Polycomb Group Gene EZH2 Research Articles

Pathogenic role of insufficiency of polycomb repressive complex in primary myelofibrosis

Primary myelofibrosis (PMF) is characterized by the clonal expansion of megakaryocytes and myeloid cells from stem cells with abnormal cytokine expression, resulting in bone marrow fibrosis, angiogenesis, and osteosclerosis. The use of next-generation sequencing revealed that both genetic and epigenetic changes are important in the pathogenesis of PMF. Several epigenetic regulator genes, including TET2, the polycomb-related gene ASXL1, and the polycomb-group gene EZH2, have been found to be targeted by somatic gene mutations in PMF patients. Among these, loss of Ezh2 has been demonstrated to disrupt the function of the polycomb repressive complex 2, promoting the development of JAK2V617F-induced myelofibrosis in mice. In this analysis, we highlight the role of PRC dysfunction in the pathogenesis of PMF.

Memory of Divisional History Directs the Continuous Process of Primitive Hematopoietic Lineage Commitment.

SummaryHematopoietic stem cells (HSCs) exist in a dormant state and progressively lose regenerative potency as they undergo successive divisions. Why this functional decline occurs and how this information is encoded is unclear. To better understand how this information is stored, we performed RNA sequencing on HSC populations differing only in their divisional history. Comparative analysis revealed that genes upregulated with divisions are enriched for lineage genes and regulated by cell-cycle-associated transcription factors, suggesting that proliferation itself drives lineage priming. Downregulated genes are, however, associated with an HSC signature and targeted by the Polycomb Repressive Complex 2 (PRC2). The PRC2 catalytic subunits Ezh1 and Ezh2 promote and suppress the HSC state, respectively, and successive divisions cause a switch from Ezh1 to Ezh2 dominance. We propose that cell divisions drive lineage priming and Ezh2 accumulation, which represses HSC signature genes to consolidate information on divisional history into memory.

Multifaceted role of the polycomb-group gene EZH2 in hematological malignancies.

Polycomb repressive complex (PRC) is a critical regulator of normal tissue homeostasis as well as tumorigenesis. EZH2, an enzymatic subunit of PRC2, is a histone H3K27 methyltransferase that functions in the regulation of gene silencing. EZH2 overexpression was first identified in prostate and breast cancers and is associated with poor clinical outcome. Subsequently, gain- and loss-of-function mutations of EZH2 have been identified in various tumors, including hematological malignancies, implicating EZH2 as either an oncogene or a tumor suppressor gene, depending on the cancer type. Molecular mechanisms underlying the multifaceted function of EZH2 have been analyzed extensively. However, because EZH2 dysregulation is functionally integrated with multiple other epigenetic events in a context-dependent manner, the precise manner in which EZH2 dysregulation impacts the pathogenesis of hematological malignancies remains to be clarified. In this perspective, we describe recent findings in pathogenic role of EZH2 in hematological malignancies, which may provide insights into the treatment of with cancers with EZH2 dysregulation and the development of novel therapies targeting epigenetic regulators.

Impact of combinatorial dysfunctions of Tet2 and Ezh2 on the epigenome in the pathogenesis of myelodysplastic syndrome

Somatic inactivating mutations in epigenetic regulators are frequently found in combination in myelodysplastic syndrome (MDS). However, the mechanisms by which combinatory mutations in epigenetic regulators promote the development of MDS remain unknown. Here we performed epigenomic profiling of hematopoietic progenitors in MDS mice hypomorphic for Tet2 following the loss of the polycomb-group gene Ezh2 (Tet2KD/KDEzh2Δ/Δ). Aberrant DNA methylation propagated in a sequential manner from a Tet2-insufficient state to advanced MDS with deletion of Ezh2. Hyper-differentially methylated regions (hyper-DMRs) in Tet2KD/KDEzh2Δ/Δ MDS hematopoietic stem/progenitor cells were largely distinct from those in each single mutant and correlated with transcriptional repression. Although Tet2 hypomorph was responsible for enhancer hypermethylation, the loss of Ezh2 induced hyper-DMRs that were enriched for CpG islands of polycomb targets. Notably, Ezh2 targets largely lost the H3K27me3 mark while acquiring a significantly higher level of DNA methylation than Ezh1 targets that retained the mark. These findings indicate that Ezh2 targets are the major targets of the epigenetic switch in MDS with Ezh2 insufficiency. Our results provide a detailed trail for the epigenetic drift in a well-defined MDS model and demonstrate that the combined dysfunction of epigenetic regulators cooperatively remodels the epigenome in the pathogenesis of MDS.

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.

A transgenic mouse model demonstrating the oncogenic role of mutations in the polycomb-group gene EZH2 in lymphomagenesis

AbstractThe histone methyltransferase EZH2 is frequently mutated in germinal center–derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2Y641F is expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2 mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2Y641F by crossing these transgenic mice with Eµ-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Myc and EZH2Y641F. The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2Y641F can collaborate with Myc to accelerate lymphomagenesis demonstrating a cooperative role of EZH2 mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments.

Deletion of the polycomb-group gene EZH2 causes myeloproleferative neoplasm in mice

Polycomb-group (PcG) proteins form the polycomb repressive complexes (PRC) 1 and 2, functioning as transcriptional repressors through histone modifications. They have been implicated in the maintenance of both hematopoietic and leukemic stem cells, by repressing the transcription of tumor suppressor genes, namely Ink4a and Arf, and thus have been characterized as oncogenes. Of great interest, inactivating mutations of EZH2 have been identified in patients with myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN), suggesting that PcG genes also have a tumor suppressor function.

Abstract 2483: Inhibition of the polycomb group gene EZH2 suppresses growth and radiosensitizes ATRT cells by promoting senescence and inhibiting the CycinD-E2F1 axis

Abstract Overexpression of the polycomb group gene Enhancer of Zeste 2 (EZH2) occurs in many tumors including prostate cancer, breast cancer and brain tumors such as Glioblastoma multiform and Medulloblastoma. Recent evidence suggests it may also have a role in rhabdoid tumors. Atypical teratoid rhabdoid tumor (ATRT) is a rare high-grade brain tumor with very poor survival that occurs most commonly in children. AT/RT tumors are characterized by deletion of the chromatin associated protein SMARCB1. Given the role of EZH2 in regulating epigenetic changes we chose to investigate the role of EZH2 in AT/RT. Here we show that targeted disruption of EZH2 by RNAi or pharmacologic means strongly impairs ATRT cell growth, induces apoptosis and potently sensitizes these cells to radiation. Using functional analysis of transcription factor activity we found the Cyclin D-E2F1 axis to be repressed upon EZH2 depletion in ATRT cells. Taken together our observations provide evidence that EZH2 regulates cell cycle progression by targeting the G1-S transition machinery and may be an important new therapeutic target particularly in combination with radiation in ATRT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2483. doi:1538-7445.AM2012-2483

ESCs Require PRC2 to Direct the Successful Reprogramming of Differentiated Cells toward Pluripotency

Embryonic stem cells (ESCs) are pluripotent, self-renewing, and have the ability to reprogram differentiated cell types to pluripotency upon cellular fusion. Polycomb-group (PcG) proteins are important for restraining the inappropriate expression of lineage-specifying factors in ESCs. To investigate whether PcG proteins are required for establishing, rather than maintaining, the pluripotent state, we compared the ability of wild-type, PRC1-, and PRC2-depleted ESCs to reprogram human lymphocytes. We show that ESCs lacking either PRC1 or PRC2 are unable to successfully reprogram B cells toward pluripotency. This defect is a direct consequence of the lack of PcG activity because it could be efficiently rescued by reconstituting PRC2 activity in PRC2-deficient ESCs. Surprisingly, the failure of PRC2-deficient ESCs to reprogram somatic cells is functionally dominant, demonstrating a critical requirement for PcG proteins in the chromatin-remodeling events required for the direct conversion of differentiated cells toward pluripotency.

Genetic Polymorphisms in the Polycomb Group Gene EZH2 and the Risk of Lung Cancer

Polycomb group (PcG) proteins play essential roles in cellular memory systems and as cell cycle regulators by maintaining homeotic genes in their silenced states. EZH1 and EZH2, the human homologues of the Drosophila gene Enhancer of Zeste (E(z)), are defined as PcG proteins and contain a highly conserved motif, called the SET (Su(var)3-9, Enhancer of Zeste, Trithorax) domain, which is required for histone methyltransferase activity. Increased expression of the transcriptional repressor EZH2 has been reported to be associated with poor prognosis in various malignancies including breast cancer and prostate cancer. Altered expression of EZH2 was also demonstrated in lung cancer, suggesting an involvement in the progression of lung cancer. All 41 polymorphisms in EZH2 were genotyped in 335 patients with lung cancer and 335 age- and gender-matched healthy controls. Finally, 26 polymorphisms were selected for the statistical analysis based on minor allele frequency (>0.05) and linkage disequilibrium. Two polymorphisms of EZH2, rs6950683 and rs3757441, showed a statistically significant association with reduced risk of lung cancer (adjusted OR [aOR] = 0.71, p = 0.007; aOR = 0.73, p = 0.015, respectively). Two copies of a haplotype (Ht2) of EZH2 also showed a significant association with reduced lung cancer risk (aOR = 0.45, 95% confidence interval = 0.23-0.87). This is the first study to show a significant association between polymorphisms of the PcG gene EZH2 and lung cancer risk. This study suggests a correlation between the genotype variants in EZH2 and reduced lung cancer risk and hence presents a possible marker for lung cancer susceptibility.

Pbx1 Regulates Self-Renewal of Long-Term Hematopoietic Stem Cells by Maintaining Their Quiescence

Self-renewal is a defining characteristic of stem cells; however, the molecular pathways underlying its regulation are poorly understood. Here, we demonstrate that conditional inactivation of the Pbx1 proto-oncogene in the hematopoietic compartment results in a progressive loss of long-term hematopoietic stem cells (LT-HSCs) that is associated with concomitant reduction in their quiescence, leading to a defect in the maintenance of self-renewal as assessed by serial transplantation. Transcriptional profiling revealed that multiple stem cell maintenance factors are perturbed in Pbx1-deficient LT-HSCs, which prematurely express a large subset of genes, including cell-cycle regulators, normally expressed in non-self-renewing multipotent progenitors. A significant proportion of Pbx1-dependent genes is associated with the TGF-beta pathway, which serves a major role in maintaining HSC quiescence. Prospectively isolated, Pbx1-deficient LT-HSCs display altered transcriptional responses to TGF-beta stimulation in vitro, suggesting a possible mechanism through which Pbx1 maintenance of stem cell quiescence may in part be achieved.

EWS-FLI-1 Expression Triggers a Ewing's Sarcoma Initiation Program in Primary Human Mesenchymal Stem Cells

Ewing's sarcoma family tumors (ESFT) express the EWS-FLI-1 fusion gene generated by the chromosomal translocation t(11;22)(q24;q12). Expression of the EWS-FLI-1 fusion protein in a permissive cellular environment is believed to play a key role in ESFT pathogenesis. However, EWS-FLI-1 induces growth arrest or apoptosis in differentiated primary cells, and the identity of permissive primary human cells that can support its expression and function has until now remained elusive. Here we show that expression of EWS-FLI-1 in human mesenchymal stem cells (hMSC) is not only stably maintained without inhibiting proliferation but also induces a gene expression profile bearing striking similarity to that of ESFT, including genes that are among the highest ESFT discriminators. Expression of EWS-FLI-1 in hMSCs may recapitulate the initial steps of Ewing's sarcoma development, allowing identification of genes that play an important role early in its pathogenesis. Among relevant candidate transcripts induced by EWS-FLI-1 in hMSCs, we found the polycomb group gene EZH2, which we show to play a critical role in Ewing's sarcoma growth. These observations are consistent with our recent findings using mouse mesenchymal progenitor cells and provide compelling evidence that hMSCs are candidate cells of origin of ESFT.

322 The different effects of neuromuscular blocking agents on adult- and fetal-types of nicotinic acetylcholine receptors

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.