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1
Fiskus, Warren, Yongchao Wang, Arun Sreekumar, Kathleen M. Buckley, Huidong Shi, Anand Jillella, Celalettin Ustun, et al. "Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells." Blood 114, no. 13 (September 24, 2009): 2733–43. http://dx.doi.org/10.1182/blood-2009-03-213496.
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Abstract The polycomb repressive complex (PRC) 2 contains 3 core proteins, EZH2, SUZ12, and EED, in which the SET (suppressor of variegation–enhancer of zeste-trithorax) domain of EZH2 mediates the histone methyltransferase activity. This induces trimethylation of lysine 27 on histone H3, regulates the expression of HOX genes, and promotes proliferation and aggressiveness of neoplastic cells. In this study, we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) depletes EZH2 levels, and inhibits trimethylation of lysine 27 on histone H3 in the cultured human acute myeloid leukemia (AML) HL-60 and OCI-AML3 cells and in primary AML cells. DZNep treatment induced p16, p21, p27, and FBXO32 while depleting cyclin E and HOXA9 levels. Similar findings were observed after treatment with small interfering RNA to EZH2. In addition, DZNep treatment induced apoptosis in cultured and primary AML cells. Furthermore, compared with treatment with each agent alone, cotreatment with DZNep and the pan-histone deacetylase inhibitor panobinostat caused more depletion of EZH2, induced more apoptosis of AML, but not normal CD34+ bone marrow progenitor cells, and significantly improved survival of nonobese diabetic/severe combined immunodeficiency mice with HL-60 leukemia. These findings indicate that the combination of DZNep and panobinostat is effective and relatively selective epigenetic therapy against AML cells.
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He, Shan, Jina Wang, Koji Kato, Fang Xie, Sooryanarayana Varambally, Shin Mineishi, Rork Kuick, et al. "Inhibition of histone methylation arrests ongoing graft-versus-host disease in mice by selectively inducing apoptosis of alloreactive effector T cells." Blood 119, no. 5 (February 2, 2012): 1274–82. http://dx.doi.org/10.1182/blood-2011-06-364422.
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Abstract Histone methylation is thought to be important for regulating Ag-driven T-cell responses. However, little is known about the effect of modulating histone methylation on inflammatory T-cell responses. We demonstrate that in vivo administration of the histone methylation inhibitor 3-deazaneplanocin A (DZNep) arrests ongoing GVHD in mice after allogeneic BM transplantation. DZNep caused selective apoptosis in alloantigen-activated T cells mediating host tissue injury. This effect was associated with the ability of DZNep to selectively reduce trimethylation of histone H3 lysine 27, deplete the histone methyltransferase Ezh2 specific to trimethylation of histone H3 lysine 27, and activate proapoptotic gene Bim repressed by Ezh2 in antigenic-activated T cells. In contrast, DZNep did not affect the survival of alloantigen-unresponsive T cells in vivo and naive T cells stimulated by IL-2 or IL-7 in vitro. Importantly, inhibition of histone methylation by DZNep treatment in vivo preserved the antileukemia activity of donor T cells and did not impair the recovery of hematopoiesis and lymphocytes, leading to significantly improved survival of recipients after allogeneic BM transplantation. Our findings indicate that modulation of histone methylation may have significant implications in the development of novel approaches to treat ongoing GVHD and other T cell–mediated inflammatory disorders in a broad context.
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Byrne, K., S. McWilliam, T. Vuocolo, C. Gondro, N. E. Cockett, and R. L. Tellam. "Genomic architecture of histone 3 lysine 27 trimethylation during late ovine skeletal muscle development." Animal Genetics 45, no. 3 (March 27, 2014): 427–38. http://dx.doi.org/10.1111/age.12145.
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Bredfeldt, Tiffany G., K. Leigh Greathouse, Stephen H. Safe, Mien-Chie Hung, Mark T. Bedford, and Cheryl L. Walker. "Xenoestrogen-Induced Regulation of EZH2 and Histone Methylation via Estrogen Receptor Signaling to PI3K/AKT." Molecular Endocrinology 24, no. 5 (May 1, 2010): 993–1006. http://dx.doi.org/10.1210/me.2009-0438.
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Abstract Although rapid, membrane-activated estrogen receptor (ER) signaling is no longer controversial, the biological function of this nongenomic signaling is not fully characterized. We found that rapid signaling from membrane-associated ER regulates the histone methyltransferase enhancer of Zeste homolog 2 (EZH2). In response to both 17β-estradiol (E2) and the xenoestrogen diethylstilbestrol, ER signaling via phosphatidylinositol 3-kinase/protein kinase B phosphorylates EZH2 at S21, reducing levels of trimethylation of lysine 27 on histone H3 in hormone-responsive cells. During windows of uterine development that are susceptible to developmental reprogramming, activation of this ER signaling pathway by diethylstilbestrol resulted in phosphorylation of EZH2 and reduced levels of trimethylation of lysine 27 on histone H3 in chromatin of the developing uterus. Furthermore, activation of nongenomic signaling reprogrammed the expression profile of estrogen-responsive genes in uterine myometrial cells, suggesting this as a potential mechanism for developmental reprogramming caused by early-life exposure to xenoestrogens. These data demonstrate that rapid ER signaling provides a direct linkage between xenoestrogen-induced nuclear hormone receptor signaling and modulation of the epigenetic machinery during tissue development.
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Mendez, Flor M., Felipe J. Núñez, Maria B. Garcia-Fabiani, Santiago Haase, Stephen Carney, Jessica C. Gauss, Oren J. Becher, Pedro R. Lowenstein, and Maria G. Castro. "Epigenetic reprogramming and chromatin accessibility in pediatric diffuse intrinsic pontine gliomas: a neural developmental disease." Neuro-Oncology 22, no. 2 (November 15, 2019): 195–206. http://dx.doi.org/10.1093/neuonc/noz218.
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Abstract Diffuse intrinsic pontine glioma (DIPG) is a rare but deadly pediatric brainstem tumor. To date, there is no effective therapy for DIPG. Transcriptomic analyses have revealed DIPGs have a distinct profile from other pediatric high-grade gliomas occurring in the cerebral hemispheres. These unique genomic characteristics coupled with the younger median age group suggest that DIPG has a developmental origin. The most frequent mutation in DIPG is a lysine to methionine (K27M) mutation that occurs on H3F3A and HIST1H3B/C, genes encoding histone variants. The K27M mutation disrupts methylation by polycomb repressive complex 2 on histone H3 at lysine 27, leading to global hypomethylation. Histone 3 lysine 27 trimethylation is an important developmental regulator controlling gene expression. This review discusses the developmental and epigenetic mechanisms driving disease progression in DIPG, as well as the profound therapeutic implications of epigenetic programming.
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Souroullas, George P., William R. Jeck, Joel S. Parker, Jeremy M. Simon, Jie-Yu Liu, Joshiawa Paulk, Jessie Xiong, et al. "An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation." Nature Medicine 22, no. 6 (May 2, 2016): 632–40. http://dx.doi.org/10.1038/nm.4092.
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Harr, Jennifer C., Teresa Romeo Luperchio, Xianrong Wong, Erez Cohen, Sarah J. Wheelan, and Karen L. Reddy. "Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins." Journal of Cell Biology 208, no. 1 (January 5, 2015): 33–52. http://dx.doi.org/10.1083/jcb.201405110.
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Nuclear organization has been implicated in regulating gene activity. Recently, large developmentally regulated regions of the genome dynamically associated with the nuclear lamina have been identified. However, little is known about how these lamina-associated domains (LADs) are directed to the nuclear lamina. We use our tagged chromosomal insertion site system to identify small sequences from borders of fibroblast-specific variable LADs that are sufficient to target these ectopic sites to the nuclear periphery. We identify YY1 (Ying-Yang1) binding sites as enriched in relocating sequences. Knockdown of YY1 or lamin A/C, but not lamin A, led to a loss of lamina association. In addition, targeted recruitment of YY1 proteins facilitated ectopic LAD formation dependent on histone H3 lysine 27 trimethylation and histone H3 lysine di- and trimethylation. Our results also reveal that endogenous loci appear to be dependent on lamin A/C, YY1, H3K27me3, and H3K9me2/3 for maintenance of lamina-proximal positioning.
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Vincek, Adam, Jigneshkumar Patel, Anbalagan Jaganathan, Antonia Green, Valerie Pierre-Louis, Vimal Arora, Jill Rehmann, et al. "Inhibitor of CBP Histone Acetyltransferase Downregulates p53 Activation and Facilitates Methylation at Lysine 27 on Histone H3." Molecules 23, no. 8 (August 2, 2018): 1930. http://dx.doi.org/10.3390/molecules23081930.
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Tumor suppressor p53-directed apoptosis triggers loss of normal cells, which contributes to the side-effects from anticancer therapies. Thus, small molecules with potential to downregulate the activation of p53 could minimize pathology emerging from anticancer therapies. Acetylation of p53 by the histone acetyltransferase (HAT) domain is the hallmark of coactivator CREB-binding protein (CBP) epigenetic function. During genotoxic stress, CBP HAT-mediated acetylation is essential for the activation of p53 to transcriptionally govern target genes, which control cellular responses. Here, we present a small molecule, NiCur, which blocks CBP HAT activity and downregulates p53 activation upon genotoxic stress. Computational modeling reveals that NiCur docks into the active site of CBP HAT. On CDKN1A promoter, the recruitment of p53 as well as RNA Polymerase II and levels of acetylation on histone H3 were diminished by NiCur. Specifically, NiCur reduces the levels of acetylation at lysine 27 on histone H3, which concomitantly increases the levels of trimethylation at lysine 27. Finally, NiCur attenuates p53-directed apoptosis by inhibiting the Caspase 3 activity and cleavage of Poly (ADP-ribose) polymerase (PARP) in normal gastrointestinal epithelial cells. Collectively, NiCur demonstrates the potential to reprogram the chromatin landscape and modulate biological outcomes of CBP-mediated acetylation under normal and disease conditions.
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Han, Xiaobin, and Zhongjie Sun. "Epigenetic Regulation of KL (Klotho) via H3K27me3 (Histone 3 Lysine [K] 27 Trimethylation) in Renal Tubule Cells." Hypertension 75, no. 5 (May 2020): 1233–41. http://dx.doi.org/10.1161/hypertensionaha.120.14642.
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Bae, W. K., I. J. Chung, S. H. Cho, and H. J. Shim. "P-024 The association between histone 3 lysine 27 trimethylation and liver fibrosis and cancer: relationship with methyltransferase." Annals of Oncology 26 (June 2015): iv7. http://dx.doi.org/10.1093/annonc/mdv233.24.
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Zhao, Liyan, Xiongtao Liu, Weina Zhu, Pei Yang, Jie Qin, Ru Gu, and Zhili Zhao. "Florofangchinoline inhibits proliferation of osteosarcoma cells via targeting of histone H3 lysine 27 trimethylation and AMPK activation." Tropical Journal of Pharmaceutical Research 19, no. 7 (November 16, 2020): 1403–9. http://dx.doi.org/10.4314/tjpr.v19i7.10.
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Purpose: To investigate the effect of florofangchinoline on osteosarcoma cell growth in vitro, and the underlying mechanism of action.Methods: Changes in the viability of KHOS and Saos-2 cells were measured using water soluble tetrazolium salt (WST) assay, while apoptosis was determined using Annexin V/PI staining and flow cytometry. Increases in mtDNA, and expressions of PGC-1α and TFAM were assayed with immunoblot analysis and quantitative real-time polymerase chain reaction (qPCR), respectively.Results: Microscopic examination of florofangchinoline-treated cells showed significant decrease in cell density, relative to control cells (p < 0.05). Treatment with 10 μM florofangchinoline increased apoptosis in KHOS and Saos-2 cells to 56.32 and 63.75 %, respectively (p < 0.05). Florofangchinoline treatment markedly enhanced cleavage of caspase-3, caspase-8, caspase-9 and PARP. It elevated Bax level and reduced Bcl-2 in KHOS and Saos-2 cells. Moreover, florofangchinoline increased p21 and p-AMPKα levels, and mtDNA counts in KHOS and Saos-2 cells (p < 0.05). Moreover, in florofangchinoline-treated KHOS cells, the expressions of EED, EZH2 and SUZ12 were significantly suppressed (p < 0.05).Conclusion: Florofangchinoline inhibits osteosarcoma cell viability by activation of apoptosis. Moreover, it activates AMPK and down-regulates histone H3 lysine 27 trimethylation in osteosarcoma cells. Therefore, florofangchinoline has potentials for development as a therapeutic drug forosteosarcoma.
Keywords: Osteosarcoma, Histone H3, Florofangchinoline, Apoptosis, Chemotherapeutic
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Paul, Thomas A., Horatiu Muresan, Emily Prentice, and Linda Wolff. "Histone Modifications Associated with DNA Methylation and Transcriptional Repression of p15INK4b in Acute Myeloid Leukemia." Blood 112, no. 11 (November 16, 2008): 3354. http://dx.doi.org/10.1182/blood.v112.11.3354.3354.
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Abstract p15INK4b is a cyclin-dependent kinase inhibitor known to regulate the G1-to-S transition of the cell cycle and to be involved in negatively regulating myeloid progenitor cell production. DNA hypermethylation leading to transcriptional silencing of p15INK4b has been reported in up to 70% of acute myeloid leukemia (AML) patient samples. In our study we sought to determine if p15INK4b DNA methylation in AML is accompanied by repressive histone modifications that contribute to the transcriptional repression of the gene at the chromatin level. Chromatin immunoprecipitation and DNA tiling microarrays (ChIP-on-chip) with 20bp resolution were utilized to assess the distribution of histone modifications over a 1.2 megabase region of human chromosome 9 including p15INK4b and adjacent tumor suppressor genes p14ARF and p16INK4a. We found that AML cell lines with p15INK4b hypermethylation (Kasumi-1, KG-1, and KG-1a) had high levels of the repressive histone modification trimethylation of lysine 27 of histone H3 (H3K27me3). Remarkably, this modification spanned the entire INK4b-ARF-INK4a region while little binding was observed in adjacent regions of chromosome 9. Binding of EZH2, the polycomb associated H3K27 histone methyltransferase, co-localized with H3K27me3 distribution over the INK locus. H3K27me3 was not identified at this region in AML cell lines without p15INK4b DNA methylation (U937 and HL-60). In contrast, histone modifications associated gene activation, trimethylation of lysine 4 of H3 (H3K4me3) and acetylation of lysine 9 (H3K9Ac), were found at the p15INK4b promoter in these cells. Enrichment of another repressive histone modification, trimethylation of histone H3 on lysine 9 (H3K9me3), did not correlate with the DNA methylation status of p15INK4b and appeared highest in exons 2 and 3 of p16INK4a in most cell lines. Since p15INK4b reactivation has been described as a component of a patient’s response to epigenetic therapies in AML treatment, we sought to determine the dynamics of histone modifications following treatment with the DNA methyltransferase (DNMT) inhibitor 5-aza-2’-deoxycytidine and histone deacetylase (HDAC) inhibitor tricostatin A. In KG-1 cells, a reduction in p15INK4b DNA methylation was observed following treatment with DNMT inhibitors. Unexpectedly, treatment with HDAC inhibitors alone was also capable of reducing p15INK4b DNA methylation suggesting that a repressive chromatin structure contributes to the DNA methylation in this cell line. Loss of DNA methylation was not sufficient for reactivation of p15INK4b expression as detectible expression was only observed following the combined treatment of DNMT and HDAC inhibitors. Reactivation was associated with an increase in the activation-associated histone modifications H3K4me3 and H3K9Ac at the promoter region and, unexpectedly, maintenance of the repressive modification H3K27me3. This “bivalent” histone modification pattern is characteristic of many developmentally poised genes in embryonic stem cells and correlates with the histone methylation status of p15INK4b we observed in CD34+ bone marrow progenitor/ stem cells. This data indicates that optimal epigenetic therapies targeted to reactivate p15INK4b expression should be designed to induce activating histone modifications in addition to reducing DNA methylation.
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Liu, Pei-Pei, Ya-Jie Xu, Zhao-Qian Teng, and Chang-Mei Liu. "Polycomb Repressive Complex 2: Emerging Roles in the Central Nervous System." Neuroscientist 24, no. 3 (December 14, 2017): 208–20. http://dx.doi.org/10.1177/1073858417747839.
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The polycomb repressive complex 2 (PRC2) is responsible for catalyzing both di- and trimethylation of histone H3 at lysine 27 (H3K27me2/3). The subunits of PRC2 are widely expressed in the central nervous system (CNS). PRC2 as well as H3K27me2/3, play distinct roles in neuronal identity, proliferation and differentiation of neural stem/progenitor cells, neuronal morphology, and gliogenesis. Mutations or dysregulations of PRC2 subunits often cause neurological diseases. Therefore, PRC2 might represent a common target of different pathological processes that drive neurodegenerative diseases. A better understanding of the intricate and complex regulatory networks mediated by PRC2 in CNS will help to develop new therapeutic approaches and to generate specific brain cell types for treating neurological diseases.
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Farber, James E., and Robert P. Lane. "Bioinformatics Discovery of Putative Enhancers within Mouse Odorant Receptor Gene Clusters." Chemical Senses 44, no. 9 (September 14, 2019): 705–20. http://dx.doi.org/10.1093/chemse/bjz043.
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Abstract Olfactory neuronal function depends on the expression and proper regulation of odorant receptor (OR) genes. Previous studies have identified 54 putative intergenic enhancers within or flanking 40 mouse OR clusters. At least 2 of these putative enhancers have been shown to regulate the expression of a small subset of proximal OR genes. In recognition of the large size of the mouse OR gene family (~1400 OR genes distributed across multiple chromosomal loci), it is likely that there remain many additional not-as-yet discovered OR enhancers. We utilized 23 of the previously identified enhancers as a training set (TS) and designed an algorithm that combines a broad range of epigenetic criteria (histone-3-lysine-4 monomethylation, histone-3-lysine-79 trimethylation, histone-3-lysine-27 acetylation, and DNase hypersensitivity) and genetic criteria (cross-species sequence conservation and transcription-factor binding site enrichment) to more broadly search OR gene clusters for additional candidates. We identified 181 new candidate enhancers located at 58 (of 68) mouse OR loci, including 25 new candidates identified by stringent search criteria whose signal strengths are not significantly different from the 23 previously characterized OR enhancers used as the TS. Additionally, we compared OR enhancer versus generic enhancer features in order to evaluate likelihoods that new enhancer candidates specifically function in OR regulation. We found that features distinguishing OR-specific function are significantly more evident for enhancer candidates located within OR clusters as compared with those in flanking regions.
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Yan, Bowen, Yanpeng Lv, Chunyu Zhao, and Xiaoxue Wang. "Knowing When to Silence: Roles of Polycomb-Group Proteins in SAM Maintenance, Root Development, and Developmental Phase Transition." International Journal of Molecular Sciences 21, no. 16 (August 15, 2020): 5871. http://dx.doi.org/10.3390/ijms21165871.
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Polycomb repressive complex 1 (PRC1) and PRC2 are the major complexes composed of polycomb-group (PcG) proteins in plants. PRC2 catalyzes trimethylation of lysine 27 on histone 3 to silence target genes. Like Heterochromatin Protein 1/Terminal Flower 2 (LHP1/TFL2) recognizes and binds to H3K27me3 generated by PRC2 activities and enrolls PRC1 complex to further silence the chromatin through depositing monoubiquitylation of lysine 119 on H2A. Mutations in PcG genes display diverse developmental defects during shoot apical meristem (SAM) maintenance and differentiation, seed development and germination, floral transition, and so on so forth. PcG proteins play essential roles in regulating plant development through repressing gene expression. In this review, we are focusing on recent discovery about the regulatory roles of PcG proteins in SAM maintenance, root development, embryo development to seedling phase transition, and vegetative to reproductive phase transition.
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Klocko, Andrew D., Tereza Ormsby, Jonathan M. Galazka, Neena A. Leggett, Miki Uesaka, Shinji Honda, Michael Freitag, and Eric U. Selker. "Normal chromosome conformation depends on subtelomeric facultative heterochromatin in Neurospora crassa." Proceedings of the National Academy of Sciences 113, no. 52 (November 16, 2016): 15048–53. http://dx.doi.org/10.1073/pnas.1615546113.
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High-throughput chromosome conformation capture (Hi-C) analyses revealed that the 3D structure of the Neurospora crassa genome is dominated by intra- and interchromosomal links between regions of heterochromatin, especially constitutive heterochromatin. Elimination of trimethylation of lysine 9 on histone H3 (H3K9me3) or its binding partner Heterochromatin Protein 1 (HP1)—both prominent features of constitutive heterochromatin—have little effect on the Hi-C pattern. It remained possible that di- or trimethylation of lysine 27 on histone H3 (H3K27me2/3), which becomes localized in regions of constitutive heterochromatin when H3K9me3 or HP1 are lost, plays a critical role in the 3D structure of the genome. We found that H3K27me2/3, catalyzed by the Polycomb Repressive Complex 2 (PRC2) member SET-7 (SET domain protein-7), does indeed play a prominent role in the Hi-C pattern of WT, but that its presence in regions normally occupied by H3K9me3 is not responsible for maintenance of the genome architecture when H3K9me3 is lost. The Hi-C pattern of a mutant defective in the PRC2 member N. crassa p55 (NPF), which is predominantly required for subtelomeric H3K27me2/3, was equivalent to that of the set-7 deletion strain, suggesting that subtelomeric facultative heterochromatin is paramount for normal chromosome conformation. Both PRC2 mutants showed decreased heterochromatin–heterochromatin contacts and increased euchromatin–heterochromatin contacts. Cytological observations suggested elimination of H3K27me2/3 leads to partial displacement of telomere clusters from the nuclear periphery. Transcriptional profiling of Δdim-5, Δset-7, Δset-7; Δdim-5, and Δnpf strains detailed anticipated changes in gene expression but did not support the idea that global changes in genome architecture, per se, led to altered transcription.
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Steiner, Laurie A., Vincent Schulz, Yelena Maksimova, Clara Wong, David Tuck, and Patrick G. Gallagher. "Patterns of Monomethylation of Histone H3 Lysine 27 Influence Gene Expression in a Cell-Type Specific Manner." Blood 114, no. 22 (November 20, 2009): 4585. http://dx.doi.org/10.1182/blood.v114.22.4585.4585.
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Abstract Abstract 4585 Post-translational histone modifications influence expression by creating a chromatin environment which is conducive to or inhibitory of transcription. Modifications such as trimethylation of histone H3 lysine 4 and acetylation of histone H3 lysine 9 are generally associated with euchromatin and gene activation, while modifications such as trimethylation of histone H3 lysine 27 are associated with regions of heterochromatin and/or gene repression. Monomethyl histone H3 lysine 27 (H3K27me1) is a poorly studied post-translational histone modification for which variable associations with mRNA expression have been observed. Initially, H3K27me1 was localized to areas of pericentric heterochromain and was thought to be a marker of gene repression. Later reports described H3K27me1 enrichment throughout the body of actively transcribing genes (Vakoc C et al. MCB 26:9185, 2006; Wang Z, 40:897 Nat Genet, 2008). Some reports describe selective depletion of H3K27Me1 at promoters and transcription start sites (TSS), implying that depletion of H3K27me1 at the TSS is necessary for active transcription, (Vakoc C et al.), while others have associated increased enrichment for H3K27me1 at the promoter with increased levels of mRNA expression (Barski A et al. Cell 129:823, 2007). We hypothesize that the relationship between H3K27me1 occupancy and gene expression varies depending on both the cell-type and the location in the gene (i.e. promoter, transcription start site (TSS) and body of the gene) and that varying H3K27me1 levels in each of these locations is associated with alterations in the level of mRNA expression. To assess the association of H3K27me1 level with mRNA expression, H3K27me1 binding was determined using chromatin immunoprecipitation on microarray analysis (ChIP-chip) and correlated with mRNA levels determined using Illumina human expression arrays. ChIP-chip was performed using an antibody specific for H3K27me1 and the resulting DNA applied the to a custom designed genomic tiling NimbleGen microarray containing over 100 erythroid expressed genes and 10-100kb of flanking DNA for each locus. Probes were tiled with 10-100bp spacing, typically ∼65bp. Regions of repetitive DNA were excluded. ChIP-chip was performed in erythroid (K562) and non-erythroid (SY5Y-neural, RD-muscle) cells and the pattern of H3K27me1 enrichment assessed. mRNA transcript analyses were performed using Illumina human V6-2 expression arrays and quantitative real time RT-PCR. H3K27me1 levels at the promoter (-1000 to +1), in the 200bp surrounding the TSS (-100 to +100), and over the body of the gene were correlated with the level of mRNA expression. Increasing levels of H3K27me1 over the body of the gene lead correlated with increased levels of gene expression (R2=0.6122), while the amount of H3K27me1 at the promoter (-1000 to +1) had no correlation with gene expression (R2=0.2769). In agreement with Vakoc et al., decreased enrichment for H3K27Me1 at the TSS (-100 to +100) correlated with increased levels of mRNA expression. This is in sharp contrast to H3K4Me3, which accumulates at the start site of active genes. H3K27me1 has not been studied in detail in transcriptionally silent genes. Interestingly, genes without H3K27Me1 enrichment had no expression, implying that H3K27me1 is a marker of active transcription. Patterns of H3K27Me1 enrichment were cell-type specific. For example, in erythroid (K562) cells, the beta-globin locus was highly enriched for H3K27me1. This enrichment was not present in non-erythroid cells (RD, SY5Y). Finally, H3K27Me1 may also mark enhancers in a cell type-specific manner. For example, in the well studied HS2 enhancer in the beta-globin LCR, there is significant enrichment for H3K27me1 in K562 cells, but not in SY5Y or RD cells. These data indicate that modulation of chromatin architecture by monomethylation of histone 3 lysine 27 influences the level of gene expression in erythroid and non-erythroid cells. Disclosures: No relevant conflicts of interest to declare.
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Tsou, Pei-Suen, Phillip Campbell, M. Asif Amin, Patrick Coit, Shaylynn Miller, David A. Fox, Dinesh Khanna, and Amr H. Sawalha. "Inhibition of EZH2 prevents fibrosis and restores normal angiogenesis in scleroderma." Proceedings of the National Academy of Sciences 116, no. 9 (February 12, 2019): 3695–702. http://dx.doi.org/10.1073/pnas.1813006116.
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Scleroderma (SSc) is a complex disease that involves activation of the immune system, vascular complications, and tissue fibrosis. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) mediates trimethylation of lysine 27 of histone 3 (H3K27me3), which acts as a repressive epigenetic mark. Both EZH2 and H3K27me3 were elevated in SSc dermal fibroblasts and endothelial cells compared with healthy controls. EZH2 inhibitor DZNep halted fibrosis both in vitro and in vivo. In SSc fibroblasts, DZNep dose-dependently reduced the expression of profibrotic genes and inhibited migratory activity of SSc fibroblasts. We show that epigenetic dysregulation and overexpression of LRRC16A explains EZH2-mediated fibroblast migration in SSc. In endothelial cells, inhibition of EZH2 restored normal angiogenesis in SSc via activating the Notch pathway, specifically by up-regulating the Notch ligand DLL4. Our results demonstrate that overexpression of EZH2 in SSc fibroblasts and endothelial cells is profibrotic and antiangiogenic. Targeting EZH2 or EZH2-regulated genes might be of therapeutic potential in SSc.
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Momparler, Richard L., Sylvie Côté, and Louise F. Momparler. "Enhancement of the Antileukemic Action of the Inhibitors of DNA and Histone Methylation: 5-Aza-2′-Deoxycytidine and 3-Deazaneplanocin-A by Vitamin C." Epigenomes 5, no. 2 (March 24, 2021): 7. http://dx.doi.org/10.3390/epigenomes5020007.
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Epigenetic gene silencing by DNA methylation and histone methylation by EZH2 play an important role in the development of acute myeloid leukemia (AML). EZH2 catalyzes the trimethylation of histone H3-lysine 27-trimethylated (H3K27me3). These epigenetic alterations silence the expression of the genes that suppress leukemogenesis. Reversal of this gene silencing by 5-aza-2′-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, and by 3-deazaneplanocin-A (DZNep), an inhibitor of EZH2, results in synergistic gene reactivation and antileukemic interaction. The objective of this study is to determine if the addition of another epigenetic agent could further enhance the antileukemic action of these inhibitors of DNA and histone methylation. Vitamin C (Vit C) is reported to enhance the antineoplastic action of 5-Aza-CdR on AML cells. The mechanism responsible for this action of Vit C is due to its function as a cofactor of alpha-ketoglutarate-dependent dioxygenases (α-KGDD). The enhancement by Vit C of the catalytic activity of α-KGDD of the ten eleven translocation (TET) pathway, as well as of the Jumonji C histone demethylases (JHDMs), is shown to result in demethylation of DNA and histones, leading to reactivation of tumor suppressor genes and an antineoplastic effect. This action of Vit C has the potential to complement the antileukemic action of 5-Aza-CdR and DZNep. We observe that Vit C remarkably increases the antineoplastic activity of 5-Aza-CdR and DZNep against myeloid leukemic cells. An important step to bring this novel epigenetic therapy to clinical trial in patients with AML is the determination of its optimal dose schedule.
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Ishii, Makoto, Haitao Wen, Callie A. S. Corsa, Tianju Liu, Ana L. Coelho, Ronald M. Allen, William F. Carson, et al. "Epigenetic regulation of the alternatively activated macrophage phenotype." Blood 114, no. 15 (October 8, 2009): 3244–54. http://dx.doi.org/10.1182/blood-2009-04-217620.
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Abstract Alternatively activated (M2) macrophages play critical roles in diverse chronic diseases, including parasite infections, cancer, and allergic responses. However, little is known about the acquisition and maintenance of their phenotype. We report that M2-macrophage marker genes are epigenetically regulated by reciprocal changes in histone H3 lysine-4 (H3K4) and histone H3 lysine-27 (H3K27) methylation; and the latter methylation marks are removed by the H3K27 demethylase Jumonji domain containing 3 (Jmjd3). We found that continuous interleukin-4 (IL-4) treatment leads to decreased H3K27 methylation, at the promoter of M2 marker genes, and a concomitant increase in Jmjd3 expression. Furthermore, we demonstrate that IL-4–dependent Jmjd3 expression is mediated by STAT6, a major transcription factor of IL-4–mediated signaling. After IL-4 stimulation, activated STAT6 is increased and binds to consensus sites at the Jmjd3 promoter. Increased Jmjd3 contributes to the decrease of H3K27 dimethylation and trimethylation (H3K27me2/3) marks as well as the transcriptional activation of specific M2 marker genes. The decrease in H3K27me2/3 and increase in Jmjd3 recruitment were confirmed by in vivo studies using a Schistosoma mansoni egg–challenged mouse model, a well-studied system known to support an M2 phenotype. Collectively, these data indicate that chromatin remodeling is mechanistically important in the acquisition of the M2-macrophage phenotype.
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Qiao, Yu, Kyuho Kang, Eugenia Giannopoulou, Celeste Fang, and Lionel B. Ivashkiv. "IFN-γ Induces Histone 3 Lysine 27 Trimethylation in a Small Subset of Promoters to Stably Silence Gene Expression in Human Macrophages." Cell Reports 16, no. 12 (September 2016): 3121–29. http://dx.doi.org/10.1016/j.celrep.2016.08.051.
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Guajardo, Laura, Rodrigo Aguilar, Fernando J. Bustos, Gino Nardocci, Rodrigo A. Gutiérrez, Brigitte van Zundert, and Martin Montecino. "Downregulation of the Polycomb-Associated Methyltransferase Ezh2 during Maturation of Hippocampal Neurons Is Mediated by MicroRNAs Let-7 and miR-124." International Journal of Molecular Sciences 21, no. 22 (November 11, 2020): 8472. http://dx.doi.org/10.3390/ijms21228472.
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Ezh2 is a catalytic subunit of the polycomb repressive complex 2 (PRC2) which mediates epigenetic gene silencing through depositing the mark histone H3 lysine 27 trimethylation (H3K27me3) at target genomic sequences. Previous studies have demonstrated that Enhancer of Zeste Homolog 2 (Ezh2) was differentially expressed during maturation of hippocampal neurons; in immature neurons, Ezh2 was abundantly expressed, whereas in mature neurons the expression Ezh2 was significantly reduced. Here, we report that Ezh2 is downregulated by microRNAs (miRs) that are expressed during the hippocampal maturation process. We show that, in mature hippocampal neurons, lethal-7 (let-7) and microRNA-124 (miR-124) are robustly expressed and can target cognate motifs at the 3′-UTR of the Ezh2 gene sequence to downregulate Ezh2 expression. Together, these data demonstrate that the PRC2 repressive activity during hippocampal maturation is controlled through a post-transcriptional mechanism that mediates Ezh2 downregulation in mature neurons.
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Kumar, Sachin, Antony Michealraj, Leo Kim, Jeremy Rich, and Michael Taylor. "EPEN-52. METABOLIC REGULATION OF THE EPIGENOME DRIVES LETHAL INFANTILE EPENDYMOMA." Neuro-Oncology 22, Supplement_3 (December 1, 2020): iii318. http://dx.doi.org/10.1093/neuonc/noaa222.185.
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Abstract PFA ependymomas are a lethal glial malignancy of the hindbrain found in infants and toddlers. Lacking any highly recurrent somatic mutations, PFAs have been proposed to be a largely epigenetically driven entity, defined by hypomethylation at the histone 3 lysine 27 residue. Unfortunately, an almost complete lack of model systems has limited the discovery of novel PFA therapies. In this study, we have identified that the PFA hypoxic microenvironment controls the availability of specific metabolites, resulting in diminished H3K27 trimethylation and increased H3K27 acetylation in vitro and in vivo. Unique to PFA cells, transient exposure to ambient oxygen results in irreversible cellular toxicity. Furthermore, perturbation of key metabolic pathways is sufficient to inhibit growth of PFA primary cultures in vitro. Although PFA tumors exhibit a low basal level of H3K27me3, inhibition of H3K27 methylation paradoxically demonstrates significant and specific activity against PFA. Thus, we propose a “Goldilocks Model” of metabolic-epigenetic regulation in PFA ependymoma, whereby increased or decreased H3K27 trimethylation results in cell death. Mapping of PFA ependymoma tumours suggests a cell of origin arising in the first trimester of human development where there is a known hypoxic microenvironment. Therefore, targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.
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Suzuki, Kei, Chitose Oneyama, Hironobu Kimura, Shoji Tajima, and Masato Okada. "Down-regulation of the Tumor Suppressor C-terminal Src Kinase (Csk)-binding Protein (Cbp)/PAG1 Is Mediated by Epigenetic Histone Modifications via the Mitogen-activated Protein Kinase (MAPK)/Phosphatidylinositol 3-Kinase (PI3K) Pathway." Journal of Biological Chemistry 286, no. 18 (March 9, 2011): 15698–706. http://dx.doi.org/10.1074/jbc.m110.195362.
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The transmembrane adaptor protein Cbp (or PAG1) functions as a suppressor of Src-mediated tumor progression by promoting the inactivation of Src. The expression of Cbp is down-regulated in Src-transformed cells and in various human cancer cells, suggesting a potential role for Cbp as a tumor suppressor. However, the mechanisms underlying the down-regulation of Cbp remain unknown. The present study shows that Cbp expression is down-regulated by epigenetic histone modifications via the MAPK/PI3K pathway. In mouse embryonic fibroblasts, transformation by oncogenic Src and Ras induced a marked down-regulation of Cbp expression. The levels of Cbp expression were inversely correlated with the activity of MEK and Akt, and Cbp down-regulation was suppressed by inhibiting MEK and PI3K. Src transformation did not affect the stability of Cbp mRNA, the transcriptional activity of the cbp promoter, or the DNA methylation status of the cbp promoter CpG islands. However, Cbp expression was restored by treatment with histone deacetylase (HDAC) inhibitors and by siRNA-mediated knockdown of HDAC1/2. Src transformation significantly decreased the acetylation levels of histone H4 and increased the trimethylation levels of histone H3 lysine 27 in the cbp promoter. EGF-induced Cbp down-regulation was also suppressed by inhibiting MEK and HDAC. Furthermore, the inhibition of MEK or HDAC restored Cbp expression in human cancer cells harboring Cbp down-regulation through promoter hypomethylation. These findings suggest that Cbp down-regulation is primarily mediated by epigenetic histone modifications via oncogenic MAPK/PI3K pathways in a subset of cancer cells.
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Iida, Atsumi, Toshiro Iwagawa, Hiroshi Kuribayashi, Shinya Satoh, Yujin Mochizuki, Yukihiro Baba, Hiromitsu Nakauchi, et al. "Histone demethylase Jmjd3 is required for the development of subsets of retinal bipolar cells." Proceedings of the National Academy of Sciences 111, no. 10 (February 26, 2014): 3751–56. http://dx.doi.org/10.1073/pnas.1311480111.
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Di- and trimethylation of lysine 27 on histone H3 (H3K27me2/3) is an important gene repression mechanism. H3K27me2/3-specific demethylase, Jmjd3, was expressed in the inner nuclear layer during late retinal development. In contrast, H3K27 methyltransferase, Ezh2, was highly expressed in the embryonic retina but its expression decreased rapidly after birth. Jmjd3 loss of function in the developing retina resulted in failed differentiation of PKC-positive bipolar cell subsets (rod-ON-BP) and reduced transcription factor Bhlhb4 expression, which is critical for the differentiation of rod-ON-BP cells. Overexpression of Bhlhb4, but not of other BP cell-related genes, such as transcription factors Neurod and Chx10, in Jmjd3-knockdown retina rescued loss of PKC-positive BP cells. Populations of other retinal cell subsets were not significantly affected. In addition, proliferation activity and apoptotic cell number during retinal development were not affected by the loss of Jmjd3. Levels of histone H3 trimethyl Lys27 (H3K27me3) in the Bhlhb4 locus were lower in Islet-1–positive BP cells and amacrine cells than in the Islet-1–negative cell fraction. The Islet-1–negative cell fraction consisted mainly of photoreceptors, suggestive of lineage-specific demethylation of H3K27me3 in the Bhlhb4 locus. We propose that lineage-specific H3K27me3 demethylation of critical gene loci by spatiotemporal-specific Jmjd3 expression is required for appropriate maturation of retinal cells.
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Bergmann, Christina, Amelie Brandt, Benita Merlevede, Ludwig Hallenberger, Clara Dees, Thomas Wohlfahrt, Sebastian Pötter, et al. "The histone demethylase Jumonji domain-containing protein 3 (JMJD3) regulates fibroblast activation in systemic sclerosis." Annals of the Rheumatic Diseases 77, no. 1 (October 25, 2017): 150–58. http://dx.doi.org/10.1136/annrheumdis-2017-211501.
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ObjectivesSystemic sclerosis (SSc) fibroblasts remain activated even in the absence of exogenous stimuli. Epigenetic alterations are thought to play a role for this endogenous activation. Trimethylation of histone H3 on lysine 27 (H3K27me3) is regulated by Jumonji domain-containing protein 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in a therapeutically targetable manner. The aim of this study was to explore H3K27me3 demethylases as potential targets for the treatment of fibrosis.MethodsJMJD3 was inactivated by small interfering RNA-mediated knockdown and by pharmacological inhibition with GSKJ4. The effects of targeted inactivation of JMJD3 were analysed in cultured fibroblasts and in the murine models of bleomycin-induced and topoisomerase-I (topoI)-induced fibrosis. H3K27me3 at the FRA2 promoter was analysed by ChIP.ResultsThe expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in experimental fibrosis in a transforming growth factor beta (TGFβ)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGFβ. Pharmacological inhibition of JMJD3 ameliorated bleomycin-induced and topoI-induced fibrosis in well-tolerated doses. JMJD3 regulated fibroblast activation in a FRA2-dependent manner: Inactivation of JMJD3 reduced the expression of FRA2 by inducing accumulation of H3K27me3 at the FRA2 promoter. Moreover, the antifibrotic effects of JMJD3 inhibition were reduced on knockdown of FRA2.ConclusionWe present first evidence for a deregulation of JMJD3 in SSc. JMJD3 modulates fibroblast activation by regulating the levels of H3K27me3 at the promoter of FRA2. Targeted inhibition of JMJD3 limits the aberrant activation of SSc fibroblasts and exerts antifibrotic effects in two murine models.
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He, Shan, Jina Wang, Koji Kato, Fang Xie, Sooryanarayana Varambally, Shin Mineishi, Rork Kuick, et al. "Inhibition of Histone Methylation Arrests Ongoing Graft-Versus-Host Diseases in Mice by Selectively Inducing Apoptosis of Alloreactive Effector T Cells." Blood 118, no. 21 (November 18, 2011): 820. http://dx.doi.org/10.1182/blood.v118.21.820.820.
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Abstract Abstract 820 Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment option for patients with hematological malignancies. However, its success is limited by life-threatening graft-versus-host disease (GVHD). Novel approaches are needed to control GVHD. Recent studies have shown the importance of histone methylation in regulating the expression of genes associated with effector T cell differentiation and proliferation. Using several mouse models of allo-HSCT, we report that in vivo administration of the histone methylation inhibitor 3-Deazaneplanocin A (DZNep) arrested ongoing GVHD while preserving graft-versus-leukemia activity (GVL). To assess the therapeutic effect of pharmacologic modulation of histone methylation on GVHD, we administered DZNep to BALB/c mice receiving major histocompatibility-mismatched C57BL/6 mouse T cells 7 days after transplantation, in which GVHD had been fully established. Notably, injection of 12 doses of DZNep controlled the disease in these recipients, with approximately 80% of them surviving long-term without significant clinical signs of GVHD. We found that in vivo administration of DZNep caused selective apoptosis in alloantigen-activated T cells, but did not impair the generation of effector T cells that produced inflammatory cytokines (e.g., TNF-α, IFN-γ and IL-17) and cytotoxic molecules (e.g., granzyme B and Fas ligand). As a result, alloreactive T cells retained potent GVL activity, leading to improved overall survival of the recipients challenged by leukemic cells. These data suggest that DZNep-mediated inhibition of GVHD may be accounted for by reduced number of alloreactive effector T cells. In vitro culture assays showed that DZNep treatment induced apoptosis in T cells activated by anti-CD3/CD28 antibodies but not in naive T cells stimulated by IL-2 or IL-7. This effect was associated with DZNep's ability to selectively reduce trimethylation of histone H3 lysine 27 (H3K27), deplete the histone methyltranferase Ezh2 that specifically catalyzes trimethylation of H3K27, and activate Ezh2-repressed pro-apoptotic gene Bim. Inactivation of Bim partially protected alloreactive T cells from DZNep-mediated apoptosis. Importantly, unlike DNA methylation inhibitors, inhibition of histone methylation by DZNep had no toxicities to hematopoietic cells or impairment on the reconstitution of hematopoiesis and thymopoiesis. Our findings indicate that modulation of histone methylation may have significant implications in the development of novel approaches to treat established GVHD and other T cell-mediated inflammatory disorders in a broad context. Disclosures: No relevant conflicts of interest to declare.
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Williams, K. J., K. R. Bondioli, and R. A. Godke. "400 KARYOTYPIC ANALYSIS AND EPIGENETIC MODIFICATIONS OF CULTURED PORCINE ADIPOSE TISSUE-DERIVED STEM CELLS." Reproduction, Fertility and Development 22, no. 1 (2010): 356. http://dx.doi.org/10.1071/rdv22n1ab400.
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The introduction of genetic modifications in donor cells for NT requires a significant number of population doublings (PD), and the deleterious effects, which may be attributed to aneuploidy or changes in DNA methylation and histone acetylation, are difficult at this time to circumvent. We hypothesize that the identification of a donor cell that is genetically stable for a long period of time in vitro such as somatic stem cells or those cells that demonstrate stem-like characteristics may be reprogrammed more completely, thus providing the key to increasing the efficiency of NT. Regulators of development in undifferentiated cells are suggested to be silenced by the presence of a bivalent domain modification pattern in which a large region of repressive histone 3 lysine 27 trimethylation (H3K27me3) contains smaller regions of activating histone 3 lysine 4 trimethylation (H3K4me3).The dual marks work to silence developmental genes in embryonic stem cells while simultaneously keeping them receptive to activation. The objectives of the current study were to determine the chromosomal stability of porcine adipose tissue-derived adult stem cells (pASC) through in vitro culture, to analyze pASC alongside fetal porcine fibroblasts (FPF) for gene expression profiles of chromatin remodeling proteins and global methylation and acetylation patterns, and to determine the presence of a co-enrichment of H3K27me3 and H3K4me3 within the promoter regions of developmentally important transcription factors. Metaphase spreads were prepared, and the presence of H3K27me3 and H3K4me3 was investigated in each of 3 individual pASC primary cultures for each analysis; whereas, gene expression and global methylation and acetylation were analyzed in each of 4 individual pASC and FPF primary cultures. Of 714 metaphases analyzed, 509 (71.3%) were aneuploid and only 205 (28.7%) were normal diploid porcine cells. For each cell population, we found a remarkable percentage of aneuploidies (43.7, 48.9, and 47.3, with a 46.6 ± 1.5 average) present immediately after the cultures were established. Chi-square analysis indicated that the percent of aneuploid cells during PD 1-10 was significantly less than that for PD 11-20 and PD 21-30. Also, porcine ASC demonstrated a consistently lower level of DNA methylation and histone acetylation through passages 2 through 7; whereas, the patterns for FPF varied. The expression levels of chromatin remodeling transcripts remained lower in pASC throughout culture when compared with FPF. Finally, porcine ASC possess a co-enrichment of H3K27me3 and H3K4me3 on the promoter region of the developmentally important transcription factor OCT-4. In vitro-cultured porcine ASC used as donor cells for NT should be chosen from early PD because of increased levels of aneuploidy at later PD. With a more complete characterization of porcine ASC, a donor cell population that can be more efficiently reprogrammed following fusion with the oocyte might be identified.
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Venneti, Sriram, Mihir T. Garimella, Lisa M. Sullivan, Daniel Martinez, Jason T. Huse, Adriana Heguy, Mariarita Santi, Craig B. Thompson, and Alexander R. Judkins. "Evaluation of Histone 3 Lysine 27 Trimethylation (H3K27me3) and Enhancer of Zest 2 (EZH2) in Pediatric Glial and Glioneuronal Tumors Shows Decreased H3K27me3 inH3F3AK27M Mutant Glioblastomas." Brain Pathology 23, no. 5 (March 6, 2013): 558–64. http://dx.doi.org/10.1111/bpa.12042.
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Kumar, Sachin, Antony Michealraj, Leo Kim, Jeremy Rich, and Michael Taylor. "ETMM-08 METABOLIC REGULATION OF THE EPIGENOME DRIVES LETHAL INFANTILE EPENDYMOMA." Neuro-Oncology Advances 3, Supplement_1 (March 1, 2021): i15—i16. http://dx.doi.org/10.1093/noajnl/vdab024.064.
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Abstract Ependymomas are malignant glial tumours that occur throughout the central nervous system. Of the nine distinct molecular subgroups of ependymoma, Posterior Fossa A (PFA), is the most prevalent, occurring in the hindbrain of infants and young children. Lacking highly recurrent somatic mutations, PFAs are thought to be a largely epigenetically driven entity, defined by hypomethylation at the histone 3 lysine 27 residue. Previous transcriptional analysis of PFAs revealed an enrichment of hypoxia signaling genes. Thus, we hypothesized that hypoxic signaling, in combination with a unique metabolic milieu, drive PFA oncogenesis through epigenetic regulation. In this study, we identified that PFA cells control the availability of specific metabolites under hypoxic conditions, resulting in diminished H3K27 trimethylation and increased H3K27 acetylation in vitro and in vivo. Unique to PFA cells, transient exposure to ambient oxygen results in irreversible cellular toxicity. Furthermore, perturbation of key metabolic pathways is sufficient to inhibit growth of PFA primary cultures in vitro. PFA cells sequester s-adenosylmethionine while upregulating EZHIP, a polycomb repressive complex 2 (PRC2) inhibitor, resulting in decreased H3K27 trimethylation. Furthermore, hypoxia fine-tunes the abundance of alpha-ketoglutarate and acetyl-CoA to fuel demethylase and acetyltransferase activity. Paradoxically, a genome-wide CRISPR knockout screen identified the core components of PRC2 as uniquely essential in PFAs. Our findings suggest that PFAs thrive in a narrow “Goldilocks” zone, whereby they must maintain a unique epigenome and deviation to increased or decreased H3K27 trimethylation results in diminished cellular fitness. Previously, we showed that PFAs have a putative cell of origin arising in the first trimester of development. Using single-cell RNAseq and metabolomics, we demonstrate that PFAs resemble the natural metabolic-hypoxic milieu of normal development. Therefore, targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.
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Cai, Demin, Yimin Jia, Jingyu Lu, Mengjie Yuan, Shiyan Sui, Haogang Song, and Ruqian Zhao. "Maternal dietary betaine supplementation modifies hepatic expression of cholesterol metabolic genes via epigenetic mechanisms in newborn piglets." British Journal of Nutrition 112, no. 9 (September 15, 2014): 1459–68. http://dx.doi.org/10.1017/s0007114514002402.
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To elucidate the effects of maternal dietary betaine supplementation on hepatic expression of cholesterol metabolic genes in newborn piglets and the involved epigenetic mechanisms, we fed gestational sows with control or betaine-supplemented diets (3 g/kg) throughout pregnancy. Neonatal piglets born to betaine-supplemented sows had higher serum methionine concentration and hepatic content of betaine, which was associated with significantly up-regulated hepatic expression of glycine N-methyltransferase. Prenatal betaine exposure increased hepatic cholesterol content and modified the hepatic expression of cholesterol metabolic genes in neonatal piglets. Sterol regulatory element-binding protein 2 was down-regulated at both mRNA and protein levels, while 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) was down-regulated at the mRNA level, but up-regulated at the protein level, in betaine-exposed piglets. The transcriptional repression of HMGCR was associated with CpG island hypermethylation and higher repressive histone mark H3K27me3 (histone H3 lysine 27 trimethylation) on the promoter, whereas increased HMGCR protein content was associated with significantly decreased expression of miR-497. Furthermore, LDL receptor was significantly down-regulated at both mRNA and protein levels in the liver of betaine-exposed piglets, which was associated with promoter CpG hypermethylation. In addition, the expression of cholesterol-27α-hydroxylase (CYP27α1) was up-regulated at both mRNA and protein levels, while the expression of cholesterol-7α-hydroxylase (CYP7α1) was increased at the mRNA level, but unchanged at the protein level associated with increased expression of miR-181. These results indicate that maternal betaine supplementation increases hepatic cholesterol content in neonatal piglets through epigenetic regulations of cholesterol metabolic genes, which involve alterations in DNA and histone methylation and in the expression of microRNA targeting these genes.
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Thakar, Sumukh, Yash T. Katakia, Shyam Kumar Ramakrishnan, Niyati Pandya Thakkar, and Syamantak Majumder. "Intermittent High Glucose Elevates Nuclear Localization of EZH2 to Cause H3K27me3-Dependent Repression of KLF2 Leading to Endothelial Inflammation." Cells 10, no. 10 (September 26, 2021): 2548. http://dx.doi.org/10.3390/cells10102548.
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Epigenetic mechanisms have emerged as one of the key pathways promoting diabetes-associated complications. Herein, we explored the role of enhancer of zeste homolog 2 (EZH2) and its product histone 3 lysine 27 trimethylation (H3K27me3) in high glucose-mediated endothelial inflammation. To examine this, we treated cultured primary endothelial cells (EC) with different treatment conditions—namely, constant or intermittent or transient high glucose. Intermittent high glucose maximally induced endothelial inflammation by upregulating transcript and/or protein-level expression of ICAM1 and P-selectin and downregulating eNOS, KLF2, and KLF4 protein levels. We next investigated the underlining epigenetic mechanisms responsible for intermittent hyperglycemia-dependent endothelial inflammation. Compared with other high glucose treatment groups, intermittent high glucose-exposed EC exhibited an increased level of H3K27me3 caused by reduction in EZH2 threonine 367 phosphorylation and nuclear retention of EZH2. Intermittent high glucose also promoted polycomb repressive complex-2 (PRC2) assembly and EZH2′s recruitment to histone H3. Abrupt enrichment of H3K27me3 on KLF2 and KLF4 gene promoters caused repression of these genes, further supporting endothelial inflammation. In contrast, reducing H3K27me3 through small molecule and/or siRNA-mediated inhibition of EZH2 rescued KLF2 level and inhibited endothelial inflammation in intermittent high glucose-challenged cultured EC and isolated rat aorta. These findings indicate that abrupt chromatin modifications cause high glucose-dependent inflammatory switch of EC.
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Shi, Yingfeng, Liuqing Xu, Min Tao, Lu Fang, Jiasun Lu, Hongwei Gu, Shuchen Ma, et al. "Blockade of enhancer of zeste homolog 2 alleviates renal injury associated with hyperuricemia." American Journal of Physiology-Renal Physiology 316, no. 3 (March 1, 2019): F488—F505. http://dx.doi.org/10.1152/ajprenal.00234.2018.
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Hyperuricemia has been identified as an independent risk factor for chronic kidney disease (CKD) and is associated with the progression of kidney diseases. It remains unknown whether enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 methyltransferase, can regulate metabolism of serum uric acid and progression of renal injury induced by hyperuricemia. In this study, we demonstrated that blockade of EZH2 with 3-DZNeP, a selective EZH2 inhibitor, or silencing of EZH2 with siRNA inhibited uric acid-induced renal fibroblast activation and phosphorylation of Smad3, epidermal growth factor receptor (EGFR), and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in cultured renal fibroblasts. Inhibition of EZH2 also suppressed proliferation of renal fibroblasts and epithelial-mesenchymal transition of tubular cells. In a mouse model of renal injury induced by hyperuricemia, EZH2 and trimethylation of histone H3 at lysine27 expression levels were enhanced, which was coincident with renal damage and increased expression of lipocalin-2 and cleaved caspase-3. Inhibition of EZH2 with 3-DZNeP blocked all these responses. Furthermore, 3-DZNeP treatment decreased the level of serum uric acid and xanthine oxidase activity, alleviated renal interstitial fibrosis, inhibited activation of transforming growth factor-β/Smad3, EGFR/ERK1/2, and nuclear factor-κB signaling pathways, as well as reduced expression of multiple chemokines/cytokines. Collectively, EZH2 inhibition can reduce the level of serum uric acid and alleviate renal injury and fibrosis through a mechanism associated with inhibition of multiple signaling pathways. Targeting EZH2 may be a novel strategy for the treatment of hyperuricemia-induced CKD.
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Vieira, Warren, Hande Sahin, Kaylee Wells, and Catherine McCusker. "Trimethylation of Histone 3 lysine 27 (H3K27me3) ChIP-PCR and transcriptional expression data of Ef1-alpha, cyp26A, HoxC10, HoxD10 and HoxD11 in the Xenopus XTC cell line." Data in Brief 15 (December 2017): 970–74. http://dx.doi.org/10.1016/j.dib.2017.10.056.
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Hübner, Jens-Martin, Torsten Müller, Dimitris N. Papageorgiou, Monika Mauermann, Jeroen Krijgsveld, Robert B. Russell, David W. Ellison, Stefan M. Pfister, Kristian W. Pajtler, and Marcel Kool. "EZHIP/CXorf67 mimics K27M mutated oncohistones and functions as an intrinsic inhibitor of PRC2 function in aggressive posterior fossa ependymoma." Neuro-Oncology 21, no. 7 (April 29, 2019): 878–89. http://dx.doi.org/10.1093/neuonc/noz058.
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Abstract Background Posterior fossa A (PFA) ependymomas are one of 9 molecular groups of ependymoma. PFA tumors are mainly diagnosed in infants and young children, show a poor prognosis, and are characterized by a lack of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark. Recently, we reported overexpression of chromosome X open reading frame 67 (CXorf67) as a hallmark of PFA ependymoma and showed that CXorf67 can interact with enhancer of zeste homolog 2 (EZH2), thereby inhibiting polycomb repressive complex 2 (PRC2), but the mechanism of action remained unclear. Methods We performed mass spectrometry and peptide modeling analyses to identify the functional domain of CXorf67 responsible for binding and inhibition of EZH2. Our findings were validated by immunocytochemistry, western blot, and methyltransferase assays. Results We find that the inhibitory mechanism of CXorf67 is similar to diffuse midline gliomas harboring H3K27M mutations. A small, highly conserved peptide sequence located in the C-terminal region of CXorf67 mimics the sequence of K27M mutated histones and binds to the SET domain (Su(var)3-9/enhancer-of-zeste/trithorax) of EZH2. This interaction blocks EZH2 methyltransferase activity and inhibits PRC2 function, causing de-repression of PRC2 target genes, including genes involved in neurodevelopment. Conclusions Expression of CXorf67 is an oncogenic mechanism that drives H3K27 hypomethylation in PFA tumors by mimicking K27M mutated histones. Disrupting the interaction between CXorf67 and EZH2 may serve as a novel targeted therapy for PFA tumors but also for other tumors that overexpress CXorf67. Based on its function, we have renamed CXorf67 as “EZH Inhibitory Protein” (EZHIP).
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Licht, Jonathan D. "Epigenetic Regulation and Therapeutic Targeting in Myeloma." Blood 132, Supplement 1 (November 29, 2018): SCI—37—SCI—37. http://dx.doi.org/10.1182/blood-2018-99-109522.
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Abstract Genetic alterations of epigenetic regulators that alter the repressive trimethylation of lysine 27 on histone H3 (H3K27me3) are a recurrent feature in cancer. The histone demethylase UTX/KDM6A Is mutated in ~5% of multiple myeloma (MM) at diagnosis and is commonly absent in MM cell lines, derived from patients with advanced disease. KDM6A forms a complex containing H3K4 specific methyltransferases KMT2D and KMT2C, the histone acetyltransferase CBP/p300 and SWI/SNF chromatin-remodelers. Collectively the complex adds activation marks on histones and removes the gene repression associated H3K37me mark at enhancers. Removal of KDM6A from MM cells using CRISPR and re-expression of KDM6A in deficient cells showed that KDM6A has tumor suppressor function and regulates genes involved in cell adhesion. ChIP-studies showed that loss of KDM6A lead to decreased H3K27 acetylation and increased H3K27me at specific loci, but at other loci changes in H3K27Ac are the major effect of changes in KDM6A expression. KDM6A loss shifts the balance of gene expression to repression and KDM6A null cells had heightened sensitivity to EZH2 inhibitors. H3K27me/Ac chromatin modifications are also deregulated in MM associated with t(4;14) and overexpression of the NSD2/MMSET histone methyltransferase. NSD2 overexpression leads to a ~10-fold genome wide increase in histone 3 lysine 36 dimethylation (H3K36me2), a chromatin mark associated with gene activation and similar loss of H3K27me3. As a result, many genes are aberrantly activated and NSD2 aberrant stimulates cell growth in part through activation of c-myc. However, some loci are repressed in the presence of high NSD2 due to redistribution of EZH2. These genes may also be important for pathogenesis and growth since NSD2 high cells also show increased sensitivity to EZH2 inhibitors. Nevertheless, the global loss of H3K27me3 makes the chromatin structure of NSD2 overexpressing MM cells more "open" and these cells demonstrated increased DNA damage in response to genotoxic agents. At the same time NSD2 overexpression increased increases DNA repair processes making cells relatively resistant to chemotherapy, potentially explaining the poor prognosis of such patients. A point mutation in NSD2, E1099K is found in some MM patients, the commonly utilized MM.1 model cell line and in up to 15% of cases of relapsed childhood acute lymphocytic leukemia (ALL). This mutation increases the activity of the enzyme and also globally increases H3K36me2 and decreases H3K27me3. Removal of the mutant allele from ALL cells lines reverses of histone methylation patterns, deceased growth, altered cellular adhesion and increased susceptibility to therapy. RNA-seq analysis showed that similar to the UTX mutation, NSD2 mutation activated a program of cell adhesion, motility and signaling. Furthermore in in ALL, the activity of mutant NSD2 blocks glucocorticoid induced expression of pro-apoptotic genes, promoting tumor relapse. In summary deregulation of histone methylation patterns in MM plays an important role in disease pathogenesis and progression. Rebalancing histone modifications using appropriate inhibitors represent a potential therapeutic strategy. Disclosures Licht: Celgene: Research Funding.
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Xue, Zhi-Gang, Zhan-Ping Shi, Juan Dong, Ting-Ting Liao, Yan-Peng Wang, Xue-Ping Sun, Zheng-Jie Yan, et al. "Evaluation of X-Inactivation Status and Cytogenetic Stability of Human Dermal Fibroblasts after Long-Term Culture." International Journal of Cell Biology 2010 (2010): 1–5. http://dx.doi.org/10.1155/2010/289653.
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Human primary fibroblasts are a popular type of somatic cells for the production of induced pluripotent stem (iPS) cells. Here we characterized biological properties of primary fibroblasts in terms of cell-growth rate, cytogenetic stability, and the number of inactive X chromosomes during long-term passaging. We produced eight lines of female human dermal fibroblasts (HDFs) and found normal karyotype and expected pattern of X chromosome inactivation (XCI) at low passages (Passage P1-5). However, four out of the eight HDF lines at high passage numbers (≥P10) exhibited duplicated hallmarks of inactive X chromosome including two punctuate signals of histone H3 lysine 27 trimethylation (H3K27me3) and X inactive-specific transcript (XIST) RNA signals in approximately 8.5–18.5% of the cells. Our data suggest that the copy number of inactive X chromosomes in a subset of female HDF is increased by a two-fold. Consistently, DNA fluorescent in situ hybridization (FISH) identified 3-4 copies of X chromosomes in one nucleus in this subset of cells with two inactive Xs. We conclude that female HDF cultures exhibit a higher risk of genetic anomalies such as carrying an increased number of X chromosomes including both active and inactive X chromosomes at a high passage (≥P10).
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Wasson, Christopher W., Giuseppina Abignano, Heidi Hermes, Maya Malaab, Rebecca L. Ross, Sergio A. Jimenez, Howard Y. Chang, Carol A. Feghali-Bostwick, and Francesco del Galdo. "Long non-coding RNA HOTAIR drives EZH2-dependent myofibroblast activation in systemic sclerosis through miRNA 34a-dependent activation of NOTCH." Annals of the Rheumatic Diseases 79, no. 4 (February 10, 2020): 507–17. http://dx.doi.org/10.1136/annrheumdis-2019-216542.
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BackgroundSystemic sclerosis (SSc) is characterised by autoimmune activation, tissue and vascular fibrosis in the skin and internal organs. Tissue fibrosis is driven by myofibroblasts, that are known to maintain their phenotype in vitro, which is associated with epigenetically driven trimethylation of lysine 27 of histone 3 (H3K27me3).MethodsFull-thickness skin biopsies were surgically obtained from the forearms of 12 adult patients with SSc of recent onset. Fibroblasts were isolated and cultured in monolayers and protein and RNA extracted. HOX transcript antisense RNA (HOTAIR) was expressed in healthy dermal fibroblasts by lentiviral induction employing a vector containing the specific sequence. Gamma secretase inhibitors were employed to block Notch signalling. Enhancer of zeste 2 (EZH2) was blocked with GSK126 inhibitor.ResultsSSc myofibroblasts in vitro and SSc skin biopsies in vivo display high levels of HOTAIR, a scaffold long non-coding RNA known to direct the histone methyltransferase EZH2 to induce H3K27me3 in specific target genes. Overexpression of HOTAIR in dermal fibroblasts induced EZH2-dependent increase in collagen and α-SMA expression in vitro, as well as repression of miRNA-34A expression and consequent NOTCH pathway activation. Consistent with these findings, we show that SSc dermal fibroblast display decreased levels of miRNA-34a in vitro. Further, EZH2 inhibition rescued miRNA-34a levels and mitigated the profibrotic phenotype of both SSc and HOTAIR overexpressing fibroblasts in vitro.ConclusionsOur data indicate that the EZH2-dependent epigenetic phenotype of myofibroblasts is driven by HOTAIR and is linked to miRNA-34a repression-dependent activation of NOTCH signalling.
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Chapman, Michael A., Jean-Philippe Brunet, Jonathan J. Keats, Angela Baker, Mazhar Adli, Anna C. Schinzel, Gregory Ahmann, et al. "HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma." Blood 114, no. 22 (November 20, 2009): 832. http://dx.doi.org/10.1182/blood.v114.22.832.832.
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Abstract Abstract 832 We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM. Disclosures: No relevant conflicts of interest to declare.
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Brunty, Sarah, Kristeena Ray Wright, Brenda Mitchell, and Nalini Santanam. "Peritoneal Modulators of EZH2-miR-155 Cross-Talk in Endometriosis." International Journal of Molecular Sciences 22, no. 7 (March 28, 2021): 3492. http://dx.doi.org/10.3390/ijms22073492.
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Activation of trimethylation of histone 3 lysine 27 (H3K27me3) by EZH2, a component of the Polycomb repressive complex 2 (PRC2), is suggested to play a role in endometriosis. However, the mechanism by which this complex is dysregulated in endometriosis is not completely understood. Here, using eutopic and ectopic tissues, as well as peritoneal fluid (PF) from IRB-approved and consented patients with and without endometriosis, the expression of PRC2 complex components, JARID2, miR-155 (known regulators of EZH2), and a key inflammatory modulator, FOXP3, was measured. A higher expression of EZH2, H3K27me3, JARID2, and FOXP3 as well as miR-155 was noted in both the patient tissues and in endometrial PF treated cells. Gain-or-loss of function of miR-155 showed an effect on the PRC2 complex but had little effect on JARID2 expression, suggesting alternate pathways. Chromatin immunoprecipitation followed by qPCR showed differential expression of PRC2 complex proteins and its associated binding partners in JARID2 vs. EZH2 pull down assays. In particular, endometriotic PF treatment increased the expression of PHF19 (p = 0.0474), a gene silencer and co-factor that promotes PRC2 interaction with its targets. Thus, these studies have identified the potential novel crosstalk between miR-155-PRC2 complex-JARID2 and PHF19 in endometriosis, providing an opportunity to test other epigenetic targets in endometriosis.
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Fiskus, Warren, Rekha Rao, Ramesh Balusu, Jianguo Tao, Eduardo M. Sotomayor, Peter Atadja, and Kapil N. Bhalla. "Efficacy of Combined Epigenetic Targeting of Histone Methyltransferase EZH2 and Histone deacetylases Against Human Mantle Cell Lymphoma Cells." Blood 116, no. 21 (November 19, 2010): 2488. http://dx.doi.org/10.1182/blood.v116.21.2488.2488.
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Abstract Abstract 2488 Lysine specific histone methylation and deacetylation are chromatin modifications that, along with DNA methylation, are involved in the epigenetic silencing of tumor suppressor genes (TSGs). This silencing is mediated by multi-protein complexes PRC (polycomb repressive complexes) 1 and 2. Of the three core protein components of PRC2, i.e., EZH2, SUZ12 and EED, EZH2 has the SET domain with its intrinsic histone methyltransferase activity, which induces the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive histone modification mediating gene repression. The PRC1 components include BMI1, MEL18, RING1 and RING2, and it serves to further compact the chromatin at PRC2 target genes. The RING1 and RING2 proteins are responsible for the ubiquitylation of K119 on H2A. We have previously reported that treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharmaceutical Corp) depletes PRC2 complex proteins EZH2 and SUZ12 and the DNA methyltransferase (DNMT) 1. We also showed that co-treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep, further depleted PRC2 complex proteins and, in combination with PS, induced synergistic apoptosis of cultured and primary AML cells (Blood 2009; 114: 2733–43). In the present studies we determined the effects of DZNep and/or PS on the expression of PRC1 and PRC2 proteins in human Mantle Cell Lymphoma (MCL) cells. Treatment with DZNep dose-dependently depleted EZH2, SUZ12 and BMI1 expression as well as inhibited K27Me3, while inducing K27 acetylation on H3. DZNep treatment also induced p21, p27 and FBXO32, while depleting the levels of cyclin D1 in the cultured MCL JeKo-1 and MO2058 cells. Similar induction of p21, p27 and FBXO32 were also observed, following siRNA knockdown of EZH2 in the cultured MCL cells. Notably, DZNep also induced similar perturbations in primary, patient-derived MCL cells. Treatment with PS alone attenuated EZH2, SUZ12 and DNMT1, as well as depleted the protein expression of BMI1, RING2 and MEL18 in the cultured MCL cells. This was associated with attenuation of H3K27Me3 and augmentation of H3K4Me3 chromatin marks. PS treatment also induced heat shock protein (hsp) 90 acetylation, and depleted the levels of hsp90 client proteins in the MCL cells, including CDK4, c-RAF and AKT. As compared to treatment with each agent alone, co-treatment with DZNep and PS caused greater depletion of EZH2, SUZ12 and BMI1, accompanied with greater induction of p21 and p27 but attenuation of cyclin D1 expression. Co-treatment with DZNep and PS also induced cell cycle growth arrest and synergistically induced apoptosis of JeKo-1 and MO2058, as well as of primary MCL cells derived from 3 patients with MCL (combination indices <1.0). Taken together these findings indicate that by targeted depletion of the PRC2 and PRC1 components and associated chromatin and other protein modifications (hsp90 acetylation), co-treatment with DZNep and PS represents a superior therapy of human MCL cells. These studies also support the in vivo testing of combined epigenetic therapies involving agents that target deregulated epigenetic mechanisms, e.g., histone deacetylases, methyl transferases and demethylases, as well as target DNMTs in the therapy of MCL. Disclosures: Atadja: Novartis: Employment.
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Tripathy, Manoj K., Mary E. M. McManamy, Brandon D. Burch, Nancie M. Archin, and David M. Margolis. "H3K27 Demethylation at the Proviral Promoter Sensitizes Latent HIV to the Effects of Vorinostat inEx VivoCultures of Resting CD4+T Cells." Journal of Virology 89, no. 16 (June 3, 2015): 8392–405. http://dx.doi.org/10.1128/jvi.00572-15.
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ABSTRACTHistone methyltransferase inhibitors (HMTis) and histone deacetylase inhibitors (HDACis) are reported to synergistically induce the expression of latent human immunodeficiency virus type 1 (HIV-1), but studies have largely been performed with cell lines. As specific and potent HMTis directed at EZH1 (enhancer of zeste 2 Polycomb repressive complex 2 subunit 1)/EZH2 are now in human testing, we wished to rigorously test such an inhibitor in a primary resting T-cell model of HIV latency. We found that GSK343, a potent and selective EZH2/EZH1 inhibitor, reduced trimethylation of histone 3 at lysine 27 (H3K27) of the HIV provirus in resting cells. Remarkably, this epigenetic change was not associated with increased proviral expression in latently infected resting cells. However, following the reduction in H3K27 at the HIV long terminal repeat (LTR), subsequent exposure to the HDACi suberoylanilide hydroxamic acid or vorinostat (VOR) resulted in increases in HIVgagRNA and HIV p24 antigen production that were up to 2.5-fold greater than those induced by VOR alone. Therefore, in primary resting CD4+T cells, true mechanistic synergy in the reversal of HIV latency may be achieved by the combination of HMTis and HDACis. Although other cellular effects of EZH2 inhibition may contribute to the sensitization of the HIV LTR to subsequent exposure to VOR, and to increase viral antigen production, this synergistic effect is directly associated with H3K27 demethylation at nucleosome 1 (Nuc-1). Based upon our findings, the combination of HMTis and HDACis should be considered for testing in animal models or clinical trials.IMPORTANCEDemethylation of H3K27 mediated by the histone methyltransferase inhibitor GSK343 in primary resting T cells is slow, occurring over 96 h, but by itself does not result in a significant upregulation of cell-associated HIV RNA expression or viral antigen production. However, following H3K27 demethylation, latent viral expression within infected primary resting CD4+T cells is synergistically increased upon exposure to the histone deacetylase inhibitor vorinostat. Demethylation at H3K27 sensitizes the HIV promoter to the effects of an HDACi and provides a proof-of-concept for the testing of combination epigenetic approaches to disrupt latent HIV infection, a necessary step toward the eradication of HIV infection.
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Fujiwara, Tohru, Haruka Saitoh, Yoko Okitsu, Noriko Fukuhara, Yasushi Onishi, Kenichi Ishizawa, Ryo Ichinohasama, and Hideo Harigae. "Regulation of Erythropoiesis by Histone Methyltransferase EZH2 Inhibitor 3-Deazaneplanocin A (DZNep)." Blood 120, no. 21 (November 16, 2012): 982. http://dx.doi.org/10.1182/blood.v120.21.982.982.
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Abstract Abstract 982 Background. EZH2, a core component of Polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through mediating trimethylation of histone H3 at lysine 27 (H3K27), and is involved in various biological processes, including hematopoiesis. Overexpression of EZH2 has been identified in a wide range of solid tumors as well as hematological malignancies. Recent studies indicated that 3-deazaneplanocin A (DZNep), an inhibitor of EZH2, preferentially induces apoptosis in cancer cells, including acute myeloid leukemia and myelodysplastic syndromes, implying that EZH2 may be a potential new target for epigenetic treatment. On the other hand, whereas PRC2 complex has been reported to participate in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation (Yu et al. Mol Cell 2009; Ross et al. MCB 2012), the impact of DZNep on erythropoiesis has not been evaluated. Method. The K562 erythroid cell line was used for the analysis. The cells were treated with DZNep at doses of 0.2 and 1 microM for 72 h. Quantitative ChIP analysis was performed using antibodies to acetylated H3K9 and GATA-1 (Abcam). siRNA-mediated knockdown of EZH2 was conducted using Amaxa nucleofection technology™ (Amaxa Inc.). For transcription profiling, SurePrint G3 Human GE 8 × 60K (Agilent) and Human Oligo chip 25K (Toray) were used for DZNep-treated and EZH2 knockdown K562 cells, respectively. Gene Ontology was analyzed using the DAVID Bioinformatics Program (http://david.abcc.ncifcrf.gov/). Results. We first confirmed that DZNep treatment decreased EZH2 protein expression without significantly affecting EZH2 mRNA levels, suggesting that EZH2 was inhibited at the posttranscriptional level. We also confirmed that DZNep treatment significantly inhibited cell growth. Interestingly, the treatment significantly induced erythroid differentiation of K562 cells, as determined by benzidine staining. Transcriptional profiling with untreated and DZNep-treated K562 cells (1 microM) revealed that 789 and 698 genes were upregulated and downregulated (> 2-fold), respectively. The DZNep-induced gene ensemble included prototypical GATA-1 targets, such as SLC4A1, EPB42, ALAS2, HBA, HBG, and HBB. Concomitantly, DZNep treatment at both 0.2 and 1 microM upregulated GATA-1 protein level as determined by Western blotting, whereas the effect on its mRNA levels was weak (1.02- and 1.43-fold induction with 0.2 and 1 microM DZNep treatment, P = 0.73 and 0.026, respectively). Furthermore, analysis using cycloheximide treatment, which blocks protein synthesis, indicated that DZNep treatment could prolong the half-life of GATA-1 protein, suggesting that DZNep may stabilize GATA-1 protein, possibly by affecting proteolytic pathways. Quantitative ChIP analysis confirmed significantly increased GATA-1 occupancy as well as increased acetylated H3K9 levels at the regulatory regions of these target genes. Next, to examine whether the observed results of DZNep treatment were due to the direct inhibition of EZH2 or hitherto unrecognized effects of the compound, we conducted siRNA-mediated transient knockdown of EZH2 in K562 cells. Quantitative RT-PCR analysis demonstrated that siRNA-mediated EZH2 knockdown had no significant effect on the expression of GATA-1 as well as erythroid-lineage related genes. Furthermore, transcription profiles of the genes in the quantitative range of the array were quite similar between control and EZH2 siRNA-treated K562 cells, with a correlation efficient of 0.977. Based on our profiling results, we are currently exploring the molecular mechanisms by which DZNep promotes erythroid differentiation of K562 cells. Conclusion. DZNep promotes erythroid differentiation of K562 cells, presumably through a mechanism not directly related to EZH2 inhibition. Our microarray analysis of DZNep-treated K562 cells may provide a better understanding of the mechanism of action of DZNep. Disclosures: No relevant conflicts of interest to declare.
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Luo, Yujie, Yuanjian Fang, Ruiqing Kang, Cameron Lenahan, Marcin Gamdzyk, Zeyu Zhang, Takeshi Okada, Jiping Tang, Sheng Chen, and John H. Zhang. "Inhibition of EZH2 (Enhancer of Zeste Homolog 2) Attenuates Neuroinflammation via H3k27me3/SOCS3/TRAF6/NF-κB (Trimethylation of Histone 3 Lysine 27/Suppressor of Cytokine Signaling 3/Tumor Necrosis Factor Receptor Family 6/Nuclear Factor-κB) in a Rat Model of Subarachnoid Hemorrhage." Stroke 51, no. 11 (November 2020): 3320–31. http://dx.doi.org/10.1161/strokeaha.120.029951.
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Background and Purpose: Neuroinflammation has been proven to play an important role in the pathogenesis of early brain injury after subarachnoid hemorrhage (SAH). EZH2 (enhancer of zeste homolog 2)-mediated H3K27Me3 (trimethylation of histone 3 lysine 27) has been recognized to play a critical role in multiple inflammatory diseases. However, there is still a lack of evidence to address the effect of EZH2 on the immune response of SAH. Therefore, the aim of this study was to determine the role of EZH2 in SAH-induced neuroinflammation and explore the effect of EZH2 inhibition with its specific inhibitor EPZ6438. Methods: The endovascular perforation method was performed on rats to induce subarachnoid hemorrhage. EPZ6438, a specific EZH2 inhibitor, was administered intraperitoneally at 1 hour after SAH. SOCS3 (Suppressor of cytokine signaling 3) siRNA and H3K27me3 CRISPR were administered intracerebroventricularly at 48 hours before SAH to explore potential mechanisms. The SAH grade, short-term and long-term neurobehavioral tests, immunofluorescence staining, and western blots were performed after SAH. Results: The expression of EZH2 and H3K27me3 peaked at 24 hours after SAH. In addition, inhibition of EZH2 with EPZ6438 significantly improved neurological deficits both in short-term and long-term outcome studies. Moreover, EPZ6438 treatment significantly decreased the levels of EZH2, H3K27Me3, pathway-related proteins TRAF6 (TNF [tumor necrosis factor] receptor family 6), NF-κB (nuclear factor-κB) p65, proinflammatory cytokines TNF-α, IL (interleukin)-6, IL-1β, but increased the expression levels of SOCS3 and anti-inflammatory cytokine IL-10. Furthermore, administration of SOCS3 siRNA and H3k27me3-activating CRISPR partly abolished the neuroprotective effect of EPZ6438, which indicated that the neuroprotective effect of EPZ6438 acted, at least partly, through activation of SOCS3. Conclusions: In summary, the inhibition of EZH2 by EPZ6438 attenuated neuroinflammation via H3K27me3/SOCS3/TRAF6/NF-κB signaling pathway after SAH in rats. By targeting EZH2, this study may provide an innovative method to ameliorate early brain injury after SAH.
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Whalen, Courtney A., Floyd J. Mattie, Cristina Florindo, Bertrand van Zelst, Neil K. Huang, Isabel Tavares de Almeida, Sandra G. Heil, Thomas Neuberger, A. Catharine Ross, and Rita Castro. "No Effect of Diet-Induced Mild Hyperhomocysteinemia on Vascular Methylating Capacity, Atherosclerosis Progression, and Specific Histone Methylation." Nutrients 12, no. 8 (July 23, 2020): 2182. http://dx.doi.org/10.3390/nu12082182.
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Hyperhomocysteinemia (HHcy) is a risk factor for atherosclerosis through mechanisms which are still incompletely defined. One possible mechanism involves the hypomethylation of the nuclear histone proteins to favor the progression of atherosclerosis. In previous cell studies, hypomethylating stress decreased a specific epigenetic tag (the trimethylation of lysine 27 on histone H3, H3K27me3) to promote endothelial dysfunction and activation, i.e., an atherogenic phenotype. Here, we conducted a pilot study to investigate the impact of mild HHcy on vascular methylating index, atherosclerosis progression and H3K27me3 aortic content in apolipoprotein E-deficient (ApoE −/−) mice. In two different sets of experiments, male mice were fed high-fat, low in methyl donors (HFLM), or control (HF) diets for 16 (Study A) or 12 (Study B) weeks. At multiple time points, plasma was collected for (1) quantification of total homocysteine (tHcy) by high-performance liquid chromatography; or (2) the methylation index of S-adenosylmethionine to S-adenosylhomocysteine (SAM:SAH ratio) by liquid chromatography tandem-mass spectrometry; or (3) a panel of inflammatory cytokines previously implicated in atherosclerosis by a multiplex assay. At the end point, aortas were collected and used to assess (1) the methylating index (SAM:SAH ratio); (2) the volume of aortic atherosclerotic plaque assessed by high field magnetic resonance imaging; and (3) the vascular content of H3K27me3 by immunohistochemistry. The results showed that, in both studies, HFLM-fed mice, but not those mice fed control diets, accumulated mildly elevated tHcy plasmatic concentrations. However, the pattern of changes in the inflammatory cytokines did not support a major difference in systemic inflammation between these groups. Accordingly, in both studies, no significant differences were detected for the aortic methylating index, plaque burden, and H3K27me3 vascular content between HF and HFLM-fed mice. Surprisingly however, a decreased plasma SAM: SAH was also observed, suggesting that the plasma compartment does not always reflect the vascular concentrations of these two metabolites, at least in this model. Mild HHcy in vivo was not be sufficient to induce vascular hypomethylating stress or the progression of atherosclerosis, suggesting that only higher accumulations of plasma tHcy will exhibit vascular toxicity and promote specific epigenetic dysregulation.
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Williamson, G. L., J. H. Pryor, K. Tessanne, M. C. Golding, and C. R. Long. "201 SUPPRESSION OF Suz12 IN BOVINE PREIMPLANTATION EMBRYOS VIA CYTOPLASMIC SMALL INTERFERING RNA INJECTION." Reproduction, Fertility and Development 23, no. 1 (2011): 200. http://dx.doi.org/10.1071/rdv23n1ab201.
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The proper removal of gametic epigenetic marks and coordinated reestablishment of the epigenome are critical to mammalian embryonic development. Suz12 is a member of Polycomb repressive complex 2, known to catalyse trimethylation of lysine 27 on histone 3 (H3K27me3; Pasini et al. 2004); Suz12 has also been shown to interact with Suv39h1 for proper trimethylation of H3K9 (de la Cruz et al. 2007). Our objective in this study was to suppress expression of Suz12 via cytoplasmic injection of small interfering RNA (siRNA) targeted at this gene, and to evaluate the effect on embryo development rates. Bovine zygotes were produced in vitro via standard laboratory procedures. Nineteen hours post-fertilization, presumptive zygotes (n = 3979) were divided into 3 treatment groups: noninjected control (CTL), or injected with a fluorescent dextran marker combined with either a nontargeting siRNA (NULL) or an Suz12-targeting siRNA (SUZ). Embryos were cultured in Bovine Evolve (Zenith Biotech, Canada) with 4 mg mL–1 of BSA (Probumin, Millipore, Billerica, MA) and collected at the 4-cell, 8-cell, morula, and blastocyst stages. Ribonucleic acid was isolated with an RNeasy® Mini Kit (Qiagen, Valencia, CA) from 3 replicates of pooled embryos at each stage (4-cell, n = 15; 8-cell, n = 20; morula, n = 10), except for the blastocyst stage [2 samples (n = 10) collected from the CTL and NULL groups, and 1 sample (n = 3) from the SUZ group]. Each RNA sample was reverse-transcribed into cDNA and diluted for use by quantitative real-time PCR. Relative gene expression levels from each sample were calculated in triplicate using the SYBR Green comparative Ct method (Applied Biosystems, Foster City, CA), adjusted according to individual PCR efficiencies for each primer pair (R2 > 0.95) and normalized to the geometric mean Ct of 3 endogenous controls (GAPDH, YWHAZ, and SDHA), to account for differences in both cell number and amount of total mRNA present in each sample (Goossens et al. 2005). Our data indicate that Suz12 expression was suppressed to undetectable levels in SUZ-treated zygotes at all embryo stages analysed. The blastocyst rate of the SUZ group was extremely low (0.88 ± 0.16% SEM) compared with the CTL (19.87 ± 0.36% SEM) and NULL (5.09 ± 0.36% SEM) groups. Morphologically, SUZ morulae appeared fragmented with fewer larger cells than expected, whereas the NULL and CTL morulae seemed to develop normally. We presume the loss of Suz12 expression during this important developmental time is detrimental to embryo morphology and results in a decreased rate of blastocyst formation. Because of this decrease, we were able to collect only 3 SUZ blastocysts from a total of 227 injected. The microinjection procedure also contributed to significant (P < 0.05) decreases in blastocyst rates of the injected groups as compared with the CTL. Future experiments will explore potential alterations in histone methylation, as well as other epigenetic modifiers in bovine preimplantation embryos, to further elucidate the role of the epigenome in early embryonic development.
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Carr, Ryan M., Terra Lasho, David Marks, Ezequiel Tolosa, Luciana L. Almada, Bonnie Alver, Moritz Binder, et al. "Clinical Categorization of Chronic Myelomonocytic Leukemia into Proliferative and Dysplastic Subtypes Correlates with Distinct Genomic, Transcriptomic and Epigenomic Signatures." Blood 134, Supplement_1 (November 13, 2019): 1710. http://dx.doi.org/10.1182/blood-2019-123877.
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Introduction: Chronic myelomonocytic leukemia (CMML), an aggressive myeloid malignancy, can be categorized into two subtypes, proliferative CMML (pCMML) and dysplastic (dCMML), based on a white blood cell (WBC) count of ≥ 13 x 109/L for the former (Arber et al. Blood 2016). While this WBC cut off is somewhat arbitrary, patients with pCMML have unique phenotypic features and a shorter survival. We carried out this study to assess the genomic, transcriptomic and epigenetic landscapes of these two CMML subtypes. Methods: Peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNC) were obtained from WHO-defined CMML patients. Next generation sequencing (NGS) using a 36-gene panel was performed on 350 patients with Illumina HiSeq4000 platform with median read depth of 400X. RNA sequencing (RNA-seq) was performed on 25 patients by bulk whole transcriptome sequencing using Illumina TruSeq. DNA immunoprecipitation sequencing (DIP-seq) was performed on 18 patients using 5-methylcystocine (5mC), 5-hydroxymethylcytosine (5hmC) and bridging monoclonal antibodies with subsequent paired-end sequencing using HiSeq4000. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed on 30 patients with Illumina HiSeq2500 to a depth of 25 million for histone 3 lysine 4 monomethylation (H3K4me1) and histone 3 lysine 4 trimethylation (H3K4me3) and 50 million reads for histone 3 lysine 27 trimethylation (H3K27me3) and Input per sample. Results: Five hundred and seventy-three patients with WHO defined CMML were included; median age 71 years (range 18-95 years), 67% males. 282 patients had pCMML (49%), while 291 (51%) had dCMML. As pre-defined, patients with pCMML were more likely to have higher absolute monocyte counts (p<0.0001), circulating immature myeloid cells (p<0.0001), PB blasts (p<0.0001), and higher lactate dehydrogenase levels (p=0.03). At last follow up 234 (41%) deaths and 70 (20%) leukemic transformations were documented. The median OS for pCMML vs dCMML in this cohort was 19 months vs 30 months (p<0.0001, Figure 1A) and validated in an independent Austrian cohort (p=0.02). Genomic profiling: NGS performed on 350 patients (BM MNC) revealed a higher frequency of NRAS (35 vs 17%, p=0.004), cumulative RAS pathway (NRAS, KRAS, CBL and PTPN11) (73 vs 47%, p=0.001), ASXL1 (p=0.003) and JAK2V617F (p=0.04) mutations in pCMML relative to dCMML (Figure 1B); while dCMML had a higher frequency of SF3B1 mutations (p=0.02). There were no differences in distribution of TET2 and SRSF2Transcriptomic analysis: RNA-seq was performed on PB MNC from RAS pathway mutant pCMML patients (n=12) and RAS pathway wildtype dCMML patients (n=13). Unsupervised clustering analysis resulted in appropriate segregation revealing distinct expression profiles between disease subtypes (Figure 1C). Compared to dCMML, 3729 genes were significantly differentially upregulated and 2658 genes were differentially downregulated in pCMML. Among genes most highly upregulated were mitotic checkpoint kinases including AURBK, PLK1, PLK2, PLK4 andEpigenetic profiling: ChIP-seq of PB and BM MNC from pCMML (n=18) and dCMML (n=12) patients and healthy, age-matched controls (n=10) revealed a global increase in H3K4me1, without significant differences in H3K4me3 or H3K27me3 occupancies (regardless of stratification by ASXL1 mutational status; 40% ASXL1mt in pCMML, 30% dCMML) in pCMML vs dCMML (Figure 1D). H3K4me1 occupancy was also increased in a sequence-specific manner at the transcription start sites of the aforementioned mitotic kinases (PLK1 and WEE1). DIP-seq was performed on PB MNC to assess global differences in 5-mC and 5-hmC levels, between pCMML (n=9) and dCMML (n=9), with no differences seen between the two subtypes (regardless of TET2 mutational status, 40% TET2mt in each subtype) (Figure 1E). Conclusions: Despite the somewhat arbitrary WBC distinction between pCMML and dCMML, clear phenotypic, genetic, transcriptomic, epigenetic and survival differences exist between the two subtypes, providing strong biological rationale for this distinction. pCMML patients have a higher frequency of oncogenic RAS pathway mutations, a unique transcriptomic profile enriched in mitotic check point kinases and a unique chromatin configuration with global and sequence specific enrichment in H3K4me1, with no significant global differences in 5mC, 5hmC, or H3K4me3 and H3K27me3 occupancy. Figure 1 Disclosures Geissler: AOP: Honoraria; Pfizer: Honoraria; AstraZeneca: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Roche: Honoraria; Abbvie: Honoraria; Ratiopharm: Honoraria; Amgen: Honoraria. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.
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Ikeda, S., M. Sugimoto, and S. Kume. "The RPMI-1640 vitamin mixture promotes bovine blastocyst development in vitro and downregulates gene expression of TXNIP with epigenetic modification of associated histones." Journal of Developmental Origins of Health and Disease 9, no. 1 (August 2, 2017): 87–94. http://dx.doi.org/10.1017/s2040174417000563.
Full textAbstract:
Diverse environmental conditions surrounding preimplantation embryos, including available nutrients, affect their metabolism and development in both short- and long-term manner. Thioredoxin-interacting protein (TXNIP) is a possible marker for preimplantation stress that is implicated in in vitro fertilization- (IVF) induced long-term DOHaD effects. B vitamins, as participants in one-carbon metabolism, may affect preimplantation embryos by epigenetic alterations of metabolically and developmentally important genes. In vitro-produced bovine embryos were cultured with or without Roswell Park Memorial Institute 1640 vitamin mixture, containing B vitamins and B vitamin-like substances, from day 3 after IVF and we evaluated blastocyst development and TXNIP messenger RNA (mRNA) expression in the blastocysts by reverse transcription-quantitative polymerase chain reaction. The degree of trimethylation of histone H3 lysine 27 (H3K27me3) at TXNIP promoter was examined semi-quantitatively by chromatin immunoprecipitation polymerase chain reaction. Total H3K27me3 were also compared between the groups by Western blot analysis. The vitamin treatment significantly increased the rates of blastocyst development (P<0.05) and their hatching (P<0.001) from the zona pellucida by day 8. The mRNA expression of TXNIP was lower (P<0.01) in blastocysts in the vitamin-mixture-treated group concomitant with higher (P<0.05) level of H3K27me3 of its promoter compared with the control group. The total H3K27me3 in the vitamin-mixture-treated group was also higher (P<0.01) than that in the control group. The epigenetic control of genes related to important metabolic processes during the periconceptional period by nutritional conditions in utero and/or in vitro may have possible implication for the developmental programming during this period that may impact the welfare and production traits of farm animals.
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Yamagata, Kazutsune, Yukiko Aikawa, Mika Shino, and Issay Kitabayashi. "Leukemia-Associated MLL-AF10 Fusion Maintains an Active State of Chromatin on Its Target Genes By Recruiting Tip60." Blood 126, no. 23 (December 3, 2015): 304. http://dx.doi.org/10.1182/blood.v126.23.304.304.
Full textAbstract:
Abstract Chromosome translocation involving the mixed lineage leukemia (MLL) gene which generates an in-frame fusion gene of the MLL 5′-region and partner genes, is a common rearrangement in acute myeloid and lymphoid leukemia that is associated with poor prognosis. Knock-in and retroviral transduction studies show that MLL-fusion results in constitutive activation of the transcription of target genes such as Hoxa9 and Meis1 during the development of leukemia. Recent studies show that several transcription regulators, such as Dot1L, Cbx8, PAF1, and AEP/EAP complexes, are required for the leukemogenic activity of MLL-fusion; however, the underlying mechanisms remain elusive. To clarify the mechanism of epigenetic regulation by MLL-fusions, we established a novel leukemia model by generating a conditional MLL-AF10 fusion gene, MLL-AF10 flox, in which the 3′-AF10 region is deleted by 4-OHT-activated Cre-ERT recombinase, resulting in inactivation of MLL-AF10 flox (Figure 1A). Mouse hematopoietic stem/progenitor cells (c-kit+) were immortalized by retroviral transduction of MLL-AF10 flox and cultured in vitro or transplanted into irradiated recipient mice to induce AML in vivo. Treatment of MLL-AF10 flox cells with 4-OHT in vitro to inactivate MLL-AF10 flox downregulated Hoxa9 expression and markedly decreased colony-forming ability. In addition, the inactivation of MLL-AF10 flox rapidly decreased the acetylation level of the histone H2A variant H2A.Z on the Hoxa9 locus. These results suggest that MLL-AF10, possibly together with a histone acetyltransferase (HAT), regulates the acetylation of H2A.Z on the Hoxa9 locus. To identify the HAT responsible for H2A.Z acetylation induced by MLL-AF10, protein complexes associated with H2A.Z-containing nucleosomes were purified, resulting in the identification of Tip60, a MYST-type HAT in a complex with H2A.Z. MLL-AF10 physically interacted with Tip60 via the AF10 C-terminal portion of MLL-AF10 (Figure 1B). ChIP analysis showed that MLL-AF10 and Tip60 co-localize on the Hoxa9 locus in MLL-AF10-transformed cells (MLL-AF10 cells). Furthermore, conditional deletion of Tip60 in MLL-AF10 (Tip60 Flox/Flox, Cre-ERT2) cells dramatically downregulated Hoxa9 expression and resulted in the accumulation of unacetylated H2A.Z on the Hoxa9 locus. Consistent with these data, in vitro acetylation analysis showed that Tip60 directly acetylates H2A.Z. To assess the role of Tip60 in leukemia development in vivo, MLL-AF10 (Tip60 Flox/Flox, Cre-ERT2) leukemia cells were injected into recipient mice. Animals receiving intraperitoneal injection of tamoxifen to delete Tip60 failed to develop MLL-AF10 leukemia (Figure 1C). These data indicate that Tip60 is required for the development of MLL-AF10 leukemia and suggest that MLL-AF10 recruits Tip60 to acetylate H2A.Z on the Hoxa9 locus. The effect of H2A.Z acetylation on Hoxa9 expression was examined by purifying nucleosomes containing acetylation-deficient 3KR H2A.Z (which mimics unacetylated H2A.Z), in which lysines 4, 7, and 11 were substituted by arginine. 3KR H2A.Z preferentially formed nucleosomes with histone H3 trimethylation at lysine 27, which is catalyzed by polycomb repressive complex 2 (PRC2). This finding suggests that nucleosomes including unacetylated H2A.Z are the preferential targets of PRC2. Loss of Tip60 in MLL-AF10 cells resulted in decreased levels of acetylated H2A.Z on the Hoxa9 locus and the recruitment of Ezh2 (a catalytic subunit of PRC2) and increased histone H3 K27 trimethylation. Taken together, these data indicate that Tip60 is a critical factor in the development of MLL-AF10 leukemia. MLL-AF10 may maintain an active chromatin state on its target genesby recruiting Tip60, which acetylates H2A.Z to prevent PRC2 recruitment and gene silencing. On the other hand, unacetylated H2A.Z may be a signal for PRC2 recruitment, which would be induced as a result of Tip60 loss or inactivation of MLL-AF10. Figure 1. Figure 1. Disclosures Kitabayashi: Daiichi Sankyo Co., Ltd.: Research Funding.
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Stomper, Julia, Ruth Meier, Tobias Ma, Dietmar Pfeifer, Annette Schmitt-Graeff, and Michael Lübbert. "Integrative Study of EZH2 Mutational Status, Copy Number, Protein Expression and H3K27 Trimethylation in AML/MDS Patients." Blood 134, Supplement_1 (November 13, 2019): 1422. http://dx.doi.org/10.1182/blood-2019-128231.
Full textAbstract:
Introduction Enhancer of Zeste Homolog 2 (EZH2), the catalytic domain of Polycomb Repressive Complex 2 (PRC2), mediates the repressive mark of trimethylation of histone H3 lysine 27 (H3K27me3). The EZH2 gene is located on chromosome (chr.) 7q36.1 (frequently deleted in AML/MDS). Its role in tumorigenesis appears to be context-dependent since both EZH2 overexpression and loss of function are associated with different types of cancer. In patients (pts) with MDS or MDS/MPN, loss-of-function EZH2 mutations (mut) are recurrently found, associated with a poor prognosis, and EZH2 is considered a tumor suppressor gene (TSG). In AML, the incidence of EZH2-mut is lower and less well-studied. We wished to determine the effects of EZH2-mut and copy number (CN) on EZH2 expression, and the consequences for H3K27me3 levels in vivo. Methods Fifty-eight pts (51 AML, 3 MDS, 4 MDS/MPN), median age 63.5 years (yr, range 20-86, almost all diagnosed between 2015 and 2017), with available sequencing data, EZH2 CN status and EZH2 expression data were studied. Mutation status was determined by next-generation sequencing (NGS) including a panel of 54 genes (Illumina Myeloid NGS panel). Metaphase cytogenetics and/or fluorescence in situ hybridization and single nucleotide polymorphism arrays (selected pts) were conducted to determine EZH2 CN status. Protein expression of EZH2 and H3K27me3 was assessed semi-quantitatively by immunohistochemistry (IHC) on formalin-fixed EDTA-decalcified paraffin-embedded bone marrow (BM) core biopsies. The Kruskal-Wallis test and Dunn's multiple comparison test were used to address whether EZH2 protein expression was associated with EZH2-mut and CN. Results The EZH2 gene showed mutations (mostly missense or nonsense, median variant allele frequency (VAF) 45%, range 7-63%) in 13/58 pts and was unmutated in 45/58 pts. EZH2-mut pts had a median of 4 mutations. Additional mutations were most frequently found in ASXL1 (10/13; median VAF 35%, range 19-48%), less frequently in TET2, RUNX1, STAG2 (3/13 each), NRAS (2/13), and DNMT3A (1/13). In contrast, EZH2-wt pts had a median of 2 mutations, most frequently in DNMT3A (12/45), followed by NRAS (10/45), IDH1 (9/45), FLT3 (8/45), and NPM1 (7/45). Regarding chr. 7, 43 pts had no detectable deletion, 15 had 7q-/-7. Notably, the incidence of EZH2-mut was similar in pts with 7q-/-7 lesions (3/15, i.e. 20%) and pts with normal chr. 7 (10/43, 23%). EZH2 expression in neoplastic BM cells ranked from no (score 0) to strong expression (score 3). While the hematopoietic BM cells of healthy donors usually showed a moderate EZH2 expression (score 2), in our cohort the score was 0 in 3, 1 in 13, 2 in 23, and 3 in 19 pts, respectively. We next asked whether EZH2 protein expression differed between pts depending on EZH2-mut and chr. 7 status. In Figure 1, 4 subgroups are depicted, showing highest expression in pts with EZH2-wt and either no chr. 7 abnormalities (group A, n=33) or 7q-/-7 (group C, n=12). In comparison, expression was significantly reduced in pts with EZH2-mut and no chr. 7 abnormalities (group B, n=10), and lowest in pts with EZH2-mut and chr. 7 abnormalities (group D, n=3), p<0.05 (indicated by asterisks). Since functional EZH2 protein is necessary for the trimethylation of H3K27, the presence of this histone mark was also determined semi-quantitatively by IHC in 40 pts. H3K27me3 levels were variable, and a test for a possible association between EZH2 and H3K27me3 levels by linear regression analysis did not show an association (R²=0.13). Sixty-two % of EZH2-mut (median age 71 yr) and 51% of EZH2-wt pts (median age 61 yr) received allografting. Median overall survival in EZH2-mut pts was 16.1 months compared to 23.5 in EZH2-wt pts (p=0.16). Conclusions Inactivating EZH2 mutations are infrequent events in AML. In our study, they were strongly associated with mutations of ASXL1 (also involved in PRC2 function). We did not observe overrepresentation of EZH2-mut in 7q-/-7 pts, in line with Bejar et al. (N Engl J Med 2011); this is in contrast to the frequent cooperative events between TSG mutations and deletions, e.g. of TP53. Functionally, EZH2 mutations appeared to have a stronger effect on decreased EZH2 expression than hemizygosity. Global H3K27me3 levels were not altered by EZH2 reduction, which could be due to compensatory upregulation of EZH1, supporting the clinical development of EZH1/2 inhibition. Disclosures No relevant conflicts of interest to declare.