Embryonic Ectoderm Development Research Articles

Expression and clinicopathological significance of EED, SUZ12 and EZH2 mRNA in colorectal cancer.

Enhancer of zeste 2 (EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 homolog (SUZ12), the key component of polycomb repressive complex 2, are of great importance in human cancer pathogenesis. This study was designed to investigate the clinical and prognostic significances of EZH2, EED and SUZ12 in colorectal cancer (CRC) patients. The expression of EZH2, EED and SUZ12 mRNA was evaluated in 82 primary CRC and paired non-cancerous mucosa samples by qRT-PCR. We found that overall EZH2, EED and SUZ12 mRNA expression in the CRC tissues was significantly increased than in the non-cancerous tissue (p < 0.05). Increased EZH2, EED and SUZ12 mRNA expression was directly correlated with primary tumor size, regional lymph node metastases, distant metastasis and AJCC stage. Furthermore, CRC patients with higher level of EED, SUZ12 or EZH2 showed a worse disease-free survival (DFS) (p < 0.01). In multivariate analysis, the increased EZH2 expression may be a risk factor for the patients' 3-year DFS (HR 2.517; 95% CI 1.104, 5.736; p = 0.028). Furthermore, the k-means cluster analysis showed that high mRNA expression of EED, SUZ12 and EZH2 was significantly correlated with the aggressive clinical behavior and poor prognosis. High expression of EED, SUZ12 and EZH2 might contribute to the CRC development/progression.

Abstract LB-132: The central role of EED in orchestration of polycomb group complexes

Abstract Polycomb Group (PcG) proteins play a vital role in establishing and maintaining gene expression patterns during cellular differentiation and development. They are epigenetic regulators that are responsible for the repression of target genes involved in development and cell fate specification. PcG proteins function as multimeric, chromatin-associated protein complexes. In mammals, there are two major Polycomb Repressive Complexes, PRC1 and PRC2 that possess histone-modifying activity crucial for their role in gene repression through methylation of histone H3K27, as well as the mono-ubiquitination of histone H2AK119. Components of both PRC1 and PRC2 have been shown to be elevated in a number of tumor types and play an important role in neoplastic progression. Major components of human PRC2 include the histone methyltransferase, Enhancer of Zeste Homolog 2, and its known binding partners, Embryonic Ectoderm Development (EED) and Suppressor of Zeste 12 (SUZ12) that collectively function as a multi-subunit complex trimethylating histone H3K27. Fundamental collaborators of PRC2 in epigenetic silencing include human PRC1 which consists of B lymphoma Mo-MLV insertion region 1 (BMI1), RING1A (also known as RING1), and RING1B (also known as RING2 or RNF2). Together, they function as a multi-protein complex to ubiquitinate histone H2A at lysine 119 (uH2A). The leading hypothesis regarding PRC2 and PRC1 interaction is that PRC2-mediated tri-methylation of H3K27 recruits PRC1 to genomic loci leading to chromatin condensation and epigenetic silencing of target genes. While the evidence linking PRC1 and PRC2 is circumstantial, a physical or molecular link between these complexes has not been established. In this study, we make an unexpected observation that EED, previously considered a critical component of PRC2, is instead a shared component of PRC2 and PRC1 that functions to interchange these epigenetic complexes at sites of histone modification. This observation markedly enhances our understanding of how PRC2 and PRC1 coordinate epigenetic regulation and may have implications in therapeutically targeting these master regulators of transcription. Citation Format: Qi Cao, Xiaoju Wang, Meng Zhao, Rendong Yang, Rohit Malik, Yuanyuan Qiao, Anton Poliakov, Xuhong Cao, Clair Harris, Felix Y. Feng, Sundeep Kalantry, Zhaohui Qin, Saravana M. Dhanasekaran, Arul M. Chinnaiyan. The central role of EED in orchestration of polycomb group complexes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-132. doi:10.1158/1538-7445.AM2014-LB-132

Polycomb protein EED is required for silencing of pluripotency genes upon ESC differentiation.

Eed (embryonic ectoderm development) is a core component of the Polycomb Repressive Complex 2 (PRC2) which catalyzes the methylation of histone H3 lysine 27 (H3K27). Trimethylated H3K27 (H3K27me3) can act as a signal for PRC1 recruitment in the process of gene silencing and chromatin condensation. Previous studies with Eed KO ESCs revealed a failure to down-regulate a limited list of pluripotency factors in differentiating ESCs. Our aim was to analyze the consequences of Eed KO for ESC differentiation. To this end we first analyzed ESC differentiation in the absence of Eed and employed in silico data to assess pluripotency gene expression and H3K27me3 patterns. We linked these data to expression analyses of wildtype and Eed KO ESCs. We observed that in wildtype ESCs a subset of pluripotency genes including Oct4, Nanog, Sox2 and Oct4 target genes progressively gain H3K27me3 during differentiation. These genes remain expressed in differentiating Eed KO ESCs. This suggests that the deregulation of a limited set of pluripotency factors impedes ESC differentiation. Global analyses of H3K27me3 and Oct4 ChIP-seq data indicate that in ESCs the binding of Oct4 to promoter regions is not a general predictor for PRC2-mediated silencing during differentiation. However, motif analyses suggest a binding of Oct4 together with Sox2 and Nanog at promoters of genes that are PRC2-dependently silenced during differentiation. In summary, our data further characterize Eed function in ESCs by showing that Eed/PRC2 is essential for the onset of ESC differentiation.

Anti-PABPC1 co-immunoprecipitation for examining the miRNAs directly targeting the 3'-UTR of EED mRNA.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate post-transcriptional gene expression by base pairing with partially complementary sequences within target messenger RNAs (mRNAs). Although the target genes and the precise biological functions of individual miRNAs remain largely unknown, miRNAs have been implicated in diverse biological processes, including both normal and pathological states. As a single stranded mRNA can be directly targeted by multiple miRNAs, and as the target sites may exist in the 3′-untranslated region (UTR), 5′-UTR, or the coding regions, it is essential to develop an effective method to identify the full-scale miRNA regulatory pattern of each particular gene. In this study, we employed a biochemical approach to identify the miRNA profiles that regulate the expression of embryonic ectoderm development (EED) protein by using anti-PABPC1 ribonucleoprotein (RNP) co-immunoprecipitation (Co-IP). The full length EED mRNA was subcloned into an expression vector and transiently transfected into a Flag-PABPC1 stable expression cell line. Subsequent to cross-linking and an anti-Flag Co-IP, the miRNAs that directly targeted EED were identified. We found that the best time point to distinguish the positive miRNAs from the background was 18 hours after the plasmid transfection. As expected, the miRNAs that directly target EED were found to interact with EED mRNA through the miRNA-induced silencing complex (miRISC). Meanwhile, the EED mRNA was bound by Flag-PABPC1. This method depends on the integrity of the miRISC complex and achieves greater efficiency when ultraviolet irradiation is used for the process of cross-linking. By using anti-PABPC1 RIP, we identified EED to be a new target gene of miR-16; a finding further confirmed using a dual-luciferase assay. In summary, our data indicate that anti-PABPC1 RIP is a validated and direct biochemical method to provide data about specific miRNA-mRNA interactions, as well as global miRNA patterns regulating the mRNAs.

Dendritic Cell-Associated miRNAs Are Modulated via Chromatin Remodeling in Response to Different Environments

IntroductionEpigenetic modification plays a critical role in regulating gene expression. To understand how epigenetic modification alters miRNA expression in monocyte-derived dendritic cells (moDCs) in different environments, we analyzed the connections between H3K4me3 and H3K27me3 modification and the expression of miRNAs in LPS- and TGF-β-conditioned moDCs.ResultsIn moDCs, H3K4me3 modification was strongly associated with the expression of activating miRNAs, whereas H3K27me3 was related to repressive miRNAs. The regulation of miRNA expression by H3K4me3 and H3K27me3 was further confirmed by silencing or inhibiting methyltransferases or methylation-associated factors in LPS- and TGF-β-conditioned moDCs. siRNAs targeting H3K4me3-associated mixed lineage leukemia (MLL) and retinoblastoma binding protein 5 (RBBP5) reduced H3K4me3 enrichment and downregulated miRNA expression; conversely, silencing H3K27me3-associated enhancer of zeste homolog 2 (EZH2) and embryonic ectoderm development (EED) genes upregulated the DC-associated miRNAs. However, LPS-mediated miRNAs were often associated with H3K4me3 redistribution from the transcription start site (TSS) to the miRNA-coding region. Silencing LPS-associated NF-κB p65 and CBP/p300 not only inhibited H3K4m3 redistribution but also reduced miRNA expression. LPS-upregulated RBBP4 and RBBP7, which are involved in chromatin remodeling, also affected the redistribution of H3K4me3 and reduced the expression of miRNAs.ConclusionIn LPS- and TGF-β-conditioned moDCs, miRNAs may be modulated not only by H3K4m3 and H3K27me3 modification but also by redistribution of H3K4me3 around the transcriptional start site of miRNAs. Thus, H3K4me3 and H3K27me3 epigenetic modification may play an important role in regulating DC differentiation and function in the presence of tumor or inflammatory environments.

Cellular senescence mediated by p16INK4A-coupled miRNA pathways

p16 is a key regulator of cellular senescence, yet the drivers of this stable state of proliferative arrest are not well understood. Here, we identify 22 senescence-associated microRNAs (SA-miRNAs) in normal human mammary epithelial cells. We show that SA-miRNAs-26b, 181a, 210 and 424 function in concert to directly repress expression of Polycomb group (PcG) proteins CBX7, embryonic ectoderm development (EED), enhancer of zeste homologue 2 (EZH2) and suppressor of zeste 12 homologue (Suz12), thereby activating p16. We demonstrate the existence of a tight positive feedback loop in which SA-miRNAs activate and re-enforce the expression of other SA-miRNA members. In contrast, PcG members restrain senescence by epigenetically repressing the expression of these SA-miRNAs. Importantly, loss of p16 leads to repression of SA-miRNA expression, intimately coupling this effector of senescence to the SA-miRNA/PcG self-regulatory loop. Taken together, our findings illuminate an important regulatory axis that underpins the transition from proliferation to cellular senescence.

Abstract B254: Targeted disruption of the EZH2-EED complex inhibits EZH2-dependent cancer.

Abstract Enhancer of zeste homolog 2 (EZH2) is the histone lysine N-methyltransferase component of the Polycomb repressive complex 2 (PRC2), which, in conjunction with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog(SUZ12), regulates cell lineage determination and homeostasis. Enzymatic hyperactivity of EZH2 has been linked to aberrant repression of tumor suppressor genes in diverse cancers. Here, we report the development of stabilized a-helix of EZH2 (SAH-EZH2) peptides that selectively inhibit H3 Lys27 trimethylation by dose-responsively disrupting the EZH2-EED complex and reducing EZH2 protein levels, a mechanism distinct from that reported for small-molecule EZH2 inhibitors targeting the enzyme catalytic domain. The essential α-helical domain of EZH2 (residues 40-68) that engages EED established the basis for designing hydrocarbon-stapled derivatives to disrupt the specific protein interaction. Non-natural amino acids with olefinic side chains were substituted at (i, i + 4) positions within the EZH2 α-helical sequence, followed by ruthenium-catalyzed olefin metathesis, to yield SAH-EZH2 peptides. Our lead, cell-permeable analog effectively targeted EED in situ, dissociated the EZH2-EED complex and impaired the function of PRC2, as reflected by reduction of the H3K27me3 and EZH2 protein levels, and increased expression of PRC2-regulated cell differentiation marker genes. In response to SAH-EZH2 treatment, murine MLL-AF9 leukemia cells, which are PRC2-dependent, manifested growth arrest and monocyte-macrophage differentiation, in accordance with the phenotypic consequences of shRNA-mediated reduction of EZH2 (or EED) or genetic ablation of Ezh2 (or Eed). In evaluating SAH-EZH2 activity alongside the small-molecule EZH2 inhibitor GSK126 in a series of B-cell lymphoma lines, we observed sequence-specific antiproliferative responses, transcriptional modulation, cellular specificity and a distinct mechanism of action for SAH-EZH2. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B254. Citation Format: Woojin Kim, Gregory H. Bird, Tobias Neff, Guoji Guo, Marc A. Kerenyi, Loren D. Walensky, Stuart H. Orkin. Targeted disruption of the EZH2-EED complex inhibits EZH2-dependent cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B254.

Early activation of nSMase2/ceramide pathway in astrocytes is involved in ischemia-associated neuronal damage via inflammation in rat hippocampi

BackgroundCeramide accumulation is considered a contributing factor to neuronal dysfunction and damage. However, the underlying mechanisms that occur following ischemic insult are still unclear.MethodsIn the present study, we established cerebral ischemia models using four-vessel occlusion and oxygen-glucose deprivation methods. The hippocampus neural cells were subjected to immunohistochemistry and immunofluorescence staining for ceramide and neutral sphingomyelinase 2 (nSMase2) levels; immunoprecipitation and immunoblot analysis for nSMase2, receptor for activated C kinase 1 (RACK1), embryonic ectoderm development (EED), p38 mitogen-activated protein kinase (p38MAPK) and phosphorylated p38MAPK expression; SMase assay for nSMase and acid sphingomyelinase (aSMase) activity; real-time reverse transcription polymerase chain reaction for cytokine expression; and Nissl, microtubule-associated protein 2 and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining.ResultsWe found considerable production of ceramide in astrocytes, but not in neurons, during early cerebral ischemia. This was accompanied by the induction of nSMase (but not aSMase) activity in the rat hippocampi. The inhibition of nSMase2 activity effectively reduced ceramide accumulation in astrocytes and alleviated neuronal damage to some extent. Meanwhile, the expression levels of proinflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6, were found to be upregulated, which may have played an import role in neuronal damage mediated by the nSMase2/ceramide pathway. Although enhanced binding of nSMase2 with RACK1 and EED were also observed after cerebral ischemia, nSMase2 activity was not blocked by the TNF-α receptor inhibitor through RACK1/EED signaling. p38MAPK, but not protein kinase Cζ or protein phosphatase 2B, was able to induce nSMase2 activation after ischemia. p38MAPK can be induced by A2B adenosine receptor (A2BAR) activity.ConclusionsThese results indicate that the inhibition of ceramide production in astrocytes by targeting A2BAR/p38MAPK/nSMase2 signaling may represent a viable approach for attenuating inflammatory responses and neuronal damage after cerebral ischemia.

Targeted disruption of the EZH2–EED complex inhibits EZH2-dependent cancer

Enhancer of zeste homolog2 (EZH2) is the histone lysine N-methyltransferase component of the Polycomb repressive complex 2 (PRC2), which in conjunction with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12), regulates cell lineage determination and homeostasis. Enzymatic hyperactivity has been linked to aberrant repression of tumor suppressor genes in diverse cancers. Here, we report the development of stabilized alpha-helix of EZH2 (SAH-EZH2) peptides that selectively inhibit H3 Lys27 trimethylation by dose-responsively disrupting the EZH2/EED complex and reducing EZH2 protein levels, a mechanism distinct from that reported for small molecule EZH2 inhibitors targeting the enzyme catalytic domain. MLL-AF9 leukemia cells, which are dependent on PRC2, undergo growth arrest and monocyte/macrophage differentiation upon treatment with SAH-EZH2, consistent with observed changes in expression of PRC2-regulated, lineage-specific marker genes. Thus, by dissociating the EZH2/EED complex, we pharmacologically modulate an epigenetic “writer” and suppress PRC2-dependent cancer cell growth.

PINK1 regulates histone H3 trimethylation and gene expression by interaction with the polycomb protein EED/WAIT1

Mutations in PTEN-induced putative kinase 1 (PINK1) gene are associated to early-onset recessive forms of Parkinson disease. PINK1 function is related to mitochondria homeostasis, but the molecular pathways in which PINK1 is involved are largely unknown. Here, we report the identification of the embryonic ectoderm development polycomb histone-methylation modulator (EED/WAIT1) as a PINK1-interacting and -regulated protein. The PINK1:EED/WAIT1 physical interaction was mediated by the PINK1 kinase domain and the EED/WAIT1 40 amino acid ending with tryptophan and aspartate (WD40)-repeat region, and PINK1 phosphorylated EED/WAIT1 in vitro. PINK1 associated with EED/WAIT1 in cells and relocated EED/WAIT1 to the mitochondria. This interaction reduced the trimethylation of lysine 27 from histone H3, which affected polycomb-regulated gene transcription during RA differentiation of SH-SY5Y human neuroblastoma cells. Our findings unveil a pathway by which PINK1 regulates histone methylation and gene expression through the polycomb repressor complex.

MicroRNA-323-3p Regulates the Activity of Polycomb Repressive Complex 2 (PRC2) via Targeting the mRNA of Embryonic Ectoderm Development (Eed) Gene in Mouse Embryonic Stem Cells

PRC2 (Polycomb repressive complex 2) mediates epigenetic gene silencing by catalyzing the triple methylation of histone H3 Lys-27 (H3K27me3) to establish a repressive epigenetic state. PRC2 is involved in the regulation of many fundamental biological processes and is especially essential for embryonic stem cells. However, how the formation and function of PRC2 are regulated is largely unknown. Here, we show that a microRNA encoded by the imprinted Dlk1-Dio3 region of mouse chromosome 12, miR-323-3p, targets Eed (embryonic ectoderm development) mRNA, which encodes one of the core components of PRC2, the EED protein. Binding of miR-323-3p to Eed mRNA resulted in reduced EED protein abundance and cellular H3K27me3 levels, indicating decreased PRC2 activity. Such regulation seems to be conserved among mammals, at least between mice and humans. We demonstrate that induced pluripotent stem cells with varied developmental abilities had different miR-323-3p as well as EED and H3K27me3 levels, indicating that miR-323-3p may be involved in the regulation of stem cell pluripotency through affecting PRC2 activity. Mouse embryonic fibroblast cells had much higher miR-323-3p expression and nearly undetectable H3K27me3 levels. These findings identify miR-323-3p as a new regulator for PRC2 and provide a new approach for regulating PRC2 activity via microRNAs.

EED Gene Polymorphism in Patients with Colorectal Cancer

Our previous work indicated that, first, the embryonic ectoderm development (EED) gene is a candidate gene associated with the pathogenesis of ulcerative colitis (UC) and, second, that the haplotypes of the EED polymorphism are one of the markers for UC susceptibility. The risk of developing colorectal cancer (CRC) increases in patients with inflammatory bowel disease. The present study aimed at determining the association between polymorphisms in the EED gene and CRC. Genotype analysis of EED single nucleotide polymorphisms (SNPs) was performed with high-resolution melting analysis, and the genotype and allele frequencies of the EED SNPs were compared between CRC patients and healthy controls. The haplotype frequencies of EED for multiple loci were estimated using the expectation maximization (EM) algorithm. Our study had a power of 76.6% at a 0.05 significance level. Genotype and allele frequencies of the SNPs and haplotype frequencies of the EED gene in CRC patients were not significantly different from those in healthy controls. Only the allele frequency of g.-1850G>C in the rectal cancer (RC) patient group was significantly different from that of the control group (p=0.04). Similarly, the genotype and allelic frequencies of the EED SNPs for either tumor site (left or right) or tumor stage were not significantly different from those in healthy controls. However, our data show an association between the g.-993G>C polymorphism in the EED gene and the presence of lymph node metastasis in CRC. These results suggest that the SNPs of the EED gene might not be associated with susceptibility to CRC. However, this study shows that the allele frequency of g.-1850G>C in the RC patient group was significantly different from that in the control group (p=0.04) and that g.-993G>C may play a role in the lymph node metastatic process of CRC.

EED and KDM6B Coordinate the First Mammalian Cell Lineage Commitment To Ensure Embryo Implantation

The first mammalian cell lineage commitment is the formation of the trophectoderm (TE) and the inner cell mass (ICM) lineages during preimplantation development. Proper development of the TE and ICM lineages is dependent upon establishment of specific transcriptional programs. However, the epigenetic mechanisms that functionally contribute to establish TE- and ICM-specific transcriptional programs are poorly understood. Here, we show that proper development of the TE and ICM lineages is coordinated via combinatorial regulation of embryonic ectoderm development (EED) and lysine-specific demethylase 6B (KDM6B). During blastocyst formation, the relative levels of EED and KDM6B expression determine altered polycomb repressor 2 (PRC2) complex recruitment and incorporation of the repressive histone H3 lysine 27 trimethylation (H3K27Me3) mark at the chromatin domains of TE-specific master regulators CDX2 and GATA3, leading to their activation in the TE lineage and repression in the ICM lineage. Furthermore, ectopic gain of EED along with depletion of KDM6B in preimplantation mouse embryos abrogates CDX2 and GATA3 expression in the nascent TE lineage. The loss of CDX2 and GATA3 in the nascent TE lineage results in improper TE development, leading to failure in embryo implantation to the uterus. Our study delineates a novel epigenetic mechanism that orchestrates proper development of the first mammalian cell lineages.

Abstract IA08: Targeting histone methylation in leukemia

Abstract Leukemias harboring rearrangements of the MLL gene carry a poor prognosis. Over the past 6 years, it has become increasingly clear that fusions of MLL induce widespread epigenetic deregulation that may mediate much of their transforming activity. The histone methyltransferase DOT1L, which methylates histone 3 on lysine 79 (H3K79), has received particular attention. Genome-wide H3K79 methylation profiles in MLL-rearranged leukemias are abnormal, and can serve to distinguish MLL-rearranged from other types of leukemias. Loss of H3K79 methylation affects expression of MLL-target loci and is detrimental to the leukemogenic activity of MLL-rearranged cells, suggesting that a DOT1L dependent, aberrant epigenetic program drives transformation in these leukemias. Small molecule DOT1L inhibitors have been developed and selectively inhibit proliferation of MLL-rearranged leukemia cells. These molecules are now in clinical trials for patients with relapsed refractory leukemia. Also, the Polycomb Repressive Complex 2 (PRC2) has been implicated in self-renewal and cancer progression, and its components are overexpressed in many cancers, but its role in cancer development and progression remains unclear. We have used conditional alleles for the PRC2 components Enhancer of Zeste 2 (Ezh2) and Embryonic Ectoderm Development (Eed) to characterize the role of PRC2 function in leukemia development and progression. Compared to wildtype leukemia, Ezh2-null MLL-AF9-mediated acute myeloid leukemia (AML) show decreased organ infiltration and failed to accelerate upon secondary transplantation. However, Ezh2-null leukemias maintained self-renewal up to the third round of transplantation, indicating that Ezh2 is not strictly required for MLL-AF9 AML. In contrast, inactivation of the essential PRC2 gene, Eed led to complete ablation of PRC2-function, which is incompatible with leukemia growth. Gene expression array analyses indicated more profound gene expression changes in Eed-null compared to Ezh2-null leukemic cells, including downregulation of Myc target genes and upregulation of PRC2 targets. These data show that histone-modifying enzymes play critical roles in leukemia and may be relevant therapeutic targets in these diseases. Citation Format: Scott A. Armstrong. Targeting histone methylation in leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA08.

CPEB1, a histone-modified hypomethylated gene, is regulated by miR-101 and involved in cell senescence in glioma

Epigenetic mechanisms have important roles in carcinogenesis. We certified that the mRNA translation-related gene cytoplasmic polyadenylation element-binding protein 1 (CPEB1) is hypomethylated and overexpressed in glioma cells and tissues. The knockdown of CPEB1 reduced cell senescence by regulating the expression or distribution of p53 in glioma cells. CPEB1 is also regulated directly by the tumor suppressor miR-101, a potential marker of glioma. It is known that the histone methyltransferase enhancer of zeste homolog 2 (EZH2) and embryonic ectoderm development (EED) are direct targets of miR-101. We demonstrated that miR-101 downregulated the expression of CPEB1 through reversing the methylation status of the CPEB1 promoter by regulating the presence on the promoter of the methylation-related histones H3K4me2, H3K27me3, H3K9me3 and H4K20me3. The epigenetic regulation of H3K27me3 on CPEB1 promoter is mediated by EZH2 and EED. EZH2 has a role in the regulation of H3K4me2. Furthermore, the downregulation of CPEB1 induced senescence in a p53-dependent manner.

Mo1155 The Polymorphisms of Embryonic Ectoderm Development Gene in Colorectal Cancer

Mo1155 The Polymorphisms of Embryonic Ectoderm Development Gene in Colorectal Cancer

Abstract 5360: Targeting H3K27 Methylation by 3-Deazaneplanocin A (DZNep) in lung and esophageal cancers.

Abstract Polycomb repressor complex-2 (PRC-2) is aberrantly expressed in a variety of human malignancies including lung and esophageal cancers. PRC-2 contains three main core proteins, enhancer of zeste 2 (EZH2), suppressor of zeste 12 (SUZ12), and embryonic ectoderm development (EED). In the present study, we sought to examine the effects of PRC-2 expression in lung and esophageal cancers, and to determine if PRC-2 is a potential therapeutic target in these malignancies. qRT-PCR analysis demonstrated significant over-expression of EZH2, SUZ12, and EED in cultured lung and esophageal cancer cells as well as primary tumors relative to normal or immortalized aerodigestive tract epithelial cells, or normal lung or esophageal tissues. Over-expression of individual PRC-2 components appeared to coincide with tumor histology; for example EZH2 was particularly over-expressed in small cell lung cancers, whereas EED appeared to be over-expressed primarily in esophageal cancers. DZNep mediated dose-dependent depletion of EZH2, EED and SUZ12, with corresponding decreases in global H3K27Me3 levels in cultured lung and esophageal cancer cells. Depletion of PRC-2 expression/activity coincided with growth arrest in these cancer cells. The growth inhibitory effects of DZNep in lung cancer cells were recapitulated byshRNA-mediated knock-down of EZH2 in these cells. DZNep induced expression of Dkk-1, a putative tumor suppressor that we have previously reported to be repressed by polycomb-mediated mechanisms in lung cancer cells and cultured normal respiratory epithelia following exposure to cigarette smoke condensate (CSC). Chromatin immunoprecipitation (ChIP) experiments demonstrated that DZNep-mediated activation of Dkk-1 coincided with decreased levels of H3K27Me3 within the Dkk-1 promoter. qRT-PCR and immunoblot experiments revealed that exposure of lung cancer cells to DZNep before or concurrent with CSC abrogated tobacco-mediated repression of Dkk-1 in vitro. Subsequent experiments demonstrated that daily intra-peritoneal (IP) injections of CSC significantly decreased Dkk-1 gene expression in subcutaneous A549 and Calu-6 xenografts, and enhanced growth of these tumors in nude mice. IP administration of DZNep (2.5 mg/kg BID q4 days every 7 days) significantly abrogated the enhancement effect of cigarette smoke on growth of lung cancer xenografts; this phenomenon coincided with decreased EZH2 and increased Dkk-1 expression in these xenografts. Collectively, these findings support development of DZNep for inhibiting PRC-2 expression in thoracic malignancies. Citation Format: Mahadev Rao, Young Hong, Scott Atay, Trevor Upham, Julie Hong, Nicole Datrice, Mary Zhang, Enrique Zudaire, Victor Marquez, David Schrump. Targeting H3K27 Methylation by 3-Deazaneplanocin A (DZNep) in lung and esophageal cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5360. doi:10.1158/1538-7445.AM2013-5360

Regional control of histone H3 lysine 27 methylation in Neurospora

Trimethylated lysine 27 on histone H3 (H3K27me3) is present in Drosophila, Arabidopsis, worms, and mammals, but is absent from yeasts that have been examined. We identified and analyzed H3K27me3 in the filamentous fungus Neurospora crassa and in other Neurospora species. H3K27me3 covers 6.8% of the N. crassa genome, encompassing 223 domains, including 774 genes, all of which are transcriptionally silent. N. crassa H3K27me3-marked genes are less conserved than unmarked genes and only ∼35% of genes marked by H3K27me3 in N. crassa are also H3K27me3-marked in Neurospora discreta and Neurospora tetrasperma. We found that three components of the Neurospora Polycomb repressive complex 2 (PRC2)--[Su-(var)3-9; E(z); Trithorax] (SET)-7, embryonic ectoderm development (EED), and SU(Z)12 (suppressor of zeste12)--are required for H3K27me3, whereas the fourth component, Neurospora protein 55 (an N. crassa homolog of p55/RbAp48), is critical for H3K27me3 only at subtelomeric domains. Loss of H3K27me3, caused by deletion of the gene encoding the catalytic PRC2 subunit, set-7, resulted in up-regulation of 130 genes, including genes in both H3K27me3-marked and unmarked regions.

Functional characterization of EZH2β reveals the increased complexity of EZH2 isoforms involved in the regulation of mammalian gene expression

BackgroundHistone methyltransferase enhancer of zeste homologue 2 (EZH2) forms an obligate repressive complex with suppressor of zeste 12 and embryonic ectoderm development, which is thought, along with EZH1, to be primarily responsible for mediating Polycomb-dependent gene silencing. Polycomb-mediated repression influences gene expression across the entire gamut of biological processes, including development, differentiation and cellular proliferation. Deregulation of EZH2 expression is implicated in numerous complex human diseases. To date, most EZH2-mediated function has been primarily ascribed to a single protein product of the EZH2 locus.ResultsWe report that the EZH2 locus undergoes alternative splicing to yield at least two structurally and functionally distinct EZH2 methyltransferases. The longest protein encoded by this locus is the conventional enzyme, which we refer to as EZH2α, whereas EZH2β, characterized here, represents a novel isoform. We find that EZH2β localizes to the cell nucleus, complexes with embryonic ectoderm development and suppressor of zeste 12, trimethylates histone 3 at lysine 27, and mediates silencing of target promoters. At the cell biological level, we find that increased EZH2β induces cell proliferation, demonstrating that this protein is functional in the regulation of processes previously attributed to EZH2α. Biochemically, through the use of genome-wide expression profiling, we demonstrate that EZH2β governs a pattern of gene repression that is often ontologically redundant from that of EZH2α, but also divergent for a wide variety of specific target genes.ConclusionsCombined, these results demonstrate that an expanded repertoire of EZH2 writers can modulate histone code instruction during histone 3 lysine 27-mediated gene silencing. These data support the notion that the regulation of EZH2-mediated gene silencing is more complex than previously anticipated and should guide the design and interpretation of future studies aimed at understanding the biochemical and biological roles of this important family of epigenomic regulators.

Abstract P2-09-06: The structure design and biological activities of inhibitory peptides, which block the interactions among polycomb repressive complex 2

Abstract Polycomb repressive complex (PRC2) contains several proteins, including embryonic ectoderm development (EED), suppressor of zeste 12 (SUZ12) and enhancer of zeste homolog 2 (EZH2). Excessive EZH2 concentrations have been reported as a marker of aggressive breast cancer and associated with invasion and cancer progression. EZH2 levels were elevated in patients with invasive breast carcinoma relative to normal or atypical hyperplasia. Except for the C-terminal SET domain, which functions as a histone-lysine N-methyltransferases, EZH2 has an N-terminal alpha helix region, which forms tight complex with EED protein. The in vivo enzymatic activity of EZH2 relies on and be tightly regulated by the interaction with EED. We designed series of artificial peptides trying to block the interaction between EZH2 and EED. By using label-free surface plasmon resonance (SPR) technology, the dynamic binding capacities of these peptides were tested. There were 3 leading structures standing out from the screening of more 80 peptides. After several round of co-crystal structure based optimization, the binding capacity of one inhibitor was reached to several nM levels, which indicated the availability for being a drug candidate. Finally, the biological activities of the inhibitory peptides were tested in several breast cancer cell lines. The PRC2 inhibitory peptides in this study are the first time reported PRC2 targeting epigenetic molecules with biological activity in breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-09-06.