Effects of T350 phospho- on gene repression activity of EZH2
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Shuai Chen
PROVIDER: E-GEOD-20433 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The Polycomb group (PcG) protein enhancer of zeste homolog 2 (EZH2) plays an essential role in histone methylation-mediated epigenetic gene silencing. We demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and 2 (CDK2) bind to and phosphorylate EZH2 at threonine 350 (T350) in an evolutionally conserved motif. T350 phosphorylation is required for EZH2 function in silencing of genes that promote differentiation and inhibit proliferation and migration. Blockage of T350 phosphorylation by generating a T350A mutation largely diminishes the global effect of EZH2 on gene silencing. Four experiments have been designed. BJ cells were transfected with si-Control, si-EZH2, si-EZH2 plus RNAi resistant lentivirus expressed EZH2, or si-EZH2 plus RNAi resistant lentivirus expressed T350A mutant for 60 hours. Each experiment was triplicated.
2010-11-10 | E-GEOD-22427 | biostudies-arrayexpress
Project description:EZH2 is frequently over-expressed in aggressive and metastatic solid tumors, including castration resistant prostate cancer (CRPC). We sought to determine EZH2-dependent gene expression programmes in prostate cancer progression, and found an intriguing functional switch of EZH2 from a repressor to an activator during CRPC development. We used microarrays to detail the global profiling of gene expression that are differentially regulated upon EZH2 depletion in two different prostate cancer cell lines. The androgen-dependent prostate cancer cell line LNCaP and the LNCaP-derived androgen-independent cell line LNCaP-abl (abl) were used for this study, as their transcription profiles strongly resemble that of clinical androgen-dependent and castration resistant prostate tumors, respectively. EZH2 was silenced by specific siRNAs in both cell lines, and total RNA was extracted and hybridized on Affymetrix microarrays.
2012-11-30 | E-GEOD-39452 | biostudies-arrayexpress
Project description:Chromatin modifications are increasingly recognized as a key mechanism in cancer. The histone methyl-transferase Enhancer of Zeste, Drosophila, Homolog 2 (EZH2) is the enzymatic subunit of the polycomb PRC2 complex and methylates histone H3K27, thereby, mediating gene silencing. Down-regulation of EZH2 by RNA interference in ET suppressed oncogenic transformation, tumor development and metastasis in a respective mouse model. Further microarray analysis of EZH2 knock down, or functionally linked HDAC-inhibitor treatment revealed an undifferentiated reversible phenotype in ET maintained by EZH2. EZH2 suppression resulted in a generalized loss of H3K27me3 as well as an increase in H3 acetylation levels. In addition, ChIP-Chip assays for H3K27me3 identified genes that had specifically lost H3K27me3 upon EZH2 silencing. These findings suggested that stemness features are preserved via epigenetic mechanisms. Taken together, the genetic EWS-Fli1 translocation is intimately linked to global and gene specific epigenetic alterations in ET biology. EZH2 mediates neuroectodermal and endothelial embryonal tumor stem cell growth and metastasic spread induced by a translocation derived chimeric transcription factor. Keywords: PNET, Ewing Tumor Epigenetic regulation A673 transiently transfected with siRNA-EZH2 and siRNA-scrambled were harvested and Chromatin-IPs of H3K27me3 or H3 followed by microarray hybridisation were carried out. For detailed procedures see Burdach et al. Epigenetic Maintenance of Stemness and Malignancy in Peripheral Neuroectodermal Tumors by EZH2.
2010-05-18 | E-GEOD-15890 | biostudies-arrayexpress
Project description:Oncogenic EZH2 is overexpressed and extensively involved in the pathophysiology of different cancers including extranodal natural killer/T-cell lymphoma (NKTL). However, the mechanisms regarding EZH2 upregulation is poorly understood, and it still remains untargetable in NKTL. In this study, we examine EZH2 protein turnover in NKTL and identify MELK kinase as a regulator of EZH2 ubiquitination and turnover. Using quantitative mass spectrometry (MS) analysis, we observed a MELK-mediated increase of EZH2 S220 phosphorylation along with a concomitant loss of EZH2 K222 ubiquitination, suggesting a phosphorylation-dependent regulation of EZH2 ubiquitination. MELK inhibition through both chemical and genetic means led to ubiquitination and destabilization of EZH2 protein. Importantly, we determine that MELK is upregulated in NKTL, and its expression correlates with EZH2 protein expression as determined by tissue microarray derived from NKTL patients. Interestingly, FOXM1, which connected MELK to EZH2 signaling in glioma, was not involved in mediating EZH2 ubiquitination. Furthermore, we identify USP36 as the deubiquitinating enzyme which deubiquitinates EZH2 at K222. These findings uncover an important role of MELK and USP36 in mediating EZH2 stability in NKTL. Moreover, MELK overexpression led to decreased sensitivity to Bortezomib treatment in NKTL based on deprivation of EZH2 ubiquitination. Therefore, modulation of EZH2 ubiquitination status by targeting MELK may be a new therapeutic strategy for NKTL patients with poor Bortezomib response.
2019-08-26 | PXD015008 | Pride
Project description:The project aimed at determining whether the Polycomb complex PRC2 has a unique composition in androgen independent prostate cancer cells and the project aimed at determining whether EZH2, the enzymatic subunit of PRC2, retains any functional role in the context of Malignant peripheral nerve sheath tumor (MPNST) where either EED or SUZ12, two essential subunits of PRC2 are inactivated.
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Project description:Enhancer of Zeste Homolog 2 (EZH2) is a histone methyltransferase that catalyzes the trimethylation of histone H3 lysine 27 (H3K27me3). EZH2 expression is significantly upregulated in MPNST. In our study, we investigated the function of EZH2 and the molecular mechanisms that are regulated by EZH2 in MPNST pathogenesis. Our findings enhance the knowledge of EZH2’s function and biology, and have the potential to provide a novel therapeutic approach for MPNST patients in the clinic. An EZH2 knockdown experiment was carried out in MPNST cells. There are one control and one siEZH2 samples for two cell lines (724 and 462) and two repeats for each cell line, so in total there are 8 samples. Two group comparison
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Project description:RAW264.7 cell was stably transfected with Tim-3 siRNA(SI) or negative control(NC) and were analyzed for microRNA expression Differerently expressed microRNA in Tim-3 knockdown RAW264.7 cells were summarized and were used for further analysis RAW264.7 cells stably transfected with Tim-3 siRNA(SI) or negative control(NC) and were used to collect microRNA without any other treatment
2016-01-14 | E-GEOD-76839 | biostudies-arrayexpress
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:The supply of soluble silicon (Si) to plants has been associated with many benefits that remain poorly explained and often contested. In this work, the effect of Si was studied on wheat plants under both control and pathogen stress (Blumeria graminis f.sp. tritici (Bgt)) conditions by conducting an exhaustive transcriptomic analysis (55,000 genes) aimed at comparing the differential response of plants under four treatments. The response to the supply of Si on control (uninfected) plants was limited to 47 genes providing little evidence of regulation of a specific metabolic process. Plants reacted to inoculation with Bgt by an up-regulation of many genes linked to stress and metabolic processes and a down-regulation of genes linked to photosynthesis. Supplying Si to inoculated plants largely prevented disease development, a phenotypic response that translated into a nearly perfect reversal of genes regulated by the effect of Bgt alone. These results suggest that Si plays a limited role on a plantâs metabolism in absence of stress, even in the case of a high-Si accumulating monocot such as wheat. On the other hand, the benefits of Si, in the form of biotic stress alleviation, were remarkably aligned with a counter-response to transcriptomic changes induced by the pathogen Bgt. Experiment Overall Design: Wheat culture in hydroponic system : Experiment Overall Design: Hydroponic systems were used to precisely control Si feeding of plants and to reduce external Si contamination. Seeds of wheat cultivar AC Drummond, chosen for its known high susceptibility to Bgt, were sown in 9-cm pots in a nylon bed consisting of nylon stockings cut into small ribbons. Hydroponic systems were set up to immerse roots for 15 minutes every 30 minutes. The plants were grown in a greenhouse (16 h light at 22°C and 8h dark at 18°C, 80% humidity). Plants were immersed in distilled water only during the first week following sowing. Experiment Overall Design: Si amendment and Bgt inoculation: Experiment Overall Design: Wheat plants were grown in the presence (Si+) or absence (Si-) of Si and inoculated (B+) or not (B-) with Bgt for a total of four treatments: Si-B-, Si+B-, Si-B+, and Si+B+. These treatments were applied in the following manner. One week after sowing, distilled water was replaced by a Hoagland solution amended or not with potassium silicate (Kasil 6, PQ Corp etc) at a concentration of 1.7 mM (Si+ Hoagland). Twice a week, Si was added to the Si+ nutrient solution in order to maintain Si concentration at 1.7 mM. Every other week, both Si- and Si+ Hoagland solutions were renewed and the pH adjusted to 5.8 in all systems at each Si addition or nutrient solution renewal. Experiment Overall Design: Four weeks after sowing (i.e. three weeks after Si amendment started), half the plants were inoculated with Bgt as described previously. Briefly, one day prior to inoculation, reservoir wheat plants heavily infected with Bgt were gently shaken to remove old spores and to stimulate the production of fresh ones. The inoculation was performed by shaking these infected wheat plants over the experimental plants . Experiment Overall Design: RNA preparation: Experiment Overall Design: Total RNA was extracted from leaves of three plants per treatment (three biological replicates) with the RNeasy plant kit (Qiagen, Hilden, Germany). Concentration and quality of RNA were assessed on a Nanodrop spectrophotometer (Nanodrop, Wilmington, De, USA) and 2100 Bioanalyzer (Agilent, Palo Alto, Ca, USA). Biotin-labeled cRNAs were synthesized using MessageAmp II-Biotin Enhanced Kit (Ambion, Austin, Tx, USA) following the manufacturerâs instructions using 1 µg total RNA. Labelled cRNA was then fragmented using 5X Array Fragmentation Buffer supplied with the MessageAmp II-Biotin Enhanced Kit. Experiment Overall Design: Microarray hybridization: Experiment Overall Design: The labeled samples were added to an Affymetrix GeneChip® Wheat Genome Array (Affymetrix, Santa Clara, CA, USA), according to the manufacturerâs instructions. The wheat chip contains probe sets representing 55,052 transcripts for all 42 chromosomes in the wheat genome. The chips were hybridized for 16 h at 45 °C in a rotisserie oven at 60 rpm. Following hybridization, the arrays were washed and stained in an Affymetrix Fluidics Station 450, according to the standard protocol from Affymetrix, and scanned using an Affymetrix Scanner 3000. Twelve chips were prepared and corresponded to three biological replications of each of the four treatments (Si-B-, Si+B-, Si-B+, and Si+B+).
2009-07-04 | E-GEOD-12936 | biostudies-arrayexpress
Project description:In lung cancer progression, p53 mutations are more often observed in invasive tumors than in non-invasive tumors, suggesting that p53 is involved in tumor invasion and metastasis. To understand the nature of p53 function as a tumor suppressor, it is crucial to elucidate the detailed mechanism of the alteration in epithelial cells, the main origin of solid tumors, following p53 inactivation. To elucidate the mechanism by which p53 loss enhances invasive and motile activities in human lung small airway epithelial cells (SAECs), we performed comprehensive expression profiling analyses between p53 knockdown and control cells using GeneChip Human Genome U133 plus 2.0 arrays. SAECs were infected with lentiviruses for the expressions of CDK4 devoid of binding ability to p16INK4A, Cyclin D1 and TERT to generate immotalized SAECs. At 18 population doublings, immortalized SAECs were transfected with the siRNAs (si-control, si-p53-#1 or si-p53-#2) and used for RNA extraction and hybridization on Affymetrix microarrays.
2013-05-29 | E-GEOD-47436 | biostudies-arrayexpress