Polycomb Proteins Research Articles

Praja1 E3 ubiquitin ligase promotes skeletal myogenesis through degradation of EZH2 upon p38\u03b1 activation

Polycomb proteins are critical chromatin modifiers that regulate stem cell differentiation via transcriptional repression. In skeletal muscle progenitors Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), contributes to maintain the chromatin of muscle genes in a repressive conformation, whereas its down-regulation allows the progression through the myogenic programme. Here, we show that p38α kinase promotes EZH2 degradation in differentiating muscle cells through phosphorylation of threonine 372. Biochemical and genetic evidence demonstrates that the MYOD-induced E3 ubiquitin ligase Praja1 (PJA1) is involved in regulating EZH2 levels upon p38α activation. EZH2 premature degradation in proliferating myoblasts is prevented by low levels of PJA1, its cytoplasmic localization and the lower activity towards unphosphorylated EZH2. Our results indicate that signal-dependent degradation of EZH2 is a prerequisite for satellite cells differentiation and identify PJA1 as a new player in the epigenetic control of muscle gene expression.

M10 - Isoform Dependent Functions of AUTS2 During Neuronal Differentiation

Background Disruptive mutations in the autism susceptibility candidate 2 (AUTS2) gene have been linked to a number of neurodevelopmental disorders. The individuals carrying AUTS2 mutations display similar phenotypes including intellectual disability, developmental delay and a number of dysmorphic features, including microcephaly. AUTS2 has two known isoforms, one with 19 exons and a shorter isoform that include only the last 11 exons. The long isoform was shown to be in a complex with polycomb proteins (PCGF3 and PCGF5) that activate transcription. AUTS2 is mainly expressed in the brain during development at a number of regions, including the developing cortex and cerebellum. Methods We used two mouse cellular models to study Auts2 during neuronal differentiation: Neuro2a (N2A) and mouse embryonic stem cells (mESC). To model the human AUTS2 mutations, we used mESC line heterozygous for a gene-trap that disrupts the two isoforms of the Auts2 gene (Auts2+/-) and compared to the parental line (wild type, WT). The N2A cells were differentiated to neurons with retinoic acid. The mESC were differentiated to cortical progenitors and neurons. Auts2 expression was measured by real-time PCR, Western blots and immunofluorescence staining. Expression arrays were used to profile the global changes in gene expression in the Auts2+/- and WT cell lines in three stages of differentiation. The influence of the two AUTS2 isoforms on transcription was tested in a luciferase reporter assay with HEK293 and N2A cells. In order to identify proteins that physically interact with AUTS2, we conducted a yeast two-hybrid (YTH) screen with the two AUTS2 isoforms, validated using the Duolink in situ proximity ligation assay (PLA) in N2A cells. Results Here, we report that the short isoform is transiently expressed during neuronal differentiation. When neuronal differentiation is initiated, there is a shift in expression from the long isoform to mutual expression of the long and short Auts2 isoform. The long isoform becomes again the dominant isoform in adult human and mouse brain. During in vitro corticogenesis, Auts2+/- cells had premature neuronal differentiation leading to increased cell death accompanied by upregulation of mesodermal genes. Reporter assays showed that AUTS2 serves as both an activator and a repressor of transcription, depending on cell type. In HEK293 cells, increase in luciferase activity was observed for both AUTS2 isoform, although the short isoform gave rise to a stronger activity. In contrast, in N2A cells the luciferase activity was decreased significantly. The polycomb group proteins, PCGF3 and PCGF5, were found to interact exclusively with the long AUTS2 isoform, whereas a splicing factor, SF3B1, with both isoforms. Discussion Taken together, our data suggest that an isoform switch of AUTS2 mediates the regulation of genes involved in brain development and differentiation timing through selective interactions with PCGF3 and SF3B1. Thus, the disruption of AUTS2 may affect different processes including the regulation of cell-type specification as well as the balance between differentiation and proliferation.

New Cross-Talk Layer between Ultraconserved Non-Coding RNAs, MicroRNAs and Polycomb Protein YY1 in Bladder Cancer.

MicroRNAs (miRNAs) are highly conserved elements in mammals, and exert key regulatory functions. Growing evidence shows that miRNAs can interact with another class of non-coding RNAs, so-called transcribed ultraconserved regions (T-UCRs), which take part in transcriptional, post-transcriptional and epigenetic regulation processes. We report here the interaction of miRNAs and T-UCRs as a network modulating the availability of these non-coding RNAs in bladder cancer cells. In our cell system, antagomiR-596 increased the expression of T-UCR 201+. Moreover, T-UCR 8+ silencing increased miR-596 expression, which in turn reduced total T-UCR 283+, showing that the perturbation of one element in this network changes the expression of other interactors. In addition, we identify the polycomb protein Yin Yang 1 (YY1) as mediator of binding between miR-596 and T-UCR 8+. These new findings describe for the first time a network between T-UCRs, miRNAs and YY1 protein, highlighting the existence of an additional layer of gene expression regulation.

Hedgehog Signaling Overcomes an EZH2-Dependent Epigenetic Barrier to Promote Cholangiocyte Expansion.

Background & AimsDevelopmental morphogens play an important role in coordinating the ductular reaction and portal fibrosis occurring in the setting of cholangiopathies. However, little is known about how membrane signaling events in ductular reactive cells (DRCs) are transduced into nuclear transcriptional changes to drive cholangiocyte maturation and matrix deposition. Therefore, the aim of this study was to investigate potential mechanistic links between cell signaling events and epigenetic regulators in DRCs.MethodsUsing directed differentiation of induced pluripotent stem cells (iPSC), isolated DRCs, and in vivo models, we examine the mechanisms whereby sonic hedgehog (Shh) overcomes an epigenetic barrier in biliary precursors and promotes both cholangiocyte maturation and deposition of fibronectin (FN).ResultsWe demonstrate, for the first time, that Gli1 influences the differentiation state and fibrogenic capacity of iPSC-derived hepatic progenitors and isolated DRCs. We outline a novel pathway wherein Shh-mediated Gli1 binding in key cholangiocyte gene promoters overcomes an epigenetic barrier conferred by the polycomb protein, enhancer of zeste homolog 2 (EZH2) and initiates the transcriptional program of cholangiocyte maturation. We also define previously unknown functional Gli1 binding sites in the promoters of cytokeratin (CK)7, CK19, and FN. Our in vivo results show that EZH2 KO mice fed the choline-deficient, ethanolamine supplemented (CDE) diet have an exaggerated cholangiocyte expansion associated with more robust ductular reaction and increased peri-portal fibrosis.ConclusionWe conclude that Shh/Gli1 signaling plays an integral role in cholangiocyte maturation in vitro by overcoming an EZH2-dependent epigenetic barrier and this mechanism also promotes biliary expansion in vivo.

EZH2 Enables the Proliferation of Germinal Center B Cells and DLBCL through a Rb-E2F1 Positive Feedback Loop Involving Repression of CDKN1A

Many B cell lymphomas arise from germinal center (GC) B cells of the humoral immune system, which are unique in their ability to replicate at an accelerated rate, which requires attenuation of replication checkpoints. Upon activation, GC B cells upregulate EZH2, a Polycomb protein that mediates transcriptional repression by trimethylating histone 3 lysine 27 (H3K27me3). Conditional deletion of EZH2 results in failure to form GCs. EZH2 is often highly expressed or affected by somatic gain of function mutations in GC B cell-derived diffuse large B cell lymphoma (DLBCL) and is required to maintain lymphoma cell proliferation and survival. Our previous research identified CDKN1A (p21Cip1) as a direct target of EZH2 in GC B cells and DLBCLs. EZH2 causes promoter H3K27 trimethylation and transcriptional repression of CDKN1A in GC B cells and DLBCL cells. Treatment of DLBCLs with a specific EZH2 inhibitor (GSK343) or EZH2 shRNA caused CDKN1A H3K27me3 demethylation and derepression. Based on these considerations we hypothesized that silencing of CDKN1Athrough H3K27me3 might explain the proliferative GC and DLBCL phenotype.To test this notion, we crossed GC-specific conditional Cg1Cre;Ezh2fl/fl mice with Cdkn1a-/- mice. We assessed GC formation after T cell-dependent immunization in double vs. single Cdkn1a or Ezh2 KO mice. Cdkn1a-/- mice manifested perfectly normal GC formation, whereas there was complete absence of GCs in Cg1Cre-Ezh2fl/fl mice. In contrast, Cg1Cre;Ezh2fl/fl;Cdkn1a-/- double KO mice exhibited normal GC formation as measured by immunohistochemistry and flow cytometry. While conditional deletion of Ezh2 in GCs abrogates immunoglobulin affinity maturation, the double KO mice manifested normal development of high affinity antibodies after specific antigen exposure (NP-KLH). Cell cycle analysis of double KO mice showed a similar proportion of GC B cells in S phase as WT or Cdkn1a-/- controls, as measured by BrdU incorporation, indicating that loss of p21 allows progression of cell cycle. These effects were linked to the methyltransferase function of EZH2 since Cdkn1a-/- also rescued the loss of GCs driven by administration of EZH2 inhibitor observed in WT mice. We observed a similar phenomenon in DLBCL cells since shRNA-mediated depletion of CDKN1A rescued the growth suppressive effect of EZH2 shRNA or specific EZH2 inhibitors. Therefore H3K27me3 and repression of CDKN1Aexplains to a large extent how EZH2 enables GC formation and maintains growth of DLBCL cells.To further understand the role of EZH2 as a driver of the cell cycle we explored its relation to the G1/2 checkpoint regulated by p21Cip1. We found that GC B cells from Cg1Cre;Ezh2fl/fl;Cdkn1a-/- double KO mice exhibited high levels of phospho Rb by IHC, similar to the levels found in WT or Cdkn1a-/- control mice. Hyperphosphorylation of Rb induces its inactivation, allowing the release of E2F transcription factors and cell cycle progression. EZH2 was previously shown to be a direct target of E2F1, E2F2 and, to a lesser extent E2F3. Among these we found that E2F1 mRNA and protein expression are especially highly expressed and upregulated in GC B cells vs. naïve B cells. By qChIP we show that E2F1 is bound to the EZH2 promoter in GC-derived DLBCL cell lines. Moreover, E2F1 gene expression is positively correlated with EZH2 (R=0.35, p<0.0001) and moderately inversely correlates with CDKN1A (R=-0.22, p<0.0001) in a cohort of 757 DLBCL patient samples. Therefore, we explored the function of E2F1 in GC formation. We found that E2f1-/- mice developed reduced number and size of GCs as compared to control mice (E2f1-/- vs. WT, p<0.01). To determine if this phenotype was due to a lack of induction of EZH2 by E2F1, we transduced bone marrow of E2f1-/- or WT donor mice with retrovirus encoding EZH2-GFP or GFP alone, transplanted them into lethally irradiated recipients and assessed the GC reaction after immunization. Notably, EZH2 expression successfully rescued E2f1-/- phenotype (E2f1-/-+GFP vs.E2f1-/-+EZH2, p<0.001), indicating that the pRb-E2F1 pathway drives the GC reaction by inducing EZH2.In summary we identified a positive feedback loop required for GC formation and DLBCL whereby EZH2 controls GC B cell proliferation by suppressing the critical cell cycle checkpoint gene CDKN1A, allowing cell cycle progression with a concomitant phosphorylation of Rb. This causes the release of E2F1, which positively regulates the expression of EZH2. DisclosuresMelnick:Janssen: Research Funding.

Regulation of Fetal Liver Hematopoietic Stem Cell Function By the Dpy30 Subunit of Set1/Mll Complexes

While stem cells undergo phenotypic and functional changes in development, the capacity of self-renewal and differentiation remains the defining property of stem cells throughout life, indicating certain fundamental regulatory mechanisms underlying these cardinal features of stem cells. A profound transition occurs to hematopoietic stem cells (HSCs) from embryonic to adult hematopoiesis, resulting in pronounced distinctions between fetal liver (FL) and adult bone marrow (BM) HSCs in many aspects. While many studies have documented a different dependence of fetal versus adult HSC function on epigenetic modulators including several Polycomb proteins, little is known about if Trithorax proteins play a similar or different role in fetal versus adult HSC function. More specifically, despite being a prominent epigenetic mark associated with gene activation, the role of H3K4 methylation (an activity of many Trithorax proteins) in different stages of HSCs remains unclear.As the major H3K4 methylases in mammals, the Set1/Mll family complexes play important roles in development and stem cell function, and are extensively associated with diseases including blood cancers. We have previously established a direct role of Dpy30, a core subunit in all Set1/Mll complexes, in facilitating genome-wide H3K4 methylation, and this allows an effective interrogation of the functional role of efficient H3K4 methylation through genetic studies of Dpy30. While dispensable for the self-renewal of embryonic stem cells (ESCs), Dpy30 is crucial for efficient differentiation of ESCs by facilitating the induction of many bivalently marked developmental genes (Jiang et al., Cell, 2011). We have then generated a Dpy30 conditional knockout mouse, and shown that Dpy30 plays a crucial role in the long term maintenance and differentiation of adult BM HSCs, and preferentially controls H3K4 methylation and expression of many hematopoiesis-associated genes in adult BM cells (Yang et al., J Exp Med, accepted). However, the role of Dpy30 and efficient H3K4 methylation in fetal HSCs is still unknown.To study the role of efficient H3K4 methylation in fetal HSCs, we deleted Dpy30 in fetal hematopoietic cells using VavCre line. VavCre; Dpy30F/- fetuses are anemic at E14.5 and E15.5, with reduced H3K4 methylation but significantly increased numbers of FL HSCs. However, these FL HSCs were functionally defective in colony formation and blood reconstitution following transplantation. Proliferation of the progenitors, but not HSCs, was significantly (but modestly) reduced. These results suggest a role of Dpy30 in differentiation of HSCs and progenitor proliferation in FL. We also competitively transplanted Mx1Cre; Dpy30F/- FL and deleted Dpy30 after stable engraftment. Our analysis at an early time point after deletion showed little effect on donor contribution to HSCs, but significant reduction of oligopotent progenitors. Analysis at a later time point after deletion, however, showed marked reduction of all hematopoietic cells including HSCs. These results support a cell-autonomous role of Dpy30 in the differentiation and long term maintenance of FL HSCs.The phenotypes of FL HSCs are largely similar to those of BM HSCs following Dpy30 loss, suggesting that Dpy30 and certain Dpy30 targets are fundamentally important in regulating HSCs regardless of the developmental stages. To identify these targets, we performed RNA-seq analyses for purified FL HSCs from VavCre; Dpy30F/- versus VavCre; Dpy30F/+ littermates. Among hundreds of genes that were significantly changed in FL HSCs, however, only a handful of genes were found to be co-downregulated in both FL and BM HSCs following Dpy30 loss, suggesting that Dpy30 may have different functional targets in different stages of HSCs. To identify Dpy30 targets fundamentally important to HSC regulation, we are now selectively investigating the function of a few common Dpy30 targets in HSCs by colony formation and potentially transplantation assays following their stable knockdown.The similar requirement of Dpy30 in both fetal and adult HSC differentiation as well as long-term maintenance underscores the fundamental importance of this epigenetic modulator in the central properties of stem cells, and studies of the common Dpy30 targets may identify new regulatory genes controlled by this modulator in fetal and adult HSC function. DisclosuresNo relevant conflicts of interest to declare.

The Role of Cbx Proteins in Human Benign and Malignant Hematopoiesis

Introduction:Hematopoietic stem cells (HSCs) need to properly balance self-renewal and differentiation to prevent malignant transformation or HSC exhaustion. We recently showed that the presence of specific Cbx proteins in the Polycomb Repressive Complex 1 (PRC1) plays a crucial role in balancing self-renewal and differentiation of murine HSCs. Whereas Cbx7 induces self-renewal, Cbx4 and Cbx8 instead induce differentiation. In this project, we investigate the role of various CBX Polycomb proteins in human normal and malignant hematopoiesis. We aim to identify genes which are controlled by CBX7 in normal and malignant human hematopoiesis.Methods:We overexpressed CBX2, -4, -6, -7 and -8 in human CD34+HSPCs. We assessed functional consequences by measuring cobblestone area-forming cell (CAFC), colony-forming unit (CFU) frequencies, and performed stem cell xenotransplantation studies in NSG mice. To identify genes which are controlled by CBX7 or CBX8, we performed RNA- and Chip-Seq in CD34+ HSPCs. To explore the role of CBX7 in leukemic cells, we performed short hairpin RNA-mediated knockdown of protein expression in leukemic cell lines.Results:Overexpression of CBX7 and CBX8 in human CD34+ HSPCs led to a significant 5-10-fold increase of week 5 CAFC (fold increase: 9,62 for CBX7; 4,92 for CBX8) and 1,5-fold increase CFU-frequencies (fold increase: 1,32 for CBX7; 1,41 for CBX8) after 14 days. In contrast, overexpression of CBX4 and CBX2 led to equal or decreased CAFC- and CFU-frequencies. Transplantation of CBX7 overexpressing human CD34+ HSPCs in NSG mice led to higher long-term engraftment in comparison to the empty vector control (p<0,05 after 18 weeks). Even after one week of in vitro culture of CBX7-overexpressing CD34+cells significant engraftment levels were obtained. Conversely, downregulation of CBX7 in various leukemic cell lines resulted in markedly decreased proliferation and strongly induced differentiation. Expression analysis showed that genes that are upregulated upon CBX7 overexpression are preferentially expressed in primitive stem cells, and are repressed in more differentiated cell types.Conclusions:Our study indicates that CBX7 and CBX8 regulate self-renewal of human HSPCs. Furthermore, our data show that repression of CBX7 markedly inhibits proliferation of leukemic cells and can release the differentiation block of leukemic cells in vitro. Collectively our results indicate that targeting CBX7 may be a viable strategy to induce terminal differentiation of leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

Destabilization of B2 RNA by EZH2 Activates the Stress Response

More than 98% of the mammalian genome is noncoding, and interspersed transposable elements account for ∼50% of noncoding space. Here, we demonstrate that a specific interaction between the polycomb protein EZH2 and RNA made from B2 SINE retrotransposons controls stress-responsive genes in mouse cells. In the heat-shock model, B2RNA binds stress genes and suppresses their transcription. Upon stress, EZH2 is recruited and triggers cleavage of B2 RNA. B2 degradation in turn upregulates stress genes. Evidence indicates that B2 RNA operates as a "speed bump" against advancement of RNA polymerase II, and temperature stress releases the brakes on transcriptional elongation. These data attribute a new function to EZH2 that is independent of its histone methyltransferase activity and reconcile how EZH2 can be associated with both gene repression and activation. Our study reveals that EZH2 and B2 together control activationof a large network of genes involved in thermal stress.

Easy Stress Relief by EZH2

While we are beginning to appreciate the cellular roles played by long noncoding RNAs, the function of transcripts emerging from repetitive genomic regions remains enigmatic. In this issue, Zovoilis etal. report that the polycomb protein EZH2, upon heat shock, facilitates transcription of stress-responsive genes by inducing the degradation of the transcriptional repressor B2 repeatRNA.

Tumorigenicity of Ewing sarcoma is critically dependent on the trithorax proteins MLL1 and menin.

Developmental transcription programs are epigenetically regulated by the competing actions of polycomb and trithorax (TrxG) protein complexes, which repress and activate genes, respectively. Ewing sarcoma is a developmental tumor that is associated with widespread de-regulation of developmental transcription programs, including HOX programs. Posterior HOXD genes are abnormally over-expressed by Ewing sarcoma and HOXD13, in particular, contributes to the tumorigenic phenotype. In MLL1 fusion-driven leukemia, aberrant activation of HOXA genes is epigenetically mediated by the TrxG complex and HOXA gene expression and leukemogenesis are critically dependent on the protein-protein interaction between the TrxG proteins MLL1 and menin. Based on these data, we investigated whether posterior HOXD gene activation and Ewing sarcoma tumorigenicity are similarly mediated by and dependent on MLL1 and/or menin. Our findings demonstrate that Ewing sarcomas express high levels of both MLL1 and menin and that continued expression of both proteins is required for maintenance of tumorigenicity. In addition, exposure of Ewing sarcoma cells to MI-503, an inhibitor of the MLL1-menin protein-protein interaction developed for MLL1-fusion driven leukemia, leads to loss of tumorigenicity and down-regulated expression of the posterior HOXD gene cluster. Together these data demonstrate an essential role for MLL1 and menin in mediating tumor maintenance and posterior HOXD gene activation in Ewing sarcoma. A critical dependency of these tumors on the MLL1-menin interaction presents a potentially novel therapeutic target.

Dynamic Protein Interactions of the Polycomb Repressive Complex 2 during Differentiation of Pluripotent Cells

Polycomb proteins assemble to form complexes with important roles in epigenetic regulation. The Polycomb Repressive Complex 2 (PRC2) modulates the di- and tri-methylation of lysine 27 on histone H3, each of which are associated with gene repression. Although three subunits, EZH1/2, SUZ12, and EED, form the catalytic core of PRC2, a wider group of proteins associate with low stoichiometry. This raises the question of whether dynamic variation of the PRC2 interactome results in alternative forms of the complex during differentiation. Here we compared the physical interactions of PRC2 in undifferentiated and differentiated states of NTERA2 pluripotent embryonic carcinoma cells. Label-free quantitative proteomics was used to assess endogenous immunoprecipitation of the EZH2 and SUZ12 subunits of PRC2. A high stringency data set reflecting the endogenous state of PRC2 was produced that included all previously reported core and associated PRC2 components, and several novel interacting proteins. Comparison of the interactomes obtained in undifferentiated and differentiated cells revealed candidate proteins that were enriched in complexes isolated from one of the two states. For example, SALL4 and ZNF281 associate with PRC2 in pluripotent cells, whereas PCL1 and SMAD3 preferentially associate with PRC2 in differentiating cells. Analysis of the mRNA and protein levels of these factors revealed that their association with PRC2 correlated with their cell state-specific expression. Taken together, we propose that dynamic changes to the PRC2 interactome during differentiation may contribute to directing its activity during cell fate transitions.

Polycomb Protein OsFIE2 Affects Plant Height and Grain Yield in Rice.

Polycomb group (PcG) proteins have been shown to affect growth and development in plants. To further elucidate their role in these processes in rice, we isolated and characterized a rice mutant which exhibits dwarfism, reduced seed setting rate, defective floral organ, and small grains. Map-based cloning revealed that abnormal phenotypes were attributed to a mutation of the Fertilization Independent Endosperm 2 (OsFIE2) protein, which belongs to the PcG protein family. So we named the mutant as osfie2-1. Histological analysis revealed that the number of longitudinal cells in the internodes decreased in osfie2-1, and that lateral cell layer of the internodes was markedly thinner than wild-type. In addition, compared to wild-type, the number of large and small vascular bundles decreased in osfie2-1, as well as cell number and cell size in spikelet hulls. OsFIE2 is expressed in most tissues and the coded protein localizes in both nucleus and cytoplasm. Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that OsFIE2 interacts with OsiEZ1 which encodes an enhancer of zeste protein previously identified as a histone methylation enzyme. RNA sequencing-based transcriptome profiling and qRT-PCR analysis revealed that some homeotic genes and genes involved in endosperm starch synthesis, cell division/expansion and hormone synthesis and signaling are differentially expressed between osfie2-1 and wild-type. In addition, the contents of IAA, GA3, ABA, JA and SA in osfie2-1 are significantly different from those in wild-type. Taken together, these results indicate that OsFIE2 plays an important role in the regulation of plant height and grain yield in rice.

Developmental Epigenetic Programming of Adult Germ Cell Death Disease: Polycomb Protein EZH2–miR-101 Pathway

The Developmental Origin of Health and Disease refers to the concept that early exposure to toxicants or nutritional imbalances during perinatal life induces changes that enhance the risk of developing noncommunicable diseases in adulthood. Patients/materials & methods: An experimental model with an adult chronic germ cell death phenotype resulting from exposure to a xenoestrogen was used. A reciprocal negative feedback loop involving decreased EZH2 protein level and increased miR-101 expression was identified. In vitro and in vivo knockdown of EZH2 induced an apoptotic process in germ cells through increased levels of apoptotic factors (BIM and BAD) and DNA repair alteration via topoisomerase 2B deregulation. The increased miR-101 levels were observed in the animal blood, meaning that miR-101 may be a part of a circulating mark of germ cell death. miR-101-EZH2 pathway deregulation could represent a novel pathophysiological epigenetic basis for adult germ cell disease with environmental and developmental origins.

MicroRNA-298 inhibits malignant phenotypes of epithelial ovarian cancer by regulating the expression of EZH2.

MicroRNA (miRNA or miR)-298 has been reported to be downregulated and to modify the expression of the polycomb protein enhancer of zeste 2 (EZH2) in recurrent epithelial ovarian cancer (EOC). To date, no functional evidence of a miR-298-EZH2 axis in EOC has been documented. The present study aimed to investigate the associations of miR-298 and/or EZH2 expression with clinicopathological features of EOC patients, and revealed their roles in cell motility based on EOC cell lines. Reverse transcription-quantitative polymerase chain reaction was performed to detect the expression levels of miR-298 and EZH2 messenger RNA in human EOC tissues and cell lines. Wound healing and transwell assays were performed to determine the function of the miR-298-EZH2 axis on cell migration and invasion, respectively. Compared with normal tissues, miR-298 expression was significantly downregulated, while EZH2 expression was significantly upregulated, in human EOC tissues (both P=0.001). In addition, miR-298 downregulation and EZH2 upregulation were significantly associated with high clinical stage (both P=0.01) and pathological grade (both P=0.02) of EOC patients. Furthermore, the ectopic expression of miR-298 could efficiently inhibit cell migration and invasion. Notably, the overexpression of EZH2 could restore the cell migration and invasion abilities suppressed by miR-298. Our data offer convincing evidence that the dysregulation of the miR-298-EZH2 axis may be important in tumor progression of EOC patients. The present study also confirmed a tumor-suppressive role of miR-298 in modulating EOC cell motility by regulating the expression of EZH2, implying its potential as a novel miRNA-based therapeutic target for the treatment of human EOC.

Differential expression of enhancer of zeste homolog 2 (EZH2) protein in small cell and aggressive B-cell non-Hodgkin lymphomas and differential regulation of EZH2 expression by p-ERK1/2 and MYC in aggressive B-cell lymphomas

AbstractEZH2, a member of the polycomb protein group, is an important methyltransferase that is overexpressed in various neoplasms. We found that in small cell B-cell lymphomas, EZH2 is expressed in <40% of neoplastic cells, with heterogenous signal intensity. In aggressive B-cell lymphomas, 70–100% of tumor cells were positive for EZH2 expression with high signal intensity, which correlated with a high proliferation rate. We investigated the potential signaling molecules that regulate EZH2 overexpression in aggressive B-cell lymphomas and found that 80% of cases of EZH2-positive diffuse large B-cell lymphoma show high p-ERK1/2 expression (average ~57% tumor cell positivity). In contrast, only a small percentage of tumor cells (~10%) show p-ERK1/2 expression in Burkitt lymphoma and double hit lymphoma. On average, 91 and 76% of neoplastic cells were positive for MYC expression in Burkitt lymphoma and double hit lymphoma, respectively, while only 20% neoplastic cells were positive for MYC expression in diffuse large B-cell lymphoma. None of the aggressive B-cell lymphomas showed significant p-STAT3 expression in EZH2-overexpressed cases. The correlation of EZH2 expression with aggressive behavior and proliferation rate in B-cell neoplasms suggests that this molecule may function as an oncogenic protein in these neoplasms, with possible regulation by different signaling cascades in different types of aggressive B-cell lymphomas: p-ERK-related signaling in diffuse large B-cell lymphoma, and MYC-related signaling in Burkitt lymphoma and double hit lymphoma. Furthermore, EZH2 and associated signaling cascades may serve as therapeutic targets for the treatment of aggressive B-cell lymphomas.

Propagation of trimethylated H3K27 regulated by polycomb protein EED is required for embryogenesis, hematopoietic maintenance, and tumor suppression

Polycomb repressive complex 2 (PRC2) catalyzes the monomethylation, dimethylation, and trimethylation of histone H3 Lys27 (H3K27) and acts as a central epigenetic regulator that marks the repressive chromatin domain. Embryonic ectoderm development (EED), an essential component of PRC2, interacts with trimethylated H3K27 (H3K27me3) through the aromatic cage structure composed of its three aromatic amino acids, Phe97, Trp364, and Tyr365. This interaction allosterically activates the histone methyltransferase activity of PRC2 and thereby propagates repressive histone marks. In this study, we report the analysis of knock-in mice harboring the myeloid disorder-associated EED Ile363Met (I363M) mutation, analogous to the EED aromatic cage mutants. The I363M homozygotes displayed a remarkable and preferential reduction of H3K27me3 and died at midgestation. The heterozygotes increased the clonogenic capacity and bone marrow repopulating activity of hematopoietic stem/progenitor cells (HSPCs) and were susceptible to leukemia. Lgals3, a PRC2 target gene encoding a multifunctional galactose-binding lectin, was derepressed in I363M heterozygotes, which enhanced the stemness of HSPCs. Thus, our work provides in vivo evidence that the structural integrity of EED to H3K27me3 propagation is critical, especially for embryonic development and hematopoietic homeostasis, and that its perturbation increases the predisposition to hematologic malignancies.

A polycomb-mediated epigenetic field defect precedes invasive cervical carcinoma.

Human papillomavirus (HPV)-associated cervical carcinoma is preceded by stages of cervical intra-epithelial neoplasia (CIN) that can variably progress to malignancy. Understanding the different molecular processes involved in the progression of pre-malignant CIN is critical to the development of improved predictive and interventional capabilities. We tested the role of regulators of transcription in both the development and the progression of HPV-associated CIN, performing the most comprehensive genomic survey to date of DNA methylation in HPV-associated cervical neoplasia, testing ~2 million loci throughout the human genome in biopsies from 78 HPV+ women, identifying changes starting in early CIN and maintained through carcinogenesis. We identified loci at which DNA methylation is consistently altered, beginning early in the course of neoplastic disease and progressing with disease advancement. While the loss of DNA methylation occurs mostly at intergenic regions, acquisition of DNA methylation is at sites involved in transcriptional regulation, with strong enrichment for targets of polycomb repression. Using an independent cohort from The Cancer Genome Atlas, we validated the loci with increased DNA methylation and found that these regulatory changes were associated with locally decreased gene expression. Secondary validation using immunohistochemistry showed that the progression of neoplasia was associated with increasing polycomb protein expression specifically in the cervical epithelium. We find that perturbations of genomic regulatory processes occur early and persist in cervical carcinoma. The results indicate a polycomb-mediated epigenetic field defect in cervical neoplasia that may represent a target for early, topical interventions using polycomb inhibitors.

Expression of polycomb protein BMI-1 maintains the plasticity of basal bronchial epithelial cells.

The airway epithelium is altered in respiratory disease and is thought to contribute to disease etiology. A caveat to disease research is that the technique of isolation of bronchial epithelial cells from patients is invasive and cells have a limited lifespan. The aim of this study was to extensively characterize the plasticity of primary human bronchial epithelial cells that have been engineered to delay cell senescence including the ability of these cells to differentiate. Cells were engineered to express BMI‐1 or hTERT using viral vector systems. Cells were characterized at passage (p) early (p5), mid (p10), and late (p15) stage for: BMI‐1, p16, and CK14 protein expression, viability and the ability to differentiate at air–liquid interface (ALI), using a range of techniques including immunohistochemistry (IHC), immunofluorescence (IF), transepithelial electrical resistance (TEER), scanning electron microscopy (SEM), MUC5AC and beta tubulin (BTUB) staining. BMI‐1‐expressing cells maintained elevated levels of the BMI‐1 protein and the epithelial marker CK14 and showed a suppression of p16. BMI‐1‐expressing cells had a viability advantage, differentiated at ALI, and had a normal karyotype. In contrast, hTERT‐expressing cells had a reduced viability, showed limited differentiation, and had an abnormal karyotype. We therefore provide extensive characterization of the plasticity of BMI‐1 expressing cells in the context of the ALI model. These cells retain properties of wild‐type cells and may be useful to characterize respiratory disease mechanisms in vitro over sustained periods.

Formation of a Polycomb-Domain in the Absence of Strong Polycomb Response Elements.

Polycomb group response elements (PREs) in Drosophila are DNA-elements that recruit Polycomb proteins (PcG) to chromatin and regulate gene expression. PREs are easily recognizable in the Drosophila genome as strong peaks of PcG-protein binding over discrete DNA fragments; many small but statistically significant PcG peaks are also observed in PcG domains. Surprisingly, in vivo deletion of the four characterized strong PREs from the PcG regulated invected-engrailed (inv-en) gene complex did not disrupt the formation of the H3K27me3 domain and did not affect inv-en expression in embryos or larvae suggesting the presence of redundant PcG recruitment mechanism. Further, the 3D-structure of the inv-en domain was only minimally altered by the deletion of the strong PREs. A reporter construct containing a 7.5kb en fragment that contains three weak peaks but no large PcG peaks forms an H3K27me3 domain and is PcG-regulated. Our data suggests a model for the recruitment of PcG-complexes to Drosophila genes via interactions with multiple, weak PREs spread throughout an H3K27me3 domain.

Repressing the Keratinocyte Genome: How the Polycomb Complex Subunits Operate in Concert to Control Skin and Hair Follicle Development

The Polycomb group proteins are transcriptional repressors that are critically important in the control of stem cell activity and maintenance of the identity ofdifferentiated cells. Polycomb proteins interact with each other to form chromatin-associated repressive complexes (Polycomb repressive complexes 1and 2) leading to chromatin compaction and gene silencing. However, the roles of the distinct components of the Polycomb repressive complex 2 in the control of skin development and keratinocyte differentiation remain obscure. Dauber etal. demonstrate the conditional ablations of three essential Polycomb repressive complex 2 subunits (EED, Suz12, or Ezh1/2) in the epidermal progenitors result in quite similar skin phenotypes including premature acquisition of a functional epidermal barrier, formation of ectopic Merkel cells, and defective postnatal hair follicle development. The reported data demonstrate that in skin epithelia, EED, Suz12, and Ezh1/2 function largely as subunits of the Polycomb repressive complex 2, which is important in the context of data demonstrating their independent activities in other cell types. The report provides an important platform for further analyses of the role of distinct Polycomb components in the control of gene expression programs in the disorders of epidermal differentiation, such as psoriasis and epidermal cancer.