The role and mechanisms of polycomb repressive complex 2 on the regulation of osteogenic and neurogenic differentiation of stem cells

Abstract The stem cells differentiate into osteoblasts or neurocytes is the key process for treatment of bone‐ or neural tissue‐related diseases which is caused by ageing, fracture, injury, inflammation, etc Polycomb group complexes (PcGs), especially the polycomb repressive complex 2 (PRC2), act as pivotal epigenetic regulators by modifying key developmental regulatory genes during stem cells differentiation. In this review, we summarize the core subunits, the variants and the potential functions of PRC2. We also highlight the underlying mechanisms of PRC2 associated with the osteogenic and neurogenic differentiation of stem cells, including its interaction with non‐coding RNAs, histone acetyltransferases, histone demethylase, DNA methyltransferase and polycomb repressive complex 1. This review provided a substantial information of epigenetic regulation mediated by PRC2 which leads to the osteogenic and neurogenic differentiation of stem cells.

example, neurogenic heterotopic ossification occurs in 25% cases of spinal cord injury, in which impaired nervous metabolic system causes chaotic bone formation surrounding neural tissue. 12 This implies that directed differentiation of stem cells requires specific initiation. Therefore, understanding the differentiation regulation of osteogenesis and neurogenesis is crucial for the required tissue repair. The fate determination of stem cells requires a careful balance of genetic and epigenetic programming. 13 Recent studies have identified that epigenetic regulation determines the stem cell-specific lineage differentiation. [14][15][16] Epigenetic mechanisms can maintain the long-term regulated effects of gene expression in response to environmental stimulation. [16][17][18][19] Therefore, understanding epigenetic regulation in osteogenic and neurogenic differentiation is of certain guidance for subsequent research and is crucial for promoting the differentiation efficiency and regeneration effect of bone or neural tissue.
Polycomb group complex (PcGs)-mediated chromatin leading to epigenetic repression is an important regulation mechanism, which caused the dynamic change of gene expression profiles during the differentiation and maturation of stem cells. 20,21 PcGs-mediated H3K27me3 is thought to inhibit inappropriate or premature differentiation and may play a key role in determining lineage differentiation of stem cells. 22,23 Human PcGs mainly consist of two subtypes, the polycomb repressive complex 1 and 2 (PRC1 and PRC2). 22 PRC1 affects chromatin compactness and can block the transcriptional elongation of RNA polymerase II, thus mediating heterochromatin inhibition. 23,24 PRC2, through its core component, enhancer of zeste homologue 2 (EZH2), keeps gene silencing by maintaining the silent form of histone H3 Lys27 (H3K27). 25,26 These two complexes are synergistic, and PRC1 function depends on the PRC2. 27 And PRC2-modified H3K27me3 is specifically recognized and bound by factors such as the PRC1 subunit CBX7 that further helps maintaining long-term gene silencing. 22 This review mainly focuses on the role and mechanism of PRC2 with EZH2 as the core unit in the osteogenic and neurogenic differentiation regulation of stem cells.

| CORE SUBUNITS OF PRC 2
The core subunits of PRC2 mainly contain four proteins: EZH1 or EZH2, suppressor of zeste 12 (SUZ12), embryo development of F I G U R E 1 The core subunits of PRC2 in Homo sapiens. (A): The core subunits of PRC2. The PRC2 complex mainly contains four core subunits: EZH1/2, SUZ12, EED and RBAP (B): The protein structure of PRC2 core subunits. EZH1/2 is the core subunit of histone H3K27me3 methyltransferase that contains the SET domain and is involved in PRC2 complex formation; SUZ12 mainly maintains the stability of PRC2 complex and assists EZH2 in exerting histone H3K27me3 methyltransferase activity; EED contains the WD repeat function domain and also assists EZH2 in exerting histone H3K27me3 methyltransferase activity; RBAP48 or RBAP46 contain WD repeat function domain involved in the formation, and subsequent histone binding anchoring of PRC2 complex the ectoderm protein (EED) and retinoblastoma binding protein (RBAP48/RBBP4 or RBAP46/RBBP7) ( Figure 1A). 28 Figure 1B). 29 SUZ12 contains the VEFS domain and the C-C-H-H zinc finger structure and mainly maintains PRC2 stability and assists EZH2 in exerting histone H3K27me3 methyltransferase activity ( Figure 1B). 30 EED contains the WD repeat function domain and also assists EZH2 in exerting histone H3K27me3 methyltransferase activity ( Figure 1B). 31 RBAP48 and RBAP46 contain WD repeat function domains and are involved in PRC2 formation and the subsequent binding to histone ( Figure 1B). 32,33

| PRC 2 VARIANTS
PRC2 plays an important role during epigenetic modification mediated lineage differentiation of stem cells. 13,33 EZH protein is the core catalytic subunit of PRC2, EZH1 and EZH2 are 65% homologous, and EZH1 is mainly expressed in the adult tissues or the undivided cells, while EZH2 is mainly expressed in the embryogenic or differentiated cells. 28 Research finds that EZH1 and EZH2 form similar PRC2 complexes but exhibit contrasting repressive roles. 34 PRC2-EZH2 complex is the classical form of PRC2, and this complex mainly contains four core subunits: EZH2, SUZ12, EED and RBAP and is assembled based on the antedate formation of EZH2-EED complex ( Figure 2A).
Its histone H3K27me3 methyltransferase activity is much lower and mainly represses transcription by directly compacting chromatin instead of methyltransferase function. 34 PRC2-EZH1 seems to switch from a methyltransferase catalytic mechanism to a non-catalytic mechanism. 35,36 Sometimes EZH1 and EZH2 may have some functional overlap in the PcGs-dependent H3K27me3 regulation. The study reveals that mouse skin appears pathologic phenotypes only when EZH1 and EZH2 both delete. 21 And EZH1 colocalizes and preferentially preserves the H3K27me3 on development-related genes to safeguard embryonic stem cells (ESCs) identity in EZH2 -/-ESCs.

F I G U R E 2
The variants of PRC2 in Homo sapiens. (A) PRC2-EZH2 complex is the classical form of PRC2, this complex mainly contains four core subunits: EZH2, SUZ12, EED, and RBAP and is assembled based on the antedate formation of EZH2-EED complex. As the classical form of PRC2, PRC2-EZH2 complex plays the mainly histone H3K27me3 methyltransferase activity. (B) PRC2-EZH1 complex contain EZH1, SUZ12, and RBAP. Compared with PRC2-EZH2, its histone H3K27me3 methyltransferase activity is much lower and mainly represses transcription by directly compacting chromatin instead of methyltransferase function. PRC2-EZH1 seems to switch from a methyltransferase catalytic mechanism to a non-catalytic mechanism. (C) PRC2-EZH2-PCL complex is PcG-like protein (PCL) binding to PRC2-EZH2 complex. This complex containing EZH2, SUZ12, EED, RBAP, and PCL. PCL includes PCL1, PCL2, and PCL3, which is not a core subunit and this combination leads complex containing two additional PHD finger structure. PCL is mainly promotes the recruitment and facilitation of classical PRC2-EZH2 binding to the targeted CpG island and further H3K27me2 to H3K27me3 enrichment Depletion of EZH1 in EZH2 -/-ESCs abolishes residual H3K27 methylation and derepresses the target genes. 37 Study shows that EZH2 deletion results in myelodysplasia, myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPNs) development in mice.
Only EZH1 deletion does not cause dysplasia in nervous system, which just happens when EZH2 and EZH1 both get deleted. This suggests that EZH1 plays partial compensatory role in hematopoietic system diseases caused by EZH2 deficiency. 38 PcG-like protein (PCL) including PCL1, PCL2 and PCL3 can also bind to PRC2 to produce another PRC2 variant and change its nature ( Figure 2C). 39 PCL is not a core subunit and is mainly involved in the recruitment and facilitation of PRC2 binding to the targeted CpG island. This complex contains two additional PHD finger structure and is mainly responsible for PRC2 recruitment and further H3K27me3 enrichment. 28 In summary, PRC2-EZH2 complex is the core catalytic complex of H3K27 histone methyltransferase and plays the central role in the epigenetic regulation of PcG complex.

| The interaction of non-coding RNAs and PRC2
The non-coding RNAs mainly includes microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Terminal mesodermal-specific lineage differentiation of stem cells is reported to be associated with upregulated miR-17, miR-21, miR-34a and miR-146a, along with lncRNAs are also regarded as a participant during the lineage commitment and maturation of the stem cell. 44 Several PcG proteins possess RNA binding activity, which is required for further PcGs binding to DNA. EZH2, the catalytic subunit of PRC2, acts as the key lncRNA nuclear target. 45 Moreover, at least half of the identified EZH2-binding lncRNAs are accompanied with a coding gene, either antisense or at the promoter region, suggesting EZH2 involved in local cis-regulation by lncRNAs. 45 The lncRNA CARMEN has been reported to co-act with both EZH2 and SUZ12 to regulate the differentiation of cardiac stem cells. 44 A lncRNA transcribed from the Kcnq1 overlapped transcript 1 (Kcnq1ot1) is highly recruited with EZH2 in the mouse brain and enhances neurogenic differentiation of mMSCs. 45,46 Similarly, systematic analysis reveals the presence of an interaction between HOTAIR and PRC2 subunit, HOTAIR interacts with PRC2 at HOXD, and knockout of HOTAIR reduces H3K27me3 at HOXD site. 47 It has been shown HOXA-AS3 serves as an epigenetic switch of PRC2-EZH2 histone methylase modification, determining the differentiation of hBMSCs, and is necessary for H3K27me3 modification on the RUNX2 promoter, and knockdown of HOXA-AS3 enhances RUNX2 expression and osteogenic ability of hBMSC. 48 Study found that lncRNA maternally expressed 3 (MEG3) could interact with EZH2 and contribute to PRC2-EZH2 recruitment and promote the transmission and aggregation of PRC2 complex to the specific target genes Glt2, Rian and Mirg and lead to the differentiation of pluripotent stem cells. 49 It has been shown that the PRC2-binding lncRNA X inactivation specific transcripts (XIST) which containing a 28 bp repetitive element repA, thus providing an example of a specific lncRNA motif that binds to the PcG complex. 50 Another study has identified a 'Crab-claw' structure which visually has two-paired 4-nt loops that act as the molecular basis for lncRNA interaction TA B L E 1 The noncoding RNA interacted with PRC2 during the osteogenic and neurogenic differentiation of stem cells with EZH2. 51,52 LncRNAs Hnf1aos1 and Gm12840 also contain the same structure that is deemed to accelerate the interaction with EZH2. 45 In conclusion, the interaction of PRC2-EZH2 and ncRNAs helps in understanding its epigenetic regulation mechanism, and further studies should investigate whether ncRNAs prevent PRC2-EZH2 target binding or lead to locus-specific recruitment (Table 1).
In addition, PRC2 is required to maintain expression of maternal miRNAs and lncRNAs from the Gtl2-Rian-Mirg locus within the Dlk1-Dio3 imprinted gene cluster in mouse ESCs. In the absence of PRC2-EZH2, the entire Gtl2-Rian-Mirg locus becomes transcriptionally silent due to gain of de novo DNA methylation at the intergenic differentially methylated regions (IG-DMRs), a critical cis-regulatory element that controls expression of maternal Gtl2-Rian-Mirg locus, and further mechanistic study shows that PRC2 prevents recruitment of Dnmt3 methyltransferases. 49 These indicate that the action of noncoding RNAs and PRC2 is reciprocal.

| The interaction of DNA, RNA, histone modification genes and PRC2
The interaction of PRC2-EZH2 subunit with DNA and other histone modifying enzymes has also been reported. PRC2 function and DNA in the functional regulation of stem cells. 56 Researchers observe the interaction between LSD1 and EZH2 proteins in MCF-7 cells. The interaction between LSD1 and EZH2 stabilizes the binding of LSD1 to IRF9 promoter, which is a key transcription factor of the interferon pathway. 57 Then, the balance between H3K4me3 activation and H3K27me3 inhibition may be considered as a rheostat of targeted genes repression. 28 During human cytomegalovirus (HCMV) infection process, study shows that EZH2 and its regulators Jumonji

| The interaction of PRC2-modified H3K27me3 and PRC1
Chromatin-modifying activities inherent to PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation and development. In addition to being directly recruited to specific target sites for H3K27me3 modification, PRC2-EZH2 also functions through other mechanisms. However, no evidence has been found to prove that H3K27 methylation causes gene silencing by changing the interaction between nucleosomes. 28 The current study notices that H3K27me3 modification in the PRC1 target genes provides recognition of binding sites and promotes the loci inner ring.  60 This suggests a linkage between the PRC subunit proteins and may complete the mechanism of PRC complexmodified H3K27me3, helping to unravel the PRC2-EZH2 regulation process, and provides intervention points for more subtle links.

| THE ROLE OF PRC 2 IN THE OS TEOG ENI C D IFFERENTIATI ON OF S TEM CELL S
It is known that craniofacial skeleton formation is crucially dependent on epigenetic regulation in senior vertebrates. 61  and further repress inflammation and bone loss in prediabetic NOD mice. 71 But EZH2 inhibition increases bone density in adult mice and alleviates bone loss in ovariectomy osteoporosis mice model. 36 In addition, GSK126 attenuates bone loss in the ovariectomy osteoporosis mouse model by inhibiting EZH2. 72 Other study discovers that conditional knockout of EZH2 increases adiposity in bone marrow and causes low trabecular bone phenotype in mice. 65  OSX Cre+/− cells can contain lipid droplets. 65,73,74 Besides, EZH2 deletion enhances hDPSCs osteogenesis while impairs adipogenesis. 66 These results hint the role of EZH2 in determining the progression of cell differentiation lineages and ultimately determine the swing between osteogenic and lipogenic differentiation.
For regulation mechanism, researchers discover that EZH2 suppresses the expression of osteogenic genes and ligand-dependent signalling pathways such as WNT, PTH and BMP to favour the adipogenic differentiation. 63,64 In other study, EZH2 deletion enhances the expression of PTH1r and WNT10b and the osteogenesis in MC3T3 pre-osteoblasts, and it also increases the BMP-dependent Smad1/5 phosphorylation by decreasing H3K27me3 near transcriptional start sites. 36   In conclusion, these discoveries suggest that PRC2-EZH2 facilitates the early mesenchymal lineage development, but suppresses the late osteogenic lineage differentiation commitment of stem cells to functional osteoblast or osteoprogenitor cells, and serves a bifunctional role during bone formation ( Figure 3 and Table 2). The long-term inhibition of EZH2 does not appear to be beneficial in adolescent or young adult osteoporosis mice model; short-term or local applications using EZH2 inhibitor in mature or engineered skeletal tissue may accelerate the maturation of differentiated osteoblasts.

| THE ROLE OF PRC 2 IN THE NEUROG ENI C D IFFERENTIATI ON OF S TEM CELL S
One study suggests that EZH2 is responsible for memory, learning, spatial patterning and cognitive functions during hippocampus development in the adult mature brain. 80 Other study has revealed that PRC2-EZH2-modified epigenetic regulation plays a critical role during spinal cord injury and repair. 81 Recently report shows that PRC2 complex forms decreased during neurogenic differentiation of hESCs. 82 Neurogenesis begins after the ectodermal cells differentiate into the neuroepithelial cells by neural induction, followed by neurulation. 83 Neurulation is followed by neural tube formation from neural plates. Then, neural tube is patterned to generate special regions and also gives rise to neural epithelial cells (NECs), mature neurons and glial cells. 84 After several rounds of proliferation, NECs generate neural progenitor cells (NPCs) or NSCs. 85 NPCs/NSCs seems to differentiate into neurons firstly and later astrocytes during the neocortical development; this neurogenic-to-astrogenic fate switch determines the final repaired neuron generation. Researchers find that EZH2 maintains NPCs self-renewal and the neurogenic-togliogenic fate switch of NPCs. 86 In addition, EZH2 represses the neurogenic and promotes the astrogenic fate transition of NPCs. 87 Another study has shown that EZH2 expression decreases during the neuronal differentiation and is completely inhibited during the differentiation into astrocytes of mNSCs. 88 86 These suggest that PRC2-EZH2 participates in the progress of neural differentiation of stem cells and functional neural structure formation by supporting the generation, maturation and maintenance of differential neural lineage.
Then, epigenetic mechanism study shows that EZH2 mediates the ubiquitination and proteasomal degradation of aggregated phospho-serine 129 (pSer129) α-Syn, thus having a neuroprotective anti-inflammatory potential, oxidative stress reduction and apoptosis prevention. 97 59 Moreover, the core subunits of PRC2 co-work during neural differentiation. EED is also essential for spinal cord development and required for neurosphere formation and NSCs proliferation in the SVZ region. 101,102 EED is highly expressed in the neural tube, and downregulation of EED causes defects in neural tube. 103 Correspondingly, EED deletion decreases the SUZ12 and H3K27me3 levels in sacral cords of rats with neural tube defects (NTDs). 104 Inactivation of PRC2-EZH2 by knockout of EZH2 or EED prolongs neurogenic phase and delays astrogenic phase of NPCs. Further, PRC2-EZH2 is found to repress the expression of Neurogenin-1 in a developmental-stage-dependent manner. 105 The above points indicate that PRC2-EZH2 acts as the temporal regulator of neurogenic fate in stem cell and inhibits the differentiation of stem cells to functional neurogenic cells, suggesting that it is the key target point for neurogenic regulation ( Figure 4 and Table 3).

| CON CLUS IONS
In summary, as the core catalytic component of H3K27 histone methyl-

ACK N OWLED G EM ENTS
This work was supported by grants from the National Natural Science Reconstruction (KFKT2019012 to L.L).

CO N FLI C T O F I NTE R E S T
The authors declare no potential conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data used to support the findings of this study are included within the article.