Mapping of EZH2-WT and EZH2-mTP5 binding sites to human promoter regions
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE39593 | GEO | 2012/11/13
SECONDARY ACCESSION(S): PRJNA171177
REPOSITORIES: GEO
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Project description:To gain genome wide information on the association of EZH2 with promoter regions in HeLa cells, DamID experiments and subsequent analysis by promoter arrays (Affymetrix GeneChip Human Promoter 1.0R ) were performed. The DamID method uses fusions of the bacterial Dam DNA methylase and the protein of interest, to direct the enzymatic activity to the proteinM-bM-^@M-^Ys genomic binding sites, where the DNA is methylated. Methylated DNA is then extracted, enriched and further analysed by microarray. EZH2 is the enzymatic subunit of the Polycomb Repressive Complex 2, which deposits the H3K27me3 mark on chromatin. This mark is associated with low gene expression, either in polycomb repressed regions or, in combination with methylation of H3K4, at poised promoters. An EZH2-T416A mutant (EZH2-mTP5) fails to bind to NIPP1, a factor implied in the regulation of PRC2 binding to a subset of target regions. To obtain a genome wide picture of differential binding of EZH2-WT and the EZH2-mTP5 mutant to promoter regions, the mutant was subjected to DamID/microanalysis as well. DamID of EZH2-WT (2 replicates) and EZH2-mTP5(T416A)(2 replicates) vs. control (Dam without fused protein)(4 samples)
2012-11-13 | E-GEOD-39593 | biostudies-arrayexpress
Project description:To gain unbiased insight into the association of NIPP1 with regulatory, coding and intergenic regions of the human genome, DamID experiments and subsequent analysis by ENCODE arrays were performed. The DamID method uses fusions of the bacterial Dam DNA methylase and the protein of interest, to direct the enzymatic activity to the protein’s genomic binding sites, where the DNA is methylated. Methylated DNA is then extracted, enriched and further analysed by microarray. The PP1 (Protein Phosphatase 1) interacting protein NIPP1 has been implicated to play a role in regulation of gene expression through PRC2 function, but also has a function in splicing. The array analysis was performed to obtain a general picture of the sites of chromatin association of NIPP1 and specifically their spatial relation to coding regions as well as to identify target regions to be analysed in more detail. In addition, a PP1 binding deficient mutant (NIPP1-RATA) was analysed, thus allowing to distinguish PP1 dependent and independent sites of association with the genome by microarray analysis. NIPP1-WT is referred to as FDN and the mutant referred to as RATA in the following descriptions.
2010-07-19 | GSE22123 | GEO
Project description:Overexpression of EZH2 in estrogen receptor negative (ER-) breast cancer promotes metastasis. EZH2 has been mainly studied as the catalytic component of the Polycomb Repressive Complex 2 (PRC2) that mediates gene repression by trimethylating histone H3 at lysine 27 (H3K27me3). However, how EZH2 drives metastasis despite the low H3K27me3 levels observed in ER- breast cancer is unknown. We have shown that in human invasive carcinomas and distant metastases, cytoplasmic EZH2 phosphorylated at T367 is significantly associated with ER- disease and low H3K27me3 levels. Here, we explore the interactome of EZH2 and of a phosphodeficient mutant EZH2_T367A. We identified novel interactors of EZH2, and identified interactions that are dependent on the phosphorylation and cellular localization of EZH2 that may play a role in EZH2 dependent metastatic progression.
2018-07-03 | PXD010073 | Pride
Project description:NIPP1 and EZH2 contribute to the silencing of a common set of genes. Since both NIPP1 and EZH2 turned out to be essential for the initiation and maintenance of global H3K27 trimethylation, a key step in the PcG-mediated transcriptional regulation of genes, we have subsequently examined by DNA-microarray analyses whether the loss of NIPP1 or EZH2 results in the expected regulation of a common set of genes. <br> <br> PC-3 cells were transfected in four independent experiments with a control siRNA or siRNA duplexes for the knockdown of EZH2 or NIPP1. To minimize indirect effects, RNA was already isolated 48h after transfection. At this time, the knockdown of NIPP1 or EZH2 was verified by quantitative qRT-PCR and amounted to 73 ± 7% and 82 ± 3% (n = 4), respectively. The isolated RNA pools were labeled with a fluorescent dye and were hybridized onto whole human genome Agilent gene chips. These chips contained 44,000 60-mer oligonucleotides, corresponding to ca 27,000 unambiguously annotated genes. Using SAM (significance analysis of microarray) analysis (P < 0.05), performed by comparing the NIPP1 or EZH2 knockdown against RNAi control, we selected 1581 and 2024 genes as significant in the NIPP1 or EZH2 knockdown, respectively. Using p<0.01, the loss of NIPP1 or EZH2 resulted in a significant upregulation of 281 genes and 319 genes, respectively, and downregulation of 409 and 175 genes respectively ). The knockdown of NIPP1 or EZH2 was confirmed in the microarray and the results of the expression array data were confirmed by qRT-PCR on a selection of affected genes, representing the entire range of fold-changes and using samples independent of those used for the microarray analysis.<br> <br> Strikingly, the list of the 50 genes that were most upregulated (SAM test, p<0.01) by the knockdown of NIPP1 were, in most cases, also upregulated following the knockdown of EZH2. In contrast, there was no obvious correlation between the 50 genes that were most downregulated after the knockdown of NIPP1 or EZH2. More detailed analysis revealed that 43% (121 genes) of the genes that were upregulated after the knockdown of NIPP1 were also upregulated by the loss of EZH2. In contrast, only 6% (25 genes) of the genes that were downregulated by the knockdown of NIPP1 were also downregulated by the loss of EZH2. Using another approach and using the list of significant genes based upon SAM test and p<0.05, we noted a significant linear correlation (R= 0.59 ± 0.01) between the extent to which genes were significantly upregulated by the loss of NIPP1 and the changes in the expression of these same genes following the knockdown of EZH2. No such correlation (R= 0.01 ± 0.01) was obtained for genes that were significantly downregulated. In the residual plot of these regressions, an obvious bend became apparent between the up and downregulated genes of the NIPP1 knockdown, confirming that we are in fact dealing with two differently regulated subpopulations. To validate this subdivision, the microarray data for a knockdown of EZH2, EED or SUZ12 which represent the core of the PRC2 complex, recently published by Bracken et al. ( 2006 ), were identically analyzed. In the residual plots, a bend between the up and downregulated genes was consistently present. We can conclude from our microarray data that NIPP1 silence a same set of genes as EZH2 and that NIPP1 also act as a transcriptional activator independent of EZH2.
2007-08-21 | E-TABM-128 | biostudies-arrayexpress
Project description:Diffuse Intrinsic Pontine Glioma (DIPG) is a rare and highly aggressive pediatric tumor. The average survival time after diagnosis is less than one year. Currently, there are no effective treatments. Characteristic of DIPG is a mutation in histone H3 which leads to a substitution of Lysine 27 to Methionine (H3K27M) which deregulates Polycomb Repressive Complex 2 (PRC2), including enzymatic activity of EZH2. Previous studies have shown that inhibition of EZH2 by chemical agents decreases DIPG cell proliferation and inhibits tumor growth in vivo. My thesis project aims to confirm that EZH2 could be a therapeutic target using chemical EZH2 inhibitors, small interfering RNAs and a CRISPR/Cas9 approach in a series of DIPG tumor cell lines and to determine underlying molecular mechanisms of action.
2022-04-01 | PXD017525 | Pride
Project description:Rap1GAP is a critical tumor suppressor gene that is down-regulated in multiple aggressive cancers such as head and neck squamous cell carcinoma, melanoma and pancreatic cancer. However, the mechanistic basis of rap1GAP down-regulation in cancers is poorly understood. By employing an integrative approach, we demonstrate polycomb-mediated repression of rap1GAP that involves EZH2, a histone methyltransferase in head and neck cancers. We further concomitant down-regulation of rap1GAP in head and neck cancers. EZH2 represses rap1GAP by facilitating the trimethylation of H3K27, a mark of gene repression, and also hypermethylation of rap1GAP promoter. These results provide a conceptual framework involving a microRNA-oncogene-tumor suppressor axis to understand head and neck cancer progression. OSCC3-siRNA Non-Targeting Vs. siRNA EZH2 with dye-swap, HOK-Adeno CMV Vs. Adeno EZH2.
2011-11-02 | E-GEOD-28501 | biostudies-arrayexpress
Project description:The Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) is an essential epigenetic modifier able to methylate lysine 27 on histone H3 (H3K27) to induce chromatin compaction, protein complex recruitment and ultimately transcriptional repression. Hematologic malignancies, including Diffuse Large B cell lymphoma (DLBCL) and Acute myeloid leukemia (AML) have shown a high EZH2-mutation frequency (>20%) associated with poor clinical outcomes. Particularly, two distinct oncogenic mutations, so-called gain-of-function (Y641F and A677G) and loss-of-function (H689A and F667I) are found in the catalytic domain of EZH2. In this study, a comprehensive multi-omics approach was employed to characterize downstream effects of H3K27me3 deposition driven by EZH2 mutations. Human embryonic kidney cells (HEK293T) were transfected to generate three stable EZH2 mutants: EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I), which were validated via immunoblotting and DIA-MS-based histone profiling assay. The histone profiling assay demonstrated a significant increase of approximately two-fold in H3.1/H3.3K27me3 for Y641F EZH2 mutant. There was a modest increase in the combinatorial PTMs H3.1/H3.3K27me3K36me1 and a significant depletion in H3.1 and H3.3 K27me2. The most depleted peptide was H3.3K27me2K36me2. For the A677G EZH2 mutant, the assay demonstrated an enrichment on H3.1/H3.3K27me3, combinatorial K27me3K36me1 and a slight increase in K27me1 and K18ac but only on H3.1. The most depleted peptide was H3.3K27me2K36me2. The H689A/F667I cell line has the most alterations in global histone PTMs. The most highly enriched were H3.1/H3.3K36me2, H3.1K9me3 and H3.3K36me1. The most depleted modifications were H3.1K23ac and H3.1K27me3.
2023-10-24 | PXD042953 | Pride
Project description:Polycomb group (PcG) proteins are transcriptional repressors important to maintain cell identity during embryonic development. Ezh2, the catalytic subunit of the Polycomb Repressive Complex 2, is responsible for placing the epigenetic repressive mark histone H3 lysine 27 trimethylation (H3K27me3). In contrast to results in mouse models, zebrafish embryos mutant for both maternal and zygotic ezh2 (MZezh2) can form a normal body plan at 1 day post fertilization (dpf) but die at 2 dpf, exhibiting pleiotropic phenotypes. To elucidate the specificity of PcG-mediated repression during early zebrafish development, we conducted in depth analysis of the transcriptome, epigenome, and proteome of the MZezh2 mutant embryos at 1 dpf. We found that, despite modifications in the epigenetic landscape, transcriptome and proteome analysis revealed only minor changes in gene and protein expression levels.
2019-09-09 | PXD010922 | Pride
Project description:Polycomb protein complexes are critical for stem cell renewal and tumorigenesis. Some components of Polycomb repressive complex 2 (PRC2) have been frequently found to be overexpressed in a variety of human cancers and contribute to tumor initiation and development partially by transcriptional silencing of tumor suppressor genes. However, the exact underlying mechanisms still remain elusive. To further characterize PRC2-directed transcriptome and explore its implication in tumorigenesis, here we performed a genome-wide profiling of EZH2-associated, a key component of PRC2, transcriptome in human gastric cancer cell lines by RNA immunoprecipitation sequencing (RIP-seq). We observed that the EZH2-interacting transcripts are distributed in protein-coding, intergenic, intronic and promoter regions and transcribed from both strands. Many transcripts are originated from cancer-related loci and transcribed from active promoters. Particularly, we demonstrated that EZH2-associated lncRNA MALAT1 tethers EZH2 to PCDH10 promoter for its transcriptional repression and contributes to gastric cellular invasion and migration.
2016-02-23 | E-MTAB-3767 | biostudies-arrayexpress
Project description:Ezh2 protein is the enzymatic component of the Polycomb Repressive Complex (PRC)-2, which represses its target genes by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and differentiation during embryonic development. Recently, hot-spot mutations of Ezh2 have been identified in diffused large B cell lymphomas (DLBCLs) and follicular lymphomas (FLs). To investigate if tumor growth is dependent on the enzymatic activity of Ezh2, we have developed a potent and selective small molecule inhibitor, EI1, which inhibits the enzymatic activity of Ezh2 through direct binding and competing with the methyl group donor S-Adenosyl methionine (SAM). EI1-treated cells exhibit genome-wide loss of H3K27 methylation. Furthermore, inhibition of Ezh2 by EI1 in DLBCL cells carrying the Y641 mutations results in decreased proliferation, cell cycle arrest and apoptosis. These results provide strong validation of Ezh2 as a potential therapeutic target for the treatment of cancer with the Ezh2 mutation. In this microarray experiment we examined the global gene expression changes in DLBCL cell line Karpas422 (Ezh2Y641N) after EI1 treatment for up to 6 days. Total RNA was prepared from KARPAS422 at 1 day, 2 days, 3 days, and 6 days after DMSO or EZH2 inhibitor (5 uM) treatment with biological triplicates and hybridized to Affimetrix U133 Plus 2 microarray chips according to the manufacturers protocol (Affymetrix, CA).
2012-10-04 | E-GEOD-41315 | biostudies-arrayexpress