enzymatic activity of EZH2. EZH2 contains a carboxy-terminal catalytic SET structural domain. During embryonic

development, EZH2 reduces chromatin accessibility and promotes silencing of HOXA and HOXD cluster genes through H3K27 trimethylation-catalyzed nucleosome compaction.108,109 EZH2 Numerous

studies have identified that Ezh2 as a key regulator of osteogenic differentiation and skeletal development.110,111 Overexpression of EZH2 facilitates lipogenic differentiation of MSCs.112

Under osteogenic induction conditions, EZH2 expression is downregulated and segregated from the RUNX2 promoter. Chromatin accessibility in osteoblasts may be maintained through the

synergistic action of Runx2 and its potential co-regulators, including Sp7 and AP-1, on enhancer modules that regulate osteoblast development.113 The conditional knockdown of Ezh2 in

undifferentiated MSCs resulted in defects in skeletal morphology and bone formation.114,115 EZH2 exerts its effects not only by directly regulating signaling modules and specific TFs, but

also through targeting of numerous novel genes(including ZBTB16, MX1, and FHL1) that mediate the osteogenic differentiation of MSCs110 As a histone methylation transferase, EZH2 enhances

migration and chemotaxis of DMSCs.116 Conversely, evidence indicates that it negatively regulates odontogenic differentiation.117,118,119 EZH2 has been observed to inhibit osteogenic

differentiation and mineralization of hDPCs through the β-catenin pathway, while simultaneously promoting their proliferation.117 Long non-coding RNAs (lncRNAs) are defined as transcripts

exceeding 200 nucleotides in length, and some lncRNAs interact with PRC2 and direct it to specific sites in chromatin.119 In contrast, EZH2, a catalytic component of PRC2, have been observed

to bind to RNA and, via EZH2-mediated trimethylation of H3K27, epigenetically silences KLF transcription, repress the expression of ALP, Osx, and OCN, and stimulate mineralization.120 A

recent study has found that the mechanosensitive lncRNA SNHG8 exerts a regulatory effect on EZH2 in PDLSCs, thereby inhibiting osteogenic differentiation.121 Mechanical force has been shown

to reduce EZH2 expression, accompanied by a delayed decrease in H3K27me3 expression and a significant decrease in H3K4me3 enrichment within the SNHG8 promoter region and 250 bp upstream of

it. The results of ChIP-seq corroborated the mutual regulation of SNHG8 and EZH2 and demonstrated that the reduction in SNHG8 expression after mechanical force stretch was attributed to the

diminution in the activation of its promoter. This indicated that the reduction in EZH2 expression was a consequence of decreased promoter activation. This indicates that the reduction of

EZH2 is a pivotal epigenetic event in the mechanical response related to the maintenance of superenhancer stability and stem cell function. CARMN is an evolutionarily conserved lncRNA that

is specific to smooth muscle cells and promotes the odontogenic differentiation of DPCs by impairing EZH2.122 KDM6A UTX/KDM6A, UTY/KDM6C, and JMJD3/KDM6B are demethylases that remove methyl

groups from H3K27me2 and H3K27me3, thereby activating the gene expression. Lysine-specific demethylase 6A (KDM6A), also known as UTX, is a member of the KDM6 family of H3K27 demethylases,

with the gene predominantly located at Xp11.3. KDM6A contains a catalytic JmjC structural domain at the C terminus and six tetrapeptide repeat (TPR) structural domains at the N terminus. The

catalytic JmjC structural domain enables KDM6A to demethylate H3K27me2/3. However, it has also been posited that the primary function of KDM6A is to recruit additional chromatin

regulators.123 KDM6A is an important tumor suppressor124 that regulates a multitude of stem cell functions, including chondrogenesis, macrophage M2 differentiation, muscle differentiation,

and neuronal differentiation. KDM6A has been shown to promote the osteogenic differentiation of PDLSCs by catalyzing H3K27me3 demethylation and enhancing the transcription of ALP, Runx2, and

OPN.125 Inhibition of EZH2 may preserve the osteogenic potential of PDLSCs following KDM6A knockdown by regulating H3K27me3.126 The utilization of pharmacological agents that target the

epigenetic regulation of the KDM6A gene has been demonstrated to effectively restore the osteogenic potential of cells. However, only a few studies have directly demonstrated alterations in

KDM6A gene expression in patients with periodontitis.52 LncRNA HOTAIRM1 has been shown to upregulate KDM6A/B expression and suppress EZH2 in a HIF-1α-dependent manner, thereby reducing the

distribution of H3K27me3 of ALP, M-CSF, Wnt-3a, Wnt-5a, Wnt-7a, β-catenin and others. This facilitates the transcription of these genes and promotes osteogenesis in hDFSCs.127 KDM6B KDM6B, a

histone demethylase with a Jmj-C structural domain, can convert H3K27me3 (repressive state) to H3K27me1 (activated state) within the central heterochromatin. Additionally, it has the

potential to regulate gene transcription through demethylases in a demethylase-dependent or -independent manner. Human KDM6B, also known as JMJD3, contains a JmjC structural domain and a

C-terminal fragment embedded in a GATA-like (GATAL) structural domain. JMJD3 counteracts the enzymatic activity of PRC2, thereby regulating the expression of specific genes. JMJD3 functions

as a transcription factor that interacts with coactivators to regulate the transcription of target genes independent of its demethylase activity. Additionally, it facilitates transcriptional

elongation and gene expression through its effect on RNA polymerase II (Pol II).128 KDM6B has been the subject of extensive studies in the context of immune diseases, cancer, and tumor

development as well as in pluripotent stem cells and fate determination.129,130 The osteogenic differentiation of BM-MSCs is a process in which KDM6B is responsible for the removal of methyl

groups from histones situated within the promoters of BMP2, BMP4, and HOXC6-1. This consequently regulates the expression of RUNX2.100 In the osteo-/odontogenic differentiation of DMSCs,

KDM6B is recruited to the BMP2 promoter, resulting in the removal of the silencing genetic marker H3K27me3 and subsequent activation of transcription of the downstream odontogenic marker

gene OSX/sp7, as well as the extracellular matrix genes BGLAP and OPN. The knockdown of KDM6B in DMSCs resulted in decreased ALP activity and mineralization.131 Postmenopausal patients with

osteoporosis exhibit a parallel decline in the levels of these markers and mineralization, indicating a significant reduction in the population of bone cells and osteoblasts, as well as

demineralization of both dense and cancellous bone. Although DPSCs primarily differentiate into dentin and BM-MSCs differentiate into bone, both dentin and bone formation share similar

mineralized matrix components. The function of KDM6B is inhibited by alcohol, which results in impaired osteo/odontogenic differentiation of DPSCs and a reduction in the expression of

several genes associated with mineralization, including BMP2, BMP4, OCN, and OPN. This indicates that excessive alcohol consumption may result in cellular damage associated with aberrant

tooth development and osteoporosis.132 During the bell stage of human tooth embryo development, miR-93-5p was identified as a differentially expressed miRNA. It was observed to target KDM6B

and regulate the H3K27me3 mark on the BMP2 promoter, thereby controlling odontogenic differentiation and dentin formation of DPSCs.133 INFLAMMATORY CONDITION EZH2 and H3K27me3 expression was

reduced in infected pulp tissues and cells. Inhibition of EZH2 suppressed the mRNA expression of IL-1β, IL-6, and IL-8 and proliferation in inflammation-stimulated hDPCs. Inhibition of EZH2

has been demonstrated to promote hDPCs mineralization through the epigenetic regulation of β-catenin expression and the activation of classical Wnt signaling pathways. LPS significantly

upregulates EZH2 and H3K27me3 expression in hPDLSCs during the inflammatory process in periodontal tissues. Inhibition of EZH2 suppresses the LPS-induced upregulation of inflammatory factors

(IL-6 and TNF-α) expression, cell proliferation and migration. In addition, the knockdown of EZH2 has been demonstrated to facilitate PDLSCs osteogenic differentiation by inhibiting the

TLR4/MyD88/NF-κB signaling pathway.118 In the initial stages of periodontitis, KDM6B is recruited to the promoters of IL-6 and IL-12β, leading to H3K27me3 removal and the activation of

target genes that regulate the inflammatory response.134 Insulin-like growth factors (IGFs) and their binding proteins (IGFBP5) have been linked to processes such as cell growth, bone

repair, and remodeling. IGFBP5 has been identified as a downstream target gene of KDM6B, which forms a protein complex with the BCL6 co-repressor (BCOR). This complex may negatively regulate

IGFBP5 transcription by promoting H3K27 methylation in the IGFBP5 promoter region. KDM6B can facilitate periodontal tissue regeneration by removing H3K27me3 from the IGFBP5 promoter region

(Table 3).135,136 Trimethylation of H3K27 is a repressive epigenetic marker essential for the expression of genes involved in tooth development. The PRC2 core subunit, EZH2, catalyzes H3K27

trimethylation and maintains gene silencing, thereby inhibiting the osteogenic differentiation of DMSCs. The specific demethylase, KDM6A, enhances the osteogenic differentiation potential of

DMSCs by removing the H3K27me3 tag at the Runx2 and Ocn transcription start sites. KDM6B is required for osteogenic differentiation, binds to BMP2 and HOX, and regulates the downstream

transcription of OSX, BGLAP, and SPP1. Ezh2 and KDM6A are epigenetic switches that function in concert to regulate the level of H3K27me3 in the promoter region of genes, thereby affecting

the expression of regulatory genes associated with adipogenesis and osteogenesis137 (Fig. 5c). ROLE OF BIVALENT CHROMATIN IN OSTEO-/ODONTOGENIC DIFFERENTIATION OF DMSCS BIVALENT CHROMATIN

Bivalent chromatin is an epigenetic state that has been identified as a mechanism for lineage commitment and the regulation of developmental gene expression. It is characterized by the

presence of H3K4me3 and H3K27me3. Chromatin immunoprecipitation demonstrated that H3K4me3 and H3K27me3 simultaneously occupy certain promoters in epithelial cells. Bivalent chromatin is

indispensable for maintaining stem cell pluripotency. It maintains a balance between promoter accessibility and long-range junctions to regulate the expression of developmental genes, such

as the regulation of VEGF-responsive angiogenesis138 and the timing of progenitor cell progression to mature neurons,139 affecting tooth organ development65. These modifications, designated

as “bivalent structural domains,” serve to maintain a stable state for the genes in question, which subsequently determines whether they are activated or repressed during the process of

differentiation. For example, under osteogenic induction conditions, H3K4me3 levels on the RUNX2 promoter in periodontal progenitor cells increased significantly 7 days after induction and

then declined rapidly at 14 days. H3K4me3 and H3K27me3 markers on the OSX promoter exhibited a continued decline during osteogenic induction, and ChIP-seq results showed that the inhibitory

histone markers H3K9me3 and H3K27me3 were higher in PDL progenitors. These data suggested that alterations in histone dynamics at the promoter level that repress key mineralizing

transcription factors regulate mineralization in DMSCs. H3K4 AND H3K27 The presence of bivalent modifications characterized by H3K4me3 and H3K27me3 may exert a pivotal influence on tooth

organogenesis by regulating cellular differentiation during tooth germ development. The expression of SET7 and EZH2, which function as methyltransferases, and KDM5B and JMJD3, which act as

demethylases, was consistent with the observed expression of H3K4me3 and H3K27me3.65 Epigenetic regulators that modulate bivalent promoters include histone methyltransferases including Ezh2

and MLL1. Inhibition of Ezh2 has been observed to results in the redistribution of bivalent structural domains in transcriptional regulators related to the WNT and Hedgehog pathways in

osteoblasts.140 Wnt5a, a significant member of the Wnt ligand family, is a lineage-specific gene that plays an important role in regulating the differentiation of DMSCs.63 It is markedly

expressed in both mouse and human dental papillae and is associated with the regulation of odontogenic differentiation and dentin layer formation during the bell stage. WNT5A is associated

with bivalent chromatin marks (H3K4me3/H3K27me3) in the stable state. Disassembly of H3K27me3 is a prerequisite for initiation of Wnt5a transcription. This process is subject to stringent

regulation by the JMJD3 and MLL complexes, which ultimately determine cell fate commitment in hDPCs. Despite speculation about the involvement of Wnt signaling in the regulation of

mineralization, there is a paucity of knowledge regarding the chromatin state of key promoters of mineralization genes, such as RUNX2 and OSX. Furthermore, the effect of these promoters on

lineage commitment and matrix secretion in mineralized tissues remains unclear.47 H3K9 AND H3K27 SATB2 is a transcription factor implicated in developmental regulation, chromatin remodeling,

and transcriptional regulation. It plays a positive regulatory role in the differentiation of osteoblasts, bone formation, and regeneration of MSCs. Mutations in this gene often result in

skeletal and dental hypoplasia.141,142 SATB2 promotes the osteogenic differentiation of PDLSCs, DPSCs, and stem cells isolated from human exfoliated deciduous teeth (SHEDs). JHDM1D/KDM7A is

a histone demethylase that specifically removes the dimethylation marks of H3K9 and H3K27 from the promoters of target genes.141,143 SATB2 may regulate odontogenic differentiation of hDPSCs

by downregulating DKK1 and activating the Wnt/β-catenin signaling pathway through the inhibition of JHDM1D expression. DKK1 is an important target of KDM6A/KDM7A,125 but the mechanism by

which DKK1 regulates JHDM1D remains unclear. H3K4 AND H3K36 It has been demonstrated that inflammation and hypoxic niches exert an influence on MSC-mediated tissue regeneration. For

instance, oxidative stress, characterized by abnormally elevated reactive oxygen species (ROS) levels, can induce mitochondrial dysfunction, leading to cell death. This, in turn, can

diminish the osteogenic and odontogenic capacity of DPCs and intensify inflammatory states.144,145 Hypoxia inhibited the accumulation of superoxide in MSCs mitochondria, upregulated membrane

potential, and internalized into damaged cells through extracellular vesicles, thus affecting metabolic status.146 KDM2A is a major member of the JmjC structural domain-containing histone

demethylases, which are involved in the demethylation of H3K36 and H3K4, thereby regulating important biological processes, including chromatin remodeling and cell development. For instance,

it is responsible for regulating the differentiation of enamel cells and odontoblasts.147 KDM2A has also been demonstrated to modify non-histone proteins, including β-catenin and NF-κB.

This enables the regulation of their stability and transcriptional activity.148 KDM2A and BCOR have been shown to form a complex that inhibits osteogenesis. This is achieved by increasing

histone H3K4/36 methylation of the epigenetic osteogenic protein (EREG) promoter. This in turn inhibits EREG transcription and subsequent OSX and DLX5 expression.149 SFRP2 is a classical

extracellular inhibitor of Wnt signaling. It can also enhance the secretion of osteo-/odontogenic related factors such as IGFBP5, IGFBP4, MMP1, and cell homing related functional proteins

CXCL5, CXCL12, CXCL6 in SCAP through paracrine effects. The demethylation process of SFRP2, catalysed by KDM2A, plays a regulatory role in the osteo-/odentigenic differentiation processes of

SCAP.150 Under conditions of inflammation and hypoxia, SFRP2 inhibits NF-κB signal transduction by inhibiting Wnt/β-Catenin pathway and enhances osteo-/odontogenic differentiation of SCAP.

In conditions of hypoxia, there is an increase in the expression of KDM2A and HIF-1, while BCOR expression remains unaltered. The transcription of SFRP2 is regulated by the demethylation of

H3K36me2 and H3K4me3 on the SFRP2 promoter, which ultimately affects the osteo-/odontogenic differentiation function of SCAPs (Table 4).151 Despite the advent of massive parallel DNA

sequencing techniques, numerous pivotal inquiries into bivalent promoters have persisted since the discovery of bivalence in pluripotent stem cells. To gain further insights into the unique

patterns of H3K4me3 and H3K27me3 repair, it is essential to develop new methods that can accurately quantify subtle changes in multiple cell types. Further investigation may elucidate the

potential links between bivalent chromatin and the osteo-/odontogenic differentiation of DMSCs, thereby advancing biomedical research on disease treatment and tissue regeneration (Fig. 6).

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL RELEVANCE OF HISTONE METHYLATION REGULATION IN DMSCS TO HEALTH AND DISEASE IN VIVO THE IMPORTANCE OF HISTONE METHYLATION REGULATION OF DMSCS IN TOOTH

DEVELOPMENT During the development of tooth embryos, bivalent modifications are characterized by the presence of H3K4me3 and H3K27me3. The spatiotemporal expression of SET7, EZH2, KDM5B, and

JMJD3 plays an instrumental role in this process.65 EZH2 has been shown to antagonize the activity of the Aridl protein, thereby regulating the process of formation and development of

dental root forks in murine models through the action of Cdkn2a, which is a critical cell cycle inhibitor.152 ASH2L plays a significant role in the regulation of the expression of key

developmental genes, including Shh and Trp63, by modulating H3K4me3 modification. Deficiencies in ASH2L have been observed to result in abnormal differentiation of tooth epithelium, which

can ultimately manifest as defects in enamel development.153 OSTEOPOROSIS The gradual accumulation of epigenetic changes associated with the process of aging can lead to abnormal regulation

of gene expression, metabolic instability, stem cell aging and/or failure, and tissue homeostasis imbalances.154 H3K4me3, H3K27me3, and H3K36me3 may have undergone ‘remodelling’ during

ageing.155 Young exosomes secreted by SHED (SHED-Exos) regulate histone methylation and suppress NF-κB, thereby reversing the senescence of aged tendon stem/progenitor cells (AT-SC) and

preserving their tenogenic capacity.156 α-ketoglutaric acid (α-KG), an intermediate derived from the tricarboxylic acid cycle, has demonstrated therapeutic potential in the treatment of

age-related osteoporosis by reducing the accumulation of H3K9me3 and H3K27me3, upregulation of BMP signaling, and Nanog expression to restore MSCs function.157 Furthermore, serine

synthesis-derived α-KG is imperative for the function of JMJD3, which catalyzes the removal of H3K27me3 at activated T nuclear factor, cytoplasmic 1 (Nfatc1) gene sites, consequently

inducing NFATc1 expression and osteoclast maturation.158 The histone demethylase KDM7A has been shown to exerts a pivotal function in regulating bone homeostasis by modulating the

differentiation of osteoblasts and osteoclasts. KDM7A has been observed to upregulate the expression of fibroblast activating protein alpha (FAP) and nuclear factor κB ligand receptor

activator (RANKL) in BM-MSCs by removing H3K9me2 and H3K27me2 markers from FAP and RANKL promoters. Inhibition of KDM7A has been observed to result in increased FAP expression and

inactivation of canonical Wnt signaling. Concurrently, this inhibition has been shown to promote osteoclast differentiation and bone resorption through enhanced RANKL expression.159

OSTEOARTHRITIS KDM6A has been shown to be involved in the process of chondrogenic differentiation of PDLSCs by demethylation of SOX9, Col2a1, and ACAN. The upregulation of KDM6A, or the

application of EZH2 inhibitors, has been observed to have the potential to enhance the process of mesenchymal stem cell-mediated cartilage regeneration in cases of inflammatory tissue

destruction, a condition that is exemplified by osteoarthritis.126 In osteoarthritis, a reduced methylation of lysine 79 on histone H3 (H3K79me) has been identified as a protective

epigenetic mechanism. The study found that preservation of H3K79 methylation with KDM7A/B inhibitors, such as daminozide, was targeted to protect bone joints in intra-articular therapy in

mice.160 KDM3A and G9A are a pair of antagonistic histone modification enzymes. KDM3A has been shown to attenuate the ubiquitination of SOX9 by demethylating lysine (K) 68 residues, thereby

enhancing the stability of SOX9. In contrast, G9A has been shown to promote the ubiquitination and subsequent degradation of SOX9 by methylating K68 residues. The highly specific G9A

inhibitor BIX-01294 has been shown to significantly induce the cartilage differentiation of DPSCs, providing a theoretical basis for enhancing the clinical application of DPSCs in cartilage

tissue engineering therapy.161 Consequently, a combination strategy that targets multiple components of epigenetic mechanisms and utilizes a combination of synthetic interactions or immune

blocking is expected to enhance the function of DMSCs. POTENTIAL THERAPEUTIC TARGETS RELATED TO HISTONE METHYLATION REGULATION OF DMSCS VITAL PULP TREATMENT As previously stated,

conventional dentistry is costly, intrusive, and based on a defunct mechanical understanding of dental diseases. In contrast, contemporary biological dentistry utilizes a cellular approach,

focusing on the stimulation of cell activity and the promotion of regeneration.162 The development of epigenetic therapy drugs for pulp capping materials currently includes HDAC inhibitors,

which have been shown to reduce inflammation and stimulate restorative dentin formation.162,163 However, there is still more that researchers need to learn about how changes to the building

blocks of histone methylation affect the regeneration of pulp dentin complex. EZH2 can be used as a potential regulator of pulpitis and regeneration,32,164 and the development of inhibitors

or small molecule agents to interfere with its function may be a viable potential therapeutic strategy. ORTHODONTIC TREATMENT It has been demonstrated that the accumulation of advanced

glycation end products (AGEs) in the periodontal tissue of type 2 diabetes patients contributes to enhanced bone fragility.16 This has been identified as a significant factor hindering

orthodontic tooth movement and compromising the efficacy of orthodontic treatment. The targeting of the AGE/RAGE pathway or the enhancement of KDM6B function has been demonstrated to

increase the antioxidant capacity of PDLSCs, inhibit cell senescence, and promote osteogenic differentiation. A combination of epigenetic or Wnt pathway modulators with RAGE blockers has the

potential to enhance the efficacy of orthodontic treatment in patients with diabetes.165 MAXILLOFACIAL BONE DEFECT The treatment of maxillofacial bone defect resulting from periodontitis,

trauma, and tumors is contingent upon the restoration of bone tissue.166 Following the degradation of bioactive microspheres, DP-Ak has been observed to release ions and activate sensory

nerve cells, resulting in the secretion of calcitonin gene-related peptide (CGRP). The reduction of H3K27me3 levels, achieved by the inhibition of EZH2 and the enhancement of KDM6A, has been

demonstrated to promote bone repair.166 The present study investigates the regulatory role of estrogen in DMSCs osteogenesis via the ERα/KDM6B/BMP2 axis in a rat cranioparietal defect

model. This process is facilitated by the recruitment of KDM6B to the BMP2 and HOXC6 promoters, resulting in the removal of the H3K27me3 marker and subsequent activation of its

transcription.167 SUMMARY AND OUTLOOK The methylation of histone lysine or arginine residues has been demonstrated to be of significant importance to gene regulation, as well as other

physiological processes. Aberrant histone methylation, which can be precipitated by gene mutation, translocation, or overexpression, frequently results in the onset of developmental defects

or diseases. MLL1/4, PRMT1/5, KDM5C, and KDM6B are essential for neurodevelopment, while EZH1/2, MLL4, KDM6B, and EED are implicated in cardiac development. LSD1, MLL1, EED, and G9a impact

the hematopoietic system.123 Genetic mutations in H3K4 methyltransferases are linked to syndromes involving bone and facial deformities, intellectual impairment, and often reduced body size

and microcephaly.123 Furthermore, it has been determined that mutations in other histone methylation regulators, such as EZH1 and NSD1, can be a causative factor for overgrowth syndrome. The

utilisation of small molecule inhibitors of histone modifying enzymes, which correct abnormal methylation, has the potential to function as both novel therapeutic interventions for these

diseases and as chemical probes for epigenetic studies.168 Nonetheless, the identification and advancement of small-molecule inhibitors of KMTs and KDMs remains in its nascent stages.

Noteworthy endeavors and notable achievements from both academic and pharmaceutical sectors have only surfaced in recent years. Further experimentation is necessary to substantiate the

pivotal function of histone methylation-related enzymes in disease processes and to establish a theoretical framework for enhancing the osteo-/odontogenic differentiation function of DMSCs

or for the treatment of other diseases. DMSCs play a pivotal role in tooth development and maintenance of oral tissue health. The capacity for differentiation and self-renewal is important

in organizational engineering and regenerative medicine. The odontogenic differentiation of DMSCs is a crucial step in dentin formation. This process is orchestrated at the molecular level

by a complex network of signaling pathways, transcription factors, and post-transcriptional and epigenetic regulatory mechanisms, which together facilitate the coordinated expression of a

multitude of genes. The histone methylation status is a significant factor in stem cell differentiation. This paper reviews the role of epigenetic histone methylation modifications in tooth

development and the potential mechanisms of histone methylation and demethylation enzymes in the regulation of the osteo-/odontogenic differentiation of DMSCs. Methylation of H3K4 and H3K36

is associated with the activation of transcription, whereas that of H3K9 and H3K27 is associated with the repression of transcription. Osteogenesis comprises three distinct phases:

proliferation, matrix maturation, and mineralization. These phases are regulated by transcription factors RUNX2 and OSX. RUNX2 and OSX are early markers of osteoblast/odontoblast

differentiation, whereas OCN functions primarily during late osteogenesis. In MSCs, the expression of these genes is negatively regulated by inhibitory epigenetic markers including H3K4me1,

H3K9me3, and H3K27me3. Simultaneous activation of the classical Wnt and BMP signaling pathways by RUNX2 and OSX is indispensable for formation and proliferation of the mature osteoblast

phenotype. H3K4me2/3 marks were enriched in the promoter regions of osteo-/odontogenic genes of BM-MSCs and DMSCs. The inhibition of histone demethylases KDM1A and KDM5A expression has been

demonstrated to facilitate the osteo-/odontogenic differentiation of DMSCs through the activation of Runx2, Osx, and DSPP gene transcription. However, under inflammatory conditions, the

accumulation of H3K4me3 may also affect the expression of inflammatory genes. H3K9 methylation is primarily associated with the repression of gene transcription. KDM4B inhibits maxillofacial

bone senescence, enhances the proliferation and migration of SCAP, and activates the expression of the osteogenic gene DLX5 by removing the H3K9me2/3 mark. The PRC2 core subunit EZH2

catalyzes the methylation of H3K27, thereby maintaining gene silencing. During the differentiation process of DMSCs, EZH2 and KDM6A/B function in concert to regulate the level of H3K27me3,

thereby controlling the expression of osteo-/odontogenic-related genes, including BMP2, OSX, OCN, DSPP, and DMP1. Bivalent histones may play a significant role in dental organ development

and lineage-specific differentiation of MSCs by functioning as regulators of cell differentiation. Co-localization of H3K4me3 and H3K27me3 marks within the promoter region has been observed

to alter the expression of certain genes. In pluripotent stem cells, the majority of H3K27me3 peaks were localized to H3K4me3-tagged promoters, indicating that genes with bivalent structural

domains are the prevailing phenomenon rather than an exception. This phenomenon also applies to osteogenic differentiation of DMSCs. The inhibitory epigenetic markers H3K9me3 and H3K27me3

exhibit a pattern of bivalent modifications in dental mesenchymal progenitor cells and are predominantly located on OSX during odontogenic differentiation. It has been hypothesized that the

histone methylases EZH2, MLL1, and MLL4, in conjunction with the histone demethylases KDM6A/B and KDM7A, may coordinately control the levels of H3K4me3, H3K9me3, and H3K27me3 in specific

gene promoters, thereby controlling downstream gene expression. Although most studies have been conducted in vitro, further in vivo studies and animal disease models are necessary to

evaluate their potential applications in disease treatment and regenerative medicine. In addition to histone modifications, a number of other epigenetic modifications, including those of RNA

and molecular chaperones, contribute to a range of biological processes, including embryonic development, cell differentiation, and maintenance of pluripotency. In conclusion, dynamic

modulation of histone methylation plays a significant and multifaceted role in the regulation of chromatin state. Modulating histone functions to control the differentiation of DMSCs is a

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_J. Hematol. Oncol._ 9, 49 (2016). Article  PubMed  PubMed Central  Google Scholar  Download references FUNDING This work was supported by grants from the National Key Research and

Development Program (2022YFA1104401), CAMS Innovation Fund for Medical Sciences (2019-I2M-5-031 to Z.P.F.) and grants from Innovation Research Team Project of Beijing Stomatological

Hospital, Capital Medical University (NO. CXTD202204 to Z.P.F.). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction,

Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, Beijing, China Meijun Hu & Zhipeng Fan * Beijing Laboratory of Oral Health, Capital Medical

University, Beijing, China Zhipeng Fan * Research Unit of Tooth Development and Regeneration, Chinese Academy of Medical Sciences, Beijing, China Zhipeng Fan Authors * Meijun Hu View author

publications You can also search for this author inPubMed Google Scholar * Zhipeng Fan View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

Conceptualization, Zhipeng Fan; writing-original draft preparation, Meijun Hu; writing-review and editing, Zhipeng Fan, Meijun Hu. All authors have read and agreed to the published version

of the paper. CORRESPONDING AUTHOR Correspondence to Zhipeng Fan. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. SUPPLEMENTARY INFORMATION

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CITE THIS ARTICLE Hu, M., Fan, Z. Role and mechanisms of histone methylation in osteogenic/odontogenic differentiation of dental mesenchymal stem cells. _Int J Oral Sci_ 17, 24 (2025).

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