Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M||IP, WB, ChIP-seq||Rb||Serum||Polyclonal Antibody|
|Application||This Anti-EZH2 Antibody is validated for use in Immunoprecipitation and Western Blotting and ChIP-seq for the detection of EZH2.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||stable for 2 years at -20°C from date of shipment|
|Material Size||200 µL|
|Anti-EZH2 (Enhancer of Zeste Homologue 2) - 1982631||1982631|
|Anti-EZH2 (Enhancer of Zeste Homologue 2) - DAM1578238||DAM1578238|
|Anti-EZH2 (Enhancer of Zeste Homologue 2) - DAM1588132||DAM1588132|
|Anti-EZH2 (Enhancer of Zeste Homologue 2) - DAM1598793||DAM1598793|
|Anti-EZH2 (Enhancer of Zeste Homologue 2) - DAM1614932||DAM1614932|
|Anti-EZH2 (Enhancer of Zeste Homologue 2) - DAM1632086||DAM1632086|
|Anti-EZH2 (Enhancer of Zeste Homologue 2) - DAM1731502||DAM1731502|
|Anti-EZH2 (Enhancer of Zeste Homologue 2) - DAM1795387||DAM1795387|
|Anti-EZH2 (Enhancer of Zeste Homologue 2) - DAM1834941||DAM1834941|
|Reference overview||Application||Species||Pub Med ID|
|Phospho-ΔNp63α/microRNA network modulates epigenetic regulatory enzymes in squamous cell carcinomas.|
Cell cycle (Georgetown, Tex.) 13 749-61 2014
The tumor protein (TP) p63/microRNAs functional network may play a key role in supporting the response of squamous cell carcinomas (SCC) to chemotherapy. We show that the cisplatin exposure of SCC-11 cells led to upregulation of miR-297, miR-92b-3p, and miR-485-5p through a phosphorylated ΔNp63α-dependent mechanism that subsequently modulated the expression of the protein targets implicated in DNA methylation (DNMT3A), histone deacetylation (HDAC9), and demethylation (KDM4C). Further studies showed that mimics for miR-297, miR-92b-3p, or miR-485-5p, along with siRNA against and inhibitors of DNMT3A, HDAC9, and KDM4C modulated the expression of DAPK1, SMARCA2, and MDM2 genes assessed by the quantitative PCR, promoter luciferase reporter, and chromatin immunoprecipitation assays. Finally, the above-mentioned treatments affecting epigenetic enzymes also modulated the response of SCC cells to chemotherapeutic drugs, rendering the resistant SCC cells more sensitive to cisplatin exposure, thereby providing the groundwork for novel chemotherapeutic venues in treating patients with SCC.
|Western Blotting, Immunoprecipitation||24394434|
|Live cell imaging of the nascent inactive X chromosome during the early differentiation process of naive ES cells towards epiblast stem cells.|
Guyochin, A; Maenner, S; Chu, ET; Hentati, A; Attia, M; Avner, P; Clerc, P
PloS one 9 e116109 2014
Random X-chromosome inactivation ensures dosage compensation in mammals through the transcriptional silencing of one of the two X chromosomes present in each female cell. Silencing is initiated in the differentiating epiblast of the mouse female embryos through coating of the nascent inactive X chromosome by the non-coding RNA Xist, which subsequently recruits the Polycomb Complex PRC2 leading to histone H3-K27 methylation. Here we examined in mouse ES cells the early steps of the transition from naive ES cells towards epiblast stem cells as a model for inducing X chromosome inactivation in vitro. We show that these conditions efficiently induce random XCI. Importantly, in a transient phase of this differentiation pathway, both X chromosomes are coated with Xist RNA in up to 15% of the XX cells. In an attempt to determine the dynamics of this process, we designed a strategy aimed at visualizing the nascent inactive X-chromosome in live cells. We generated transgenic female XX ES cells expressing the PRC2 component Ezh2 fused to the fluorescent protein Venus. The fluorescent fusion protein was expressed at sub-physiological levels and located in nuclei of ES cells. Upon differentiation of ES cell towards epiblast stem cell fate, Venus-fluorescent territories appearing in interphase nuclei were identified as nascent inactive X chromosomes by their association with Xist RNA. Imaging of Ezh2-Venus for up to 24 hours during the differentiation process showed survival of some cells with two fluorescent domains and a surprising dynamics of the fluorescent territories across cell division and in the course of the differentiation process. Our data reveal a strategy for visualizing the nascent inactive X chromosome and suggests the possibility for a large plasticity of the nascent inactive X chromosome.
|Corepressor-dependent silencing of fetal hemoglobin expression by BCL11A.|
Xu, J; Bauer, DE; Kerenyi, MA; Vo, TD; Hou, S; Hsu, YJ; Yao, H; Trowbridge, JJ; Mandel, G; Orkin, SH
Proceedings of the National Academy of Sciences of the United States of America 110 6518-23 2013
Reactivation of fetal hemoglobin (HbF) in adults ameliorates the severity of the common β-globin disorders. The transcription factor BCL11A is a critical modulator of hemoglobin switching and HbF silencing, yet the molecular mechanism through which BCL11A coordinates the developmental switch is incompletely understood. Particularly, the identities of BCL11A cooperating protein complexes and their roles in HbF expression and erythroid development remain largely unknown. Here we determine the interacting partner proteins of BCL11A in erythroid cells by a proteomic screen. BCL11A is found within multiprotein complexes consisting of erythroid transcription factors, transcriptional corepressors, and chromatin-modifying enzymes. We show that the lysine-specific demethylase 1 and repressor element-1 silencing transcription factor corepressor 1 (LSD1/CoREST) histone demethylase complex interacts with BCL11A and is required for full developmental silencing of mouse embryonic β-like globin genes and human γ-globin genes in adult erythroid cells in vivo. In addition, LSD1 is essential for normal erythroid development. Furthermore, the DNA methyltransferase 1 (DNMT1) is identified as a BCL11A-associated protein in the proteomic screen. DNMT1 is required to maintain HbF silencing in primary human adult erythroid cells. DNMT1 haploinsufficiency combined with BCL11A deficiency further enhances γ-globin expression in adult animals. Our findings provide important insights into the mechanistic roles of BCL11A in HbF silencing and clues for therapeutic targeting of BCL11A in β-hemoglobinopathies.
|A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells.|
Johnsson, P; Ackley, A; Vidarsdottir, L; Lui, WO; Corcoran, M; Grandér, D; Morris, KV
Nature structural & molecular biology 20 440-6 2013
PTEN is a tumor-suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1-encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, α and β. The α isoform functions in trans, localizes to the PTEN promoter and epigenetically modulates PTEN transcription by the recruitment of DNA methyltransferase 3a and Enhancer of Zeste. In contrast, the β isoform interacts with PTENpg1 through an RNA-RNA pairing interaction, which affects PTEN protein output through changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell-cycle arrest and sensitizes cells to doxorubicin, which suggests a biological function for the respective PTENpg1 expressed asRNAs.
|Disruption of the MYC-miRNA-EZH2 loop to suppress aggressive B-cell lymphoma survival and clonogenicity.|
Zhao, X; Lwin, T; Zhang, X; Huang, A; Wang, J; Marquez, VE; Chen-Kiang, S; Dalton, WS; Sotomayor, E; Tao, J
Leukemia 27 2341-50 2013
c-MYC (hereafter MYC) overexpression has been recognized in aggressive B-cell lymphomas and linked to adverse prognosis. MYC activation results in widespread repression of micro-RNA (miRNA) expression and associated with lymphoma aggressive progression. Our recent study identified a MYC-miRNA-EZH2 feed-forward loop linking overexpression of MYC, EZH2 and miRNA repression. Here, using a novel small-molecule BET bromodomain inhibitor, JQ1, and the EZH2 inhibitor, DZNep, we demonstrated that combined treatment of JQ1 and DZNep cooperatively disrupted MYC activation, resulting in a greater restoration of miR-26a expression and synergistically suppressed lymphoma growth and clonogenicity in aggressive lymphoma cells. Furthermore, CHIP assay demonstrated that MYC recruited EZH2 to miR-26a promoter and cooperatively repressed miR-26a expression in aggressive lymphoma cell lines, as well as primary lymphoma cells. Loss- or gain-of-function approaches revealed that miR-26a functioned as a tumor suppressor miRNA and mediated the combinatorial effects of JQ1 and DZNep. These findings represent a novel promising approach for silencing MYC-miRNA-EZH2 amplification loop for combinatorial therapy of aggressive B-cell lymphomas.
|Heterogeneity of chromatin modifications in testicular spermatocytic seminoma point toward an epigenetically unstable phenotype.|
Dina G Kristensen,Olga Mlynarska,John E Nielsen,Grete K Jacobsen,Ewa Rajpert-De Meyts,Kristian Almstrup
Cancer genetics 205 2012
Testicular spermatocytic seminoma (SS) is a rare tumor type predominantly found in elderly men. It is thought to originate from spermatogonia and shows cytological and genetic heterogeneity. In this study, we performed for the first time a comprehensive analysis of epigenetic modifications in a series of 36 SS samples. We assessed by immunohistochemistry tumor DNA methylation levels, the expression of methyltransferases DNMT3A, DNMT3B and DNMT3L as well as levels of histone modifications H3K9me2, H3K27me3, H3K4me1, H3K4me2/3, H3K9ac, and H2A.Z. We did not identify any epigenetic marks that matched the pattern of the supposed cell-of-origin, the spermatogonia, and found no correlation between specific marks and the size of the SS cells. The emerging epigenetic picture of SS is a heterogeneous salt-and-pepper-like pattern, with neighboring cells displaying very variable levels of epigenetic marks. We conclude that SS cells display apparent epigenetic heterogeneity and instability, with loss of the organized manner typical for normal germ cell maturation in the adult testis, likely due to the lack of regulatory signals from the absent somatic cell niche.
|Sodium arsenite represses the expression of myogenin in C2C12 mouse myoblast cells through histone modifications and altered expression of Ezh2, Glp, and Igf-1.|
Hong, GM; Bain, LJ
Toxicology and applied pharmacology 260 250-9 2012
Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site of myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic-exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (~2-fold) to the myogenin promoter at the transcription start site (-40 to +42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (-40 to +42).
|ALDH1A1 is a novel EZH2 target gene in epithelial ovarian cancer identified by genome-wide approaches.|
Li, H; Bitler, BG; Vathipadiekal, V; Maradeo, ME; Slifker, M; Creasy, CL; Tummino, PJ; Cairns, P; Birrer, MJ; Zhang, R
Cancer prevention research (Philadelphia, Pa.) 5 484-91 2012
Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy in the United States. EZH2 silences gene expression through trimethylating lysine 27 on histone H3 (H3K27Me3). EZH2 is often overexpressed in EOC and has been suggested as a target for EOC intervention. However, EZH2 target genes in EOC remain poorly understood. Here, we mapped the genomic loci occupied by EZH2/H3K27Me3 using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and globally profiled gene expression in EZH2-knockdown EOC cells. Cross-examination of gene expression and ChIP-seq revealed a list of 60 EZH2 direct target genes whose expression was upregulated more than 1.5-fold upon EZH2 knockdown. For three selected genes (ALDH1A1, SSTR1, and DACT3), we validated their upregulation upon EZH2 knockdown and confirmed the binding of EZH2/H3K27Me3 to their genomic loci. Furthermore, the presence of H3K27Me3 at the genomic loci of these EZH2 target genes was dependent upon EZH2. Interestingly, expression of ALDH1A1, a putative marker for EOC stem cells, was significantly downregulated in high-grade serous EOC (n = 53) compared with ovarian surface epithelial cells (n = 10, P less than 0.001). Notably, expression of ALDH1A1 negatively correlated with expression of EZH2 (n = 63, Spearman r = -0.41, P less than 0.001). Thus, we identified a list of 60 EZH2 target genes and established that ALDH1A1 is a novel EZH2 target gene in EOC cells. Our results suggest a role for EZH2 in regulating EOC stem cell equilibrium via regulation of ALDH1A1 expression.
|PRC1 and PRC2 are not required for targeting of H2A.Z to developmental genes in embryonic stem cells.|
Illingworth, RS; Botting, CH; Grimes, GR; Bickmore, WA; Eskeland, R
PloS one 7 e34848 2012
The essential histone variant H2A.Z localises to both active and silent chromatin sites. In embryonic stem cells (ESCs), H2A.Z is also reported to co-localise with polycomb repressive complex 2 (PRC2) at developmentally silenced genes. The mechanism of H2A.Z targeting is not clear, but a role for the PRC2 component Suz12 has been suggested. Given this association, we wished to determine if polycomb functionally directs H2A.Z incorporation in ESCs. We demonstrate that the PRC1 component Ring1B interacts with multiple complexes in ESCs. Moreover, we show that although the genomic distribution of H2A.Z co-localises with PRC2, Ring1B and with the presence of CpG islands, H2A.Z still blankets polycomb target loci in the absence of Suz12, Eed (PRC2) or Ring1B (PRC1). Therefore we conclude that H2A.Z accumulates at developmentally silenced genes in ESCs in a polycomb independent manner.
|ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression.|
Abdel-Wahab, O; Adli, M; LaFave, LM; Gao, J; Hricik, T; Shih, AH; Pandey, S; Patel, JP; Chung, YR; Koche, R; Perna, F; Zhao, X; Taylor, JE; Park, CY; Carroll, M; Melnick, A; Nimer, SD; Jaffe, JD; Aifantis, I; Bernstein, BE; Levine, RL
Cancer cell 22 180-93 2012
Recurrent somatic ASXL1 mutations occur in patients with myelodysplastic syndrome, myeloproliferative neoplasms, and acute myeloid leukemia, and are associated with adverse outcome. Despite the genetic and clinical data implicating ASXL1 mutations in myeloid malignancies, the mechanisms of transformation by ASXL1 mutations are not understood. Here, we identify that ASXL1 mutations result in loss of polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) tri-methylation. Through integration of microarray data with genome-wide histone modification ChIP-Seq data, we identify targets of ASXL1 repression, including the posterior HOXA cluster that is known to contribute to myeloid transformation. We demonstrate that ASXL1 associates with the PRC2, and that loss of ASXL1 in vivo collaborates with NRASG12D to promote myeloid leukemogenesis.
|White Paper - The Message in the Marks: Deciphering Cancer Epigenetics|