Loss of Polycomb Repressive Complex 2 Function Alters Digestive Organ Homeostasis and Neuronal Differentiation in Zebrafish.

Polycomb repressive complex 2 (PRC2) mediates histone H3K27me3 methylation and the stable transcriptional repression of a number of gene expression programs involved in the control of cellular identity during development and differentiation. Here, we report on the generation and on the characterization of a zebrafish line harboring a null allele of eed, a gene coding for an essential component of the PRC2. Homozygous eed-deficient mutants present a normal body plan development but display strong defects at the level of the digestive organs, such as reduced size of the pancreas, hepatic steatosis, and a loss of the intestinal structures, to die finally at around 10-12 days post fertilization. In addition, we found that PRC2 loss of function impairs neuronal differentiation in very specific and discrete areas of the brain and increases larval activity in locomotor assays. Our work highlights that zebrafish is a suited model to study human pathologies associated with PRC2 loss of function and H3K27me3 decrease.

Differential requirement for the Polycomb repressor complex 2 in dendritic cell and tissue-resident myeloid cell homeostasis.

Dendritic cells (DCs) and macrophages are at the forefront of immune responses, modifying their transcriptional programs in response to their tissue environment or immunological challenge. Posttranslational modifications of histones, such as histone H3 lysine-27 trimethylation (H3K27me3) by the Polycomb repressive complex 2 (PRC2), are tightly associated with epigenetic regulation of gene expression. To explore whether H3K27me3 is involved in either the establishment or function of the mononuclear phagocyte system, we selectively deleted core components of PRC2, either EZH2 or SUZ12, in CD11c-expressing myeloid cells. Unexpectedly, EZH2 deficiency neither prevented the deposition and maintenance of H3K27me3 in DCs nor hindered DC/macrophage homeostasis. In contrast, SUZ12 deficiency markedly impaired the capacity of DCs and macrophages to maintain H3K27me3. SUZ12 ablation induced a rapid loss of the alveolar macrophage and Langerhans cell networks under both steady state and inflammatory conditions because these cells could no longer proliferate to facilitate their self-renewal. Despite the reduced H3K27me3, DC development and function were unaffected by SUZ12 ablation, suggesting that PRC2-mediated gene repression was dispensable for DC homeostasis. Thus, the role of SUZ12 highlights the fundamentally different homeostatic mechanisms used by tissue-resident myeloid cells versus DCs.

Polycomb regulation is coupled to cell cycle transition in pluripotent stem cells.

When self-renewing pluripotent cells receive a differentiation signal, ongoing cell duplication needs to be coordinated with entry into a differentiation program. Accordingly, transcriptional activation of lineage specifier genes and cell differentiation is confined to the G1 phase of the cell cycle by unknown mechanisms. We found that Polycomb repressive complex 2 (PRC2) subunits are differentially recruited to lineage specifier gene promoters across cell cycle in mouse embryonic stem cells (mESCs). Jarid2 and the catalytic subunit Ezh2 are markedly accumulated at target promoters during S and G2 phases, while the transcriptionally activating subunits EPOP and EloB are enriched during G1 phase. Fluctuations in the recruitment of PRC2 subunits promote changes in RNA synthesis and RNA polymerase II binding that are compromised in Jarid2 -/- mESCs. Overall, we show that differential recruitment of PRC2 subunits across cell cycle enables the establishment of a chromatin state that facilitates the induction of cell differentiation in G1 phase.

Conversion of random X-inactivation to imprinted X-inactivation by maternal PRC2.

Imprinted X-inactivation silences genes exclusively on the paternally-inherited X-chromosome and is a paradigm of transgenerational epigenetic inheritance in mammals. Here, we test the role of maternal vs. zygotic Polycomb repressive complex 2 (PRC2) protein EED in orchestrating imprinted X-inactivation in mouse embryos. In maternal-null (Eedm-/-) but not zygotic-null (Eed-/-) early embryos, the maternal X-chromosome ectopically induced Xist and underwent inactivation. Eedm-/- females subsequently stochastically silenced Xist from one of the two X-chromosomes and displayed random X-inactivation. This effect was exacerbated in embryos lacking both maternal and zygotic EED (Eedmz-/-), suggesting that zygotic EED can also contribute to the onset of imprinted X-inactivation. Xist expression dynamics in Eedm-/- embryos resemble that of early human embryos, which lack oocyte-derived maternal PRC2 and only undergo random X-inactivation. Thus, expression of PRC2 in the oocyte and transmission of the gene products to the embryo may dictate the occurrence of imprinted X-inactivation in mammals.

Ezh1 arises from Ezh2 gene duplication but its function is not required for zebrafish development.

Trimethylation on H3K27 mediated by Polycomb Repressive Complex 2 (PRC2) is required to control gene repression programs involved in development, regulation of tissue homeostasis or maintenance and lineage specification of stem cells. In Drosophila, the PRC2 catalytic subunit is the single protein E(z), while in mammals this function is fulfilled by two proteins, Ezh1 and Ezh2. Based on database searches, we propose that Ezh1 arose from an Ezh2 gene duplication that has occurred in the common ancestor to elasmobranchs and bony vertebrates. Expression studies in zebrafish using in situ hybridization and RT-PCR followed by the sequencing of the amplicon revealed that ezh1 mRNAs are maternally deposited. Then, ezh1 transcripts are ubiquitously distributed in the entire embryo at 24 hpf and become more restricted to anterior part of the embryo at later developmental stages. To unveil the function of ezh1 in zebrafish, a mutant line was generated using the TALEN technology. Ezh1-deficient mutant fish are viable and fertile, but the loss of ezh1 function is responsible for the earlier death of ezh2 mutant larvae indicating that ezh1 contributes to zebrafish development in absence of zygotic ezh2 gene function. Furthermore, we show that presence of ezh1 transcripts from the maternal origin accounts for the delayed lethality of ezh2-deficient larvae.

Polycomb Repressive Complex 2 is essential for development and maintenance of a functional TEC compartment.

Thymic epithelial cells (TEC) are essential for thymocyte differentiation and repertoire selection. Despite their indispensable role in generating functional T cells, the molecular mechanisms that orchestrate TEC development from endodermal progenitors in the third pharyngeal pouch (3rd PP) are not fully understood. We recently reported that the T-box transcription factor TBX1 negatively regulates TEC development. Although initially expressed throughout the 3rd PP, Tbx1 becomes downregulated in thymus-fated progenitors and when ectopically expressed impairs TEC progenitor proliferation and differentiation. Here we show that ectopic Tbx1 expression in thymus fated endoderm increases expression of Polycomb repressive complex 2 (PRC2) target genes in TEC. PRC2 is an epigenetic modifier that represses gene expression by catalyzing trimethylation of lysine 27 on histone H3. The increased expression of PRC2 target genes suggests that ectopic Tbx1 interferes with PRC2 activity and implicates PRC2 as an important regulator of TEC development. To test this hypothesis, we used Foxn1Cre to delete Eed, a PRC2 component required for complex stability and function in thymus fated 3rd PP endoderm. Proliferation and differentiation of fetal and newborn TEC were disrupted in the conditional knockout (EedCKO) mutants leading to severely dysplastic adult thymi. Consistent with PRC2-mediated transcriptional silencing, the majority of differentially expressed genes (DEG) were upregulated in EedCKO TEC. Moreover, a high frequency of EedCKO DEG overlapped with DEG in TEC that ectopically expressed Tbx1. These findings demonstrate that PRC2 plays a critical role in TEC development and suggest that Tbx1 expression must be downregulated in thymus fated 3rd PP endoderm to ensure optimal PRC2 function.

Inducible CRISPR genome editing platform in naive human embryonic stem cells reveals JARID2 function in self-renewal.

To easily edit the genome of naïve human embryonic stem cells (hESC), we introduced a dual cassette encoding an inducible Cas9 into the AAVS1 site of naïve hESC (iCas9). The iCas9 line retained karyotypic stability, expression of pluripotency markers, differentiation potential, and stability in 5iLA and EPS pluripotency conditions. The iCas9 line induced efficient homology-directed repair (HDR) and non-homologous end joining (NHEJ) based mutations through CRISPR-Cas9 system. We utilized the iCas9 line to study the epigenetic regulator, PRC2 in early human pluripotency. The PRC2 requirement distinguishes between early pluripotency stages, however, what regulates PRC2 activity in these stages is not understood. We show reduced H3K27me3 and pluripotency markers in JARID2 2iL-I-F hESC mutants, indicating JARID2 requirement in maintenance of hESC 2iL-I-F state. These data suggest that JARID2 regulates PRC2 in 2iL-I-F state and the lack of PRC2 function in 5iLA state may be due to lack of sufficient JARID2 protein.

Regulation of embryonic haematopoietic multipotency by EZH1.

All haematopoietic cell lineages that circulate in the blood of adult mammals derive from multipotent haematopoietic stem cells (HSCs). By contrast, in the blood of mammalian embryos, lineage-restricted progenitors arise first, independently of HSCs, which only emerge later in gestation. As best defined in the mouse, 'primitive' progenitors first appear in the yolk sac at 7.5 days post-coitum. Subsequently, erythroid-myeloid progenitors that express fetal haemoglobin, as well as fetal lymphoid progenitors, develop in the yolk sac and the embryo proper, but these cells lack HSC potential. Ultimately, 'definitive' HSCs with long-term, multilineage potential and the ability to engraft irradiated adults emerge at 10.5 days post-coitum from arterial endothelium in the aorta-gonad-mesonephros and other haemogenic vasculature. The molecular mechanisms of this reverse progression of haematopoietic ontogeny remain unexplained. We hypothesized that the definitive haematopoietic program might be actively repressed in early embryogenesis through epigenetic silencing, and that alleviating this repression would elicit multipotency in otherwise lineage-restricted haematopoietic progenitors. Here we show that reduced expression of the Polycomb group protein EZH1 enhances multi-lymphoid output from human pluripotent stem cells. In addition, Ezh1 deficiency in mouse embryos results in precocious emergence of functional definitive HSCs in vivo. Thus, we identify EZH1 as a repressor of haematopoietic multipotency in the early mammalian embryo.

Incorporating genomic, transcriptomic and clinical data: a prognostic and stem cell-like MYC and PRC imbalance in high-risk neuroblastoma.

BACKGROUND: Previous studies suggested that cancer cells possess traits reminiscent of the biological mechanisms ascribed to normal embryonic stem cells (ESCs) regulated by MYC and Polycomb repressive complex 2 (PRC2). Several poorly differentiated adult tumors showed preferentially high expression levels in targets of MYC, coincident with low expression levels in targets of PRC2. This paper will reveal this ESC-like cancer signature in high-risk neuroblastoma (HR-NB), the most common extracranial solid tumor in children. METHODS: We systematically assembled genomic variants, gene expression changes, priori knowledge of gene functions, and clinical outcomes to identify prognostic multigene signatures. First, we assigned a new, individualized prognostic index using the relative expressions between the poor- and good-outcome signature genes. We then characterized HR-NB aggressiveness beyond these prognostic multigene signatures through the imbalanced effects of MYC and PRC2 signaling. We further analyzed Retinoic acid (RA)-induced HR-NB cells to model tumor cell differentiation. Finally, we performed in vitro validation on ZFHX3, a cell differentiation marker silenced by PRC2, and compared cell morphology changes before and after blocking PRC2 in HR-NB cells. RESULTS: A significant concurrence existed between exons with verified variants and genes showing MYCN-dependent expression in HR-NB. From these biomarker candidates, we identified two novel prognostic gene-set pairs with multi-scale oncogenic defects. Intriguingly, MYC targets over-represented an unfavorable component of the identified prognostic signatures while PRC2 targets over-represented a favorable component. The cell cycle arrest and neuronal differentiation marker ZFHX3 was identified as one of PRC2-silenced tumor suppressor candidates. Blocking PRC2 reduced tumor cell growth and increased the mRNA expression levels of ZFHX3 in an early treatment stage. This hypothesis-driven systems bioinformatics work offered novel insights into the PRC2-mediated tumor cell growth and differentiation in neuroblastoma, which may exert oncogenic effects together with MYC regulation. CONCLUSION: Our results propose a prognostic effect of imbalanced MYC and PRC2 moderations in pediatric HR-NB for the first time. This study demonstrates an incorporation of genomic landscapes and transcriptomic profiles into the hypothesis-driven precision prognosis and biomarker discovery. The application of this approach to neuroblastoma, as well as other cancer more broadly, could contribute to reduced relapse and mortality rates in the long term.

Knockdown of JARID2 inhibits the proliferation and invasion of ovarian cancer through the PI3K/Akt signaling pathway.

Protein Jumonji (JARID2), a member of the family of JmjC domain-containing proteins, has been reported to serve an important role in tumor growth and metastasis. However, the expression pattern and role of JARID2 in ovarian cancer remains unclear. Therefore, in the present study, the role of JARID2 in ovarian cancer was investigated, as well as the underlying mechanisms. The results of the present study demonstrated that the expression of JARID2 is upregulated in human ovarian cancer cell lines. Furthermore, downregulation of JARID2 significantly suppressed proliferation, migration, invasion and epithelial­mesenchymal transition in human ovarian cancer cells. Mechanistically, downregulation of JARID2 decreased the protein expression levels of phosphorylated phosphoinositide 3­kinase (PI3K) and protein kinase B (Akt) in ovarian cancer cells. In conclusion the observations suggested that knockdown of JARID2 inhibited proliferation, migration and invasion in vitro through the inactivation of the PI3K/Akt signaling pathway. Therefore, JARID2 may represent a potential therapeutic target for the treatment of ovarian cancer.

MiR-155 contributes to Th17 cells differentiation in dextran sulfate sodium (DSS)-induced colitis mice via Jarid2.

MicroRNAs (miRNAs) play an important role in regulating immune system function by mRNA destabilisation or inhibition of translation. Recently, miR-155 was detected to be significantly up-regulated in colonic tissues of patients with active UC. However, it is unknown whether miR-155 is involved in the pathogenesis of UC and how it influences immune response in dextran sulfate sodium (DSS)-induced colitis mice. Here, we investigated the role of miR-155 in UC. Firstly, through bioinformatics analysis and luciferase report assay, we found Jarid2 was a direct target of miR-155; then, we carried out in situ hybridization, immunofluorescence and flow cytometry, and revealed that miR-155 levels were increased, Jarid2 levels were decreased and the frequency of Th17 cells was elevated in DSS-induced mice; we also used lentiviral vector to deliver miR-155 inhibition sequences to silence miR-155 that was effectively taken up by epithelial cells. MiR-155 inhibition attenuated DSS-induced colonic damage and inhibited Th17 cells differentiation. This study suggests that miR-155 plays a host-damaging role during DSS-induced colitis mice and induces Th17 differentiation by targeting Jarid2.

Divergent Requirements for EZH1 in Heart Development Versus Regeneration.

RATIONALE: Polycomb repressive complex 2 is a major epigenetic repressor that deposits methylation on histone H3 on lysine 27 (H3K27me) and controls differentiation and function of many cells, including cardiac myocytes. EZH1 and EZH2 are 2 alternative catalytic subunits with partial functional redundancy. The relative roles of EZH1 and EZH2 in heart development and regeneration are unknown. OBJECTIVE: We compared the roles of EZH1 versus EZH2 in heart development and neonatal heart regeneration. METHODS AND RESULTS: Heart development was normal in Ezh1-/- (Ezh1 knockout) and Ezh2f/f::cTNT-Cre (Ezh2 knockout) embryos. Ablation of both genes in Ezh1-/-::Ezh2f/f::cTNT-Cre embryos caused lethal heart malformations, including hypertrabeculation, compact myocardial hypoplasia, and ventricular septal defect. Epigenome and transcriptome profiling showed that derepressed genes were upregulated in a manner consistent with total EZH dose. In neonatal heart regeneration, Ezh1 was required, but Ezh2 was dispensable. This finding was further supported by rescue experiments: cardiac myocyte-restricted re-expression of EZH1 but not EZH2 restored neonatal heart regeneration in Ezh1 knockout. In myocardial infarction performed outside of the neonatal regenerative window, EZH1 but not EZH2 likewise improved heart function and stimulated cardiac myocyte proliferation. Mechanistically, EZH1 occupied and activated genes related to cardiac growth. CONCLUSIONS: Our work unravels divergent mechanisms of EZH1 in heart development and regeneration, which will empower efforts to overcome epigenetic barriers to heart regeneration.

Stage- and subunit-specific functions of polycomb repressive complex 2 in bladder urothelial formation and regeneration.

Urothelium is the protective lining of the urinary tract. The mechanisms underlying urothelial formation and maintenance are largely unknown. Here, we report the stage-specific roles of PRC2 epigenetic regulators in embryonic and adult urothelial progenitors. Without Eed, the obligatory subunit of PRC2, embryonic urothelial progenitors demonstrate reduced proliferation with concomitant dysregulation of genes including Cdkn2a (p16), Cdkn2b (p15) and Shh. These mutants display premature differentiation of keratin 5-positive (Krt5+) basal cells and ectopic expression of squamous-like differentiation markers. Deletion of Ezh2, the major enzymatic component of PRC2, causes upregulation of Upk3a+ superficial cells. Unexpectedly, Eed and Eed/Ezh2 double mutants exhibit delayed superficial cell differentiation. Furthermore, Eed regulates the proliferative and regenerative capacity of adult urothelial progenitors and prevents precocious differentiation. Collectively, these findings uncover the epigenetic mechanism by which PRC2 controls urothelial progenitor cell fate and the timing of differentiation, and further suggest an epigenetic basis of urothelial maintenance and regeneration.

Polycomb repressive complex 2 (PRC2) silences genes responsible for neurodegeneration.

Normal brain function depends on the interaction between highly specialized neurons that operate within anatomically and functionally distinct brain regions. Neuronal specification is driven by transcriptional programs that are established during early neuronal development and remain in place in the adult brain. The fidelity of neuronal specification depends on the robustness of the transcriptional program that supports the neuron type-specific gene expression patterns. Here we show that polycomb repressive complex 2 (PRC2), which supports neuron specification during differentiation, contributes to the suppression of a transcriptional program that is detrimental to adult neuron function and survival. We show that PRC2 deficiency in striatal neurons leads to the de-repression of selected, predominantly bivalent PRC2 target genes that are dominated by self-regulating transcription factors normally suppressed in these neurons. The transcriptional changes in PRC2-deficient neurons lead to progressive and fatal neurodegeneration in mice. Our results point to a key role of PRC2 in protecting neurons against degeneration.

Deletion of Polycomb Repressive Complex 2 From Mouse Intestine Causes Loss of Stem Cells.

BACKGROUND & AIMS: The polycomb repressive complex 2 (PRC2) regulates differentiation by contributing to repression of gene expression and thereby stabilizing the fate of stem cells and their progeny. PRC2 helps to maintain adult stem cell populations, but little is known about its functions in intestinal stem cells. We studied phenotypes of mice with intestine-specific deletion of the PRC2 proteins embryonic ectoderm development (EED) (a subunit required for PRC2 function) and enhancer of zeste homolog 2 (EZH2) (a histone methyltransferase). METHODS: We performed studies of AhCre;EedLoxP/LoxP (EED knockout) mice and AhCre;Ezh2LoxP/LoxP (EZH2 knockout) mice, which have intestine-specific disruption in EED and EZH2, respectively. Small intestinal crypts were isolated and subsequently cultured to grow organoids. Intestines and organoids were analyzed by immunohistochemical, in situ hybridization, RNA sequence, and chromatin immunoprecipitation methods. RESULTS: Intestines of EED knockout mice had massive crypt degeneration and lower numbers of proliferating cells compared with wild-type control mice. Cdkn2a became derepressed and we detected increased levels of P21. We did not observe any differences between EZH2 knockout and control mice. Intestinal crypts from EED knockout mice had signs of aberrant differentiation of uncommitted crypt cells-these differentiated toward the secretory cell lineage. Furthermore, crypts from EED-knockout mice had impaired Wnt signaling and concomitant loss of intestinal stem cells, this phenotype was not reversed upon ectopic stimulation of Wnt and Notch signaling in organoids. Analysis of gene expression patterns from intestinal tissues of EED knockout mice showed dysregulation of several genes involved in Wnt signaling. Wnt signaling was regulated directly by PRC2. CONCLUSIONS: In intestinal tissues of mice, PRC2 maintains small intestinal stem cells by promoting proliferation and preventing differentiation in the intestinal stem cell compartment. PRC2 controls gene expression in multiple signaling pathways that regulate intestinal homeostasis. Sequencing data are available in the genomics data repository GEO under reference series GSE81578; RNA sequencing data are available under subseries GSE81576; and ChIP sequencing data are available under subseries GSE81577.

Combined inhibition of EZH2 and histone deacetylases as a potential epigenetic therapy for non-small-cell lung cancer cells.

Recent discoveries have revealed that human cancer involves aberrant epigenetic alterations. We and others have previously shown that the histone methyltransferase EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), is frequently overexpressed in non-small-cell lung cancer (NSCLC) and that an EZH2 inhibitor, 3-deazaneplanocin A, inhibits the proliferation of NSCLC cells. Transcriptional silencing by EZH2 was recently shown to be required for the activity of histone deacetylases (HDACs) that interact with another PRC2 protein, EED. To develop a more effective epigenetic therapy for NSCLC, we determined the effects of co-treatment with 3-deazaneplanocin A and the HDAC inhibitor vorinostat (SAHA) in NSCLC cells. The co-treatment synergistically suppressed the proliferation of all tested NSCLC cell lines, regardless of their epidermal growth factor receptor (EGFR) status. The synergistic effect was associated with slightly decreased histone H3 lysine 27 trimethylation, modestly increased histone acetylation, and the depletion of EZH2 and other PRC2 proteins. The co-treatment resulted in an accumulation of p27Kip1, decrease in cyclin A, and increased apoptotic fraction in an additive/synergistic manner. Interestingly, the co-treatment strongly suppressed EGFR signaling, not only in EGFR-wild-type NSCLC cells, but also in EGFR-mutant cells, mainly through dephosphorylation of EGFR. Furthermore, the co-treatment suppressed the in vivo tumor growth of EGFR-mutant, EGFR-tyrosine kinase-resistant H1975 cells more effectively than did each agent alone, without visible toxicity. These results suggest that the combined pharmacological targeting of EZH2 and HDACs may provide more effective epigenetic therapeutics for NSCLC.

Ezh2 Controls an Early Hematopoietic Program and Growth and Survival Signaling in Early T Cell Precursor Acute Lymphoblastic Leukemia.

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of ALL distinguished by stem-cell-associated and myeloid transcriptional programs. Inactivating alterations of Polycomb repressive complex 2 components are frequent in human ETP-ALL, but their functional role is largely undefined. We have studied the involvement of Ezh2 in a murine model of NRASQ61K-driven leukemia that recapitulates phenotypic and transcriptional features of ETP-ALL. Homozygous inactivation of Ezh2 cooperated with oncogenic NRASQ61K to accelerate leukemia onset. Inactivation of Ezh2 accentuated expression of genes highly expressed in human ETP-ALL and in normal murine early thymic progenitors. Moreover, we found that Ezh2 contributes to the silencing of stem-cell- and early-progenitor-cell-associated genes. Loss of Ezh2 also resulted in increased activation of STAT3 by tyrosine 705 phosphorylation. Our data mechanistically link Ezh2 inactivation to stem-cell-associated transcriptional programs and increased growth/survival signaling, features that convey an adverse prognosis in patients.

Ezh2 regulates the Lin28/let-7 pathway to restrict activation of fetal gene signature in adult hematopoietic stem cells.

Fetal liver hematopoietic stem cells (HSCs) seed bone marrow (BM) and undergo reprograming into adult-type HSCs that are largely quiescent and restricted in their self-renewal activity. Here we report that in the absence of the polycomb-group gene Ezh2, a cohort of fetal-specific genes, including let-7 target genes, were activated in BM hematopoietic stem/progenitor cells (HSPCs), leading to acquisition of fetal phenotypes by BM HSPCs, such as enhanced self-renewal activity and production of fetal-type lymphocytes. The Lin28b/let-7 pathway determines developmentally timed changes in HSPC programs. Of note, many of the fetal-specific let-7 target genes, including Lin28, appear to be transcriptionally repressed by Ezh2-mediated H3K27me3 in BM HSPCs, and Ezh2 loss results in their ectopic expression, particularly in hematologic malignancies that develop in the absence of Ezh2. These findings suggest that Ezh2 cooperates with let-7 microRNAs in silencing the fetal gene signature in BM HSPCs and restricts their transformation.

Enhancer of zeste homolog 2 depletion induces cellular senescence via histone demethylation along the INK4/ARF locus.

Polycomb group proteins are epigenetic transcriptional repressors that function through recognition and modification of histone methylation and chromatin structure. As a member of PcG proteins, enhancer of zeste homolog 2 (EZH2) targets cell cycle regulatory proteins which govern cell cycle progression and cellular senescence. In previous work, we reported that EZH2 depletion functionally induced cellular senescence in human gastric cancer cells with mutant p53. However, whether EZH2 expression contributes to the change of key cell cycle regulators and the mechanism involved are still unclear. To address this issue, we investigated the effects of EZH2 depletion on alteration of histone methylation pattern. In gastric cancer cells, INK4/ARF locus was activated to certain extent in consequence of a decrease of H3K27me3 along it caused by EZH2 silence, which contributed substantially to an increase in the expression of p15(INK4b), p14(ARF) and p16(INK4a) and resulted in cellular senescence ultimately. Furthermore, MKN28 cells, which did not express p16(INK4a) and p21(cip), could be induced to senescence via p15(INK4b) activation and suppression of p15(INK4b) reversed senescence progression induced by EZH2 downregulated. These data unravel a crucial role of EZH2 in the regulation of INK4/ARF expression and senescence procedure in gastric cancer cells, and show that the cellular senescence could just depend on the activation of p15(INK4b)/Rb pathway, suggesting the cell-type and species specificity involved in the mechanisms of senescence inducement.

EZH2-induced H3K27me3 is associated with epigenetic repression of the ARHI tumor-suppressor gene in ovarian cancer.

Epithelial ovarian cancer (EOC) is the second leading cause of death from gynecological malignancies worldwide. Enhancer of zeste homology 2 (EZH2), participating in gene expression silencing by trimethylating histone 3 lysine 27 (H3K27me3), is often up-regulated in EOC. ARHI, an imprinted tumor-suppressor gene, is markedly down-regulated or even undetectable in the majority of EOC. To explore the correlation between EZH2 and ARHI expression in EOC as well as the possible mechanism of EZH2-ARH1 interaction. We used immunohistochemical staining to evaluate the expression of EZH2 and ARHI in EOC and normal ovarian tissue specimens; western blotting, shRNA, and chromatin immunoprecipitation were used to study the expression correlation of EZH2 and ARHI in EOC and normal ovarian epithelial cells and to further explore the mechanism of EZH2 regulation of ARHI expression. Cell viability assay was used to evaluate the influence of these two genes on cell survival. (1) The expression of EZH2 inversely correlated with ARHI expression levels and predicted shorter overall survival in EOC patients; (2) EZH2 promoted repression of ARHI by catalyzing trimethylation on H3K27; (3) ARHI was synergistically silenced by DNA methylation and histone modification; and (4) DZNep, an inhibitor of EZH2, significantly reduced survival rate of EOC cells by restoring ARHI expression. EZH2-″induced H3K27me3 is associated with epigenetic repression of the ARHI tumor-suppressor gene in EOC. Suppression of EZH2 by DZNep, as a way of restoring the expression of ARHI, could be a potential treatment modality to EOC.