Epigenetic regulation of embryonic ectoderm development in stem cell differentiation and transformation during ontogenesis

Abstract Dynamic chromatin accessibility regulates stem cell fate determination and tissue homeostasis via controlling gene expression. As a histone‐modifying enzyme that predominantly mediates methylation of lysine 27 in histone H3 (H3K27me1/2/3), Polycomb repressive complex 2 (PRC2) plays the canonical role in targeting developmental regulators during stem cell differentiation and transformation. Embryonic ectoderm development (EED), the core scaffold subunit of PRC2 and as an H3K27me3‐recognizing protein, has been broadly implicated with PRC2 stabilization and allosterically stimulated PRC2. Accumulating evidences from experimental data indicate that EED‐associating epigenetic modifications are indispensable for stem cell maintenance and differentiation into specific cell lineages. In this review, we discuss the most updated advances to summarize the structural architecture of EED and its contributions and underlying mechanisms to mediating lineage differentiation of different stem cells during epigenetic modification to expand our understanding of PRC2.

(H3K27me3), respectively. 11 PRC2 is composed of four core subunits, enhancer of zeste homologue 1/2 (EZH1/2), embryonic ectoderm development (EED), suppressor of Zeste 12 (SUZ12) and RB-binding protein 4 or 7 (RBBP4/RBAP48 or RBBP7/RBAP46). 7 EZH1/2 are homologous analogs, and EZH1 can partially compensate for EZH2 function in some developmental cells. [12][13][14][15] The SET-domain containing protein EZH2 endows the Polycomb PRC2 complex with histone lysine methyltransferase activity, but its activity requires the participation of the other three subunits of the core complex. 16,17 EZH2 displays an autoinhibited state and exhibits little histone methyltransferase activity when on its own. 18 In mammals, The WDrepeat-containing protein EED is present as four distinct isoforms, which are believed to be mediated by utilizing four in-frame start codons of a single EED mRNA transcript. 19 One common critical function of the EED isoforms is stabilizing EZH2 in the PRC2 complex. In addition, the PRC2 complex binds to H3K27me3 through the aromatic cage of EED, which has specific recognition of defined (repressive) trimethylated-lysine residues. 20,21 SUZ12 stabilizes PRC2 by interacting with EZH2 via its VRN2-EMF2-FIS2-Suz12 box (VEFS) domain. 22,23 Furthermore, SUZ12 is responsible for the methylation of lysine 9 of histone 3. 24 As the fourth core subunit of PRC2, the histone-binding protein RbAp46/48 is essential for PRC2 binding with histone tails. 25 Recruitment of the PRC2 complex to chromatin is mediated by interaction with non-core subunits such as Zinc finger protein AE binding protein 2 (AEBP2), Polycomb-like homologues (PCLs), PRC2-associated LCOR isoform 1 or 2 (PALI1/2), Polycomb repressive complex 2-associated protein (EPOP), and Jumonji and ATrich interaction domain containing 2 (JARID2). 7,[26][27][28][29] Non-core subunits compete to bind to the N-terminal region of SUZ12, which defines the PRC2.1 and PRC2.2 subcomplexes. PRC2.1 contains one of the three PCLs with either EPOP or PALI1/2, while PRC2.2 contains AEBP2 and JARID2 (Figures 1 and 2A). [28][29][30][31] A vast number of studies report that the core subunits of the PRC2 complex show spatiotemporal specific expression patterns, indicating that they may have distinct functions. 32-35 EZH2 is highly expressed in proliferating cells, while EZH1 is at high levels in the differentiated tissues. 15,36 EED is generally regarded as a gene silencing regulator that maintains pluripotency of embryonic stem cells (ESCs) and cell proliferation. Furthermore, increasing evidence suggests that EED contributes to stem cell maintenance and lineage specification in ontogenesis. 34,[37][38][39][40][41] This review provides a comprehensive overview of EED in regulating stem cell fate and the underlying regulatory mechanisms during various organ morphogenesis, refining the framework for understanding the functions and mechanisms of PRC2-mediated regulation of gene expression.

| THE STRUCTURAL BASIS OF EED
As a WD-40 repeat family protein, EED contains seven WD40-repeat motifs at its C terminus preceded by an extended N-terminal F I G U R E 1 Subunits composition, chromatin association and protein domain structure of Polycomb repressive complex 2 (PRC2). (A) Schematic drawing of PRC2.1 and PRC2.2 core subunits and accessory subunits. Single-headed arrows represent the deposition of histone marks, and double-headed arrows depict chromatin binding. (B) Domain structure of PRC2 core subunits. Linker regions are omitted. segment. 20 A WD40 repeat also called a WD or β-transducin repeat is a short, $40-residue motif. 42,43 The WD40 repeats of EED were multimerized to fold into a canonical seven-blade β-propeller structure.
The seven blades are radially arranged around a central axis to form a peptide-binding pocket in the centre of the β-propeller structure. 20 EED interacts with the N-terminal domain of EZH2 through its larger bottom surface of WD40 repeat motifs, which in turn modulates EZH2's histone methyltransferase activity. 44 An aromatic cage is a common trait for most methyllysine-binding motifs, 45 and EED recognizes H3K27me3 through the aromatic cage located on the top surface of its WD40 repeat domain. 46,47 Through the trimethyllysine, the histone peptide binds to EED and is recognized by the aromatic cage to form EED-H3K27me3 peptide complex structure. 20 Thus, EED acts not only as a critical molecule to compose the PRC2 complex but also as an 'epigenetic exchange factor' to modulate methylation on histones.

| EED MODULATES ALLOSTERIC ACTIVATION AND RECRUITMENT OF PRC2
EZH2 adopts an autoinhibitory conformation through crystal structures of the inactive isolated catalytic domain, suggesting that structural rearrangement of EZH2 is likely required for PRC2 activation. 18,47 Moreover, a range of crystallographic structures of EED in complex with EZH2, trimethylated histone and nonhistone peptides highlight the pivotal role of EED in mediating EZH2 binding and triggering PRC2 an allosteric activation of catalysis. 20,44,48,49 After PRC2 complex assembly, the activation loop from the N-terminal portion of EZH2 is moved by EED to the neighbouring catalytic SET domain ( Figure 2B). 45 The EEDbinding domain (EBD) of EZH2 occupies the bottom face of the EED WD40 domain, and then three β strands are added to EED to form the β-addition motif (BAM) and maintain EED in a stable position to allow H3K27me3 binding to the top WD40-repeat domain of EED ( Figure 3A). 47,50 The SET activation loop (SAL) is formed after the BAM migrates away from the EED surface to the back of the SET domain of the catalytic moiety. Then the H3K27me3 peptide is sandwiched between EED and an exposed EZH2 motif, referred to as the stimulation-responsive motif (SRM), and immediately follows the SAL of EZH2. SRM of the sandwich-like assembly interacts extensively with two others and transforms itself into a fully ordered α-helix-loop structure. 47 Finally, the interaction between SAL and the newly formed SRM helix of EZH2 stabilizes the active conformation of the EZH2 SET, resulting in enhanced histone methyltransferase activity of PRC2. In addition, EED along with the SAL and SET regions of EZH2 get glued together by the N-terminal loop region of SUZ12 (VEFS). Additionally, the SANT1-binding domain (SBD) of EZH2 contacts with the DNA in the H3K27me3-marked nucleosome after binding by EED. 47,51 Through a highly complex series of EED, EZH2, and H3K27me3 interactions, EED allosterically regulates PRC2 histone methyltransferase activity and PRC2 on-chromatin spreading. 20,52,53 The PRC2 crystal structures provide fundamental insights into the interaction with each subunit, substrate recognition, and allosteric activation of its enzymatic activity. Moreover, PRC2 is more active on di-nucleosomes and higher-order chromatin structures than on mononucleosomes or histone tails, 50,54,55 indicating the crucial role of interaction with the chromatin in PRC2 activation. A response to allosteric stimulation of PRC2 results in it catalysing H3K27me3 on neighbouring nucleosomes, causing the formation of broad H3K27me3 domains. 56 Recently, a cryo-EM structure of PRC2 in di-nucleosomes revealed that EED engages with one H3K27me3-modified nucleosome to allow the H3K27me3 thread into the aromatic cage of EED and locates the SET domain present in EZH2 to methylate an unmodified H3 tail on the other adjacent nucleosome. 50 EED and EZH2 consist of the dominant interface to interaction with di-nucleosome, while SUZ12, RbAp46/48, AEBP2 and JARID2 could also potentially assist nucleosome interaction. 57 Figure 3B) which has an allosteric stimulatory effect on PRC2 histone methyltransferase activity. 61 It is noteworthy that JARID2-K116me3 promotes the allosteric activation of PRC2 but does not participate in H3K27me3, which is invoked as one of a mechanism for PRC2 deposition to unmodified nucleosomes. 62 When H2AK119ub1 enriches at CpG islands (CGIs), EED-mediated JARID2 cooperation with H2AK119ub1 could contribute to the CGI preference of PRC2. 63,64 More recently, the trimethylated state of PALI1 has also been shown can allosterically activate the PRC2 complex when bound to EED through a similar mechanism to that proposed for H3K27me3 ( Figure 3C,D). 65

| THE INTERACTION OF EED AND H3K27ME3
Mammalian heterochromatin contains great repressive chromatin domains, including H3K9me2/3-modified constitutive heterochromatin and H3K27me2/3-decorated facultative heterochromatin. The two categories of histone modifications are catalysed by Suv39h1/2 and PRC2, respectively. 66,67 PRC2 deposits the repressive histone mark H3K27me3 through an aromatic cage of EED and establishes the direct interaction with its target genes. 20 Furthermore, PRC2 binding or catalytic activity is also affected by histone modifications in the chromatin region, one of which is H3K27me3. 68 Aranda et al. report that EED has high-affinity binding for histone methylations correlated with transcriptional repression. 69

EED can bind H3K27me3
when H3K27me3 is present on two adjacent nucleosomes, and the presence of H3K27me3 stimulates PRC2 enzyme activity, thus generating a positive feedback loop. Mutations of the EED aromatic cage or EED absence will disrupt this interaction and lead to a global loss of H3K27me3. 47,52,70,71 Other repressive chromatin marks that can be recognized by EED, such as H3K9me3, H4K20me3 and H1K26me3, indicate that recognition of repressive histone marks via EED could serve as a mechanism of PRC2 recruitment to silenced loci. 20,48,72

| EED FUNCTIONS IN ONTOGENESIS
For pluripotent stem cells to expand and differentiate into one or more specialized and committed cell types in the body, critical cell fate decisions and lineage commitment must be made during growth and development. As differentiating stem cells undergo the cascade of lineage decisions on branching points through epigenetic-threshold modulation, specific genes must be switched on and genes associated with alternative lineages need to be repressed in a dynamic and timely manner. Next, we will dissect the dynamic roles of EED during ontogenesis to further insight into the PRC2 and its role in the specificity and diversity of lineage specification (Figure 4).

| Embryonic development
ESCs possess the ability to differentiate into all the cell types of tissues and organs. 73 ESCs pluripotency is governed by a gene regulatory network composed of various TFs and chromatin-modifying enzymes. 74 PRC2 complex is essential for early embryonic development and has been implicated in pluripotency as well as cell fate determination in ESCs. In human and murine ESCs, PRC2 localizes to the promoters of repressed genes, which encode TFs for specification later during development, and EED mutations cause their premature expression. 75 EED is also required for regulating normal murine embryogenesis. When mice are knocked out of EED, early embryonic lethality occurs because they fail to properly gastrulate and produce embryonic mesoderm. [76][77][78] Montgomery et al. demonstrate that EED À/À embryos result in a genome-wide decrease in H3K27me1, H3K27me2 and H3K27me3. 19 ESCs absent EED results in inactivating histone methyltransferase and lack the H3K27me3 modification. 21,79 EED and H3K27 methylation are also involved in the gene regulation of undifferentiated and differentiating cells, facilitating ESCs differentiation towards a specific lineage. Through performing gene expression and chimera analyses on both low and high-passage EED null ESCs, Chamberlain et al. identify that EED null ESCs fail to differentiate properly in vitro, but can contribute to chimeras. 80 ESCs without EED can maintain pluripotency markers and self-renew, but fail to execute differentiation programmes promptly. 81 These results focus on studying undifferentiated ESCs. By contrast, Obier et al. perform global gene expression analysis in both undifferentiated ESCs and embryoid body formation of EED knocked out, and results demonstrated that EED is required to silence the pluripotency network during differentiation. 82 Galonska et al. find both H3K27me3 and DNA F I G U R E 4 Embryonic ectoderm development (EED) protein is reside in various organ morphogenesis, including embryonic development, spermatogenesis, oogenesiscy, neurogenesis, and so on. Polycomb repressive complex 2 absent EED results in multiple developmental abnormalities. methylation absence when EED-deficient ESCs culture in the two inhibitors (2i) conditions. 83 But the latest research shows that genome-wide DNA methylation and H4 acetylation are increased in the EED-deficient ESCs culture in the 2i conditions. 84 Hence, EED is critical for ESCs as it regulates both developmental control genes and a subset of canonically imprinted genes.

| Spermatogenesis and oogenesis
The primordial germ cells (PGCs) are precursors of the oocyte and sperm, which transmit significant epigenetic information to the offspring. [85][86][87] High EED expression is concurrent with a high level of H3K27me3 enrichment in XX and XY PGCs during development. 88,89 EED is a key effector of oocyte meiosis and spermatogonia differenti- that sporadic sub-fertility of lacking EED in the paternal germline produces sub-fertile male offspring, involving de-repression of both LINE elements and retrotransposed pseudogenes. 94 Impressively, Oocytespecific deletion of EED results in H3K27me3 absent and a significant overgrowth of offspring, which involves both adiposity and bone mineral density increase. 95 Recent research has shown that loss of EED in somatic and germ cells leads to abnormal ovaries in adult mutant females, but the mutants are fertile. 96 Overall, epigenetic inheritance altered by EED is important to spermatogenesis and oogenesis, especially regulating repetitive sequences in the paternal germline.

| Neurogenesis
During the development of the nervous system, the PRC2 complex plays a vital role in maintaining the self-renewal and proliferation capacities of neural stem/progenitor cells (NSPCs). 97 Embryonic neurogenesis is initiated in the neuroepithelial cells of the ventricular zone (VZ), and the subventricular zone (SVZ) differentiates into radial glial cells (RGCs). RGCs can either directly produce neurons or generate neuronal intermediate progenitor cells, which differentiate into neurons, astrocytes and oligodendrocytes. While in the adult brain, NSPs are in the SVZ and the subgranular zone of the hippocampal dentate gyrus (DG). 98

EED is indispensable for spinal cord development and
NSPCs proliferation in the SVZ region. Partial mutations in genes encoding the EZH2 or EED subunits lead to Weaver syndrome, 99 characterized by variable intellectual disability and distinctive facial features. EED is highly expressed in the brain and involved in the differentiation and maturation process of the central nervous system cells. Downregulation of EED in the spinal cord and neural tube inevitably leads to spina bifida and neural tube defects. 100 An early study has been done to characterize EED as a key regulator of neurogenesis using EED-deficient mice. 101 Conditional knocked out (cKO) of EED in neural progenitors of the neocortex leads to a prolonged neurogenic phase and deferring astrocytes differentiation. 97 Sun et al. report that EED promotes TF Gata6 expression and reduces p21 protein level when EED is deleted in neural stem cells of the SVZ, indicating the importance of EED to NSPCs proliferation and neurogenesis in the SVZ. 102 Besides, the proper formation of DG is also required for EED.
In the absence of EED in the NSPCs, cyclin-dependent kinase inhibitor 2 A (Cdkn2a) expression is increased and critical gene SRY-box transcription factor 11 (Sox11) for neural differentiation is repressed, which ultimately leads to shorter and smaller dentate gyrus formation, indicating that EED primarily acts as an activator for maintaining proliferation and differentiation. 39 EED is essential for oligodendrocyte (OL) remyelination, while it is dispensable for myelin maintenance.
EED deletion in OL progenitors results in H3K27me3 absent and OL lineage abnormalities. 40 Yaghmaeian and colleagues also find H3K27me3 disruption in the developing mouse hypothalamus when EED is cKO, which triggers reduced cell proliferation, ectopic expression of posteriorly expressed regulators and increased expression of cell-cycle regulators. 103 In addition to NSPCs and oligodendrocytes, recently, microglial EED has been demonstrated to be essential for synaptic pruning during normal postnatal brain development. 104 Furthermore, deletion of EED in the forebrain leads to the upper-layer neuron numbers being significantly decreased and abnormal cortical architecture. Genomic and transcriptomic network analyses indicate that abnormal acetylation of H3K27 (H3K27ac) accumulation is associated with the decrease of H3K27me3 and the recruitment of RNA-Pol2. 41 Consequently, EED has multiple functions in neurogenesis, and it plays dynamic roles in NSPCs maintenance and the stage of gliogenesis.

| Cardiogenesis
The development and function of the cardiovascular system are vulnerable to epigenetic insults, one of which is epigenetic repressors PRC2. [105][106][107] In the murine hearts, alterations of chromatin landscape have repeatedly been linked to both cardiomyopathy and structural heart disease in postmitotic cardiomyocytes. 37,106,108,109 Moreover, the inactivation of EED in murine foetal cardiomyocytes leads to cardiac fibrosis and significant systolic impairment. During early heart development, loss of H3K27me3 is complete upon cardiac-specific inactivation of EED, resulting in lethal heart malformations. 105 The previous study has shown that EED interacts with significant amounts of proteins in the Endothelin-1-induced cardiomyocyte proteome. 110 EED also interacts with phospholipase neutral sphingomyelinase 2 (N-SMase2) through coupling TNF-R1 to N-SMase2, thus mediating heart failure and atherosclerosis. 111 Using Tie2Cre to inactivate the floxed murine EED results in absence of blood-perfused vasculature via exhausting the haematopoietic stem cells pool. 112 Ai et al. highlight that in EED cKO mice, significant loss of H3K27me3 can be observed, but it does not directly regulate the upregulation genes, which include upregulated skeletal muscle genes. In contrast, abnormal H3K27ac accumulation is associated with these upregulated genes, indicating that EED complexes with and stimulates HDAC deacetylase activity to silence the slow-twitch myofiber gene programme to orchestrate heart maturation. 37

| Intestinal morphogenesis
Intestinal stem cells (ISCs), located at the bottom of the crypts, are a cell population self-renew extensively and differentiates into all cell types within crypts and villi. 34,[115][116][117] In the developmental and adult intestine, PRC2-mediated H3K27me3 deposition is required for transitions from ISCs to transit-amplifying progenitors and postmitotic villus cells. 34,116,118 It is estimated that about 2000 genes are marked by H3K27me3 in both crypt and villus cells. 119  Surprisingly, genes essential for intestinal development are silenced by H3K27me3 in the adult intestinal epithelium but reactivated without PRC2 action, which is associated with functional interactions of H3K27me3 with H3K4me3. 120 A novel observation reveals that the absence of EZH2 and H3K27me3 in EED-null villi cells results in stunted and dysmorphic villi. 121 By performing absenting EED in distinct cell compartments of the intestinal epithelium, the further investigation reveals that H3K27me3 loss occurs as a result of replicational dilution, which occurs proportional to the frequency of cell division.

| Skin and hair follicle morphogenesis
The skin is the first barrier that protects mammals against external insults and dehydration. Epidermal lineages derive from a single layer of multipotent skin progenitors named basal cells, which attach to underlying basement membranes separating the epidermis from the dermis. 122,123 The basal cells generate the hair follicles, the sebaceous glands, the interfollicular epidermis, and the Merkel cells. 124,125 Among these, the hair follicles contain stem cells of the dermal and epidermal lineages. 126

Regulated function References
Embryonic development Target genes are de-repressed Faust et al. 76 Genome-wide decrease in H3K27me1, H3K27me2 and H3K27me3 Montgomery et al. 19 Decrease in Ezh2 protein levels. Disrupted axial patterning Chamberlain et al. 80 Fail to properly gastrulate and to produce embryonic mesoderm Leeb et al. 81 EED null ESCs fail to differentiate properly in vitro, but can contribute to chimeras Obier et al. 82 EED is required to silence the pluripotency network during differentiation van Mierlo et al. 84 Genome-wide of DNA methylation and H4 acetylation are increased in the EED À/À 2i ESCs Regulates proliferation in the telencephalon Sun et al. 102 Shorter and smaller dentate gyrus Yaghmaeian et al. 103 Mediates oligodendrocyte remyelination; maintains normal synaptic and cognitive functions Liu et al. 39 Controls embryonic cortical neurogenesis Wang et al. 40 Wang et al. 104 Zhang et al. 41 Cardiogenesis Mediates heart failure and atherosclerosis Philipp et al. 111 Lethal heart malformations He et al. 108 Participates in ET-1 induced cardiomyocyte terminal differentiation Shin et al. 110 Abnormal H3K27ac accumulation Ai et al. 37 Regulates lifetime of cardiomyocytes Li et al. 113 Enhances cardiac differentiation Liu et al. 114 Intestinal morphogenesis Crypt-villus architecture disorders Chiacchiera et al. 116 Uncommitted crypt cells in an aberrant differentiation and reduced cell proliferation Koppens et al. 34 Jadhav et al. 120 Significant weight loss and severely degraded crypt Jadhav et al. 121 Stunted and dysmorphic villi Skin and hair follicle morphogenesis Premature epidermal barrier development, ectopic Merkel cell formation, and postnatal hair follicle developmental hurdle Dauber et al. 129 Decreases the proliferation of hair follicle progenitor cells Perdigoto et al. 130 Cannot induce HFSCs activation or fate switch; epidermal pigmentation Cohen et al. 131 Flora et al. 132 Li et al. 133 Haematopoiesis Haematopoiesis defects Lessard et al. 141 Increasing susceptibility to hematologic malignancies of EED heterozygous mutants Majewski et al. 144 Neonatal pale and hypocellular Xie et al. 138 Perturbs the BM HSCs differentiation into restricted lineage progenitor cells Yu et al. 112 Ueda et al. 143

| CONCLUSIONS
In this study, we comprehensively and systematically reviewed the research advances on EED/PRC2 function regulating ontogenesis (Table 1)

Regulated function References
Osteogenesis Weaver syndrome Cohen et al. 150 Regulates vertebra development Cooney et al. 151 Causes severely deformed thoracic spine, and shortening the long bones Kim et al. 153 Mirzamohammadi et al. 35 Shows few gonarthrotic symptoms in collagen-induced arthritis Lian et al. 154 concise understanding of the role of EED-mediated epigenetic regula-

CONFLICT OF INTEREST STATEMENT
The authors declare no potential conflicts of interest.