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1
Yan, Dongsheng, Yong Zhang, Lifang Niu, Yi Yuan, and Xiaofeng Cao. "Identification and characterization of two closely related histone H4 arginine 3 methyltransferases in Arabidopsis thaliana." Biochemical Journal 408, no. 1 (October 29, 2007): 113–21. http://dx.doi.org/10.1042/bj20070786.
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Arginine methylation of histone H3 and H4 plays important roles in transcriptional regulation in eukaryotes such as yeasts, fruitflies, nematode worms, fish and mammals; however, less is known in plants. In the present paper, we report the identification and characterization of two Arabidopsis thaliana protein arginine N-methyltransferases, AtPRMT1a and AtPRMT1b, which exhibit high homology with human PRMT1. Both AtPRMT1a and AtPRMT1b methylated histone H4, H2A, and myelin basic protein in vitro. Site-directed mutagenesis of the third arginine (R3) on the N-terminus of histone H4 to lysine (H4R3N) completely abolished the methylation of histone H4. When fused to GFP (green fluorescent protein), both methyltransferases localized to the cytoplasm as well as to the nucleus. Consistent with their subcellular distribution, GST (glutathione transferase) pull-down assays revealed an interaction between the two methyltransferases, suggesting that both proteins may act together in a functional unit. In addition, we demonstrated that AtFib2 (Arabidopsis thaliana fibrillarin 2), an RNA methyltransferase, is a potential substrate for AtPRMT1a and AtPRMT1b, and, furthermore, uncovered a direct interaction between the protein methyltransferase and the RNA methyltransferase. Taken together, our findings implicate AtPRMT1a and AtPRMT1b as H4-R3 protein arginine N-methyltransferases in Arabidopsis and may be involved in diverse biological processes inside and outside the nucleus.
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Poreba, Elzbieta, Krzysztof Lesniewicz, and Julia Durzynska. "Aberrant Activity of Histone–Lysine N-Methyltransferase 2 (KMT2) Complexes in Oncogenesis." International Journal of Molecular Sciences 21, no. 24 (December 8, 2020): 9340. http://dx.doi.org/10.3390/ijms21249340.
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KMT2 (histone-lysine N-methyltransferase subclass 2) complexes methylate lysine 4 on the histone H3 tail at gene promoters and gene enhancers and, thus, control the process of gene transcription. These complexes not only play an essential role in normal development but have also been described as involved in the aberrant growth of tissues. KMT2 mutations resulting from the rearrangements of the KMT2A (MLL1) gene at 11q23 are associated with pediatric mixed-lineage leukemias, and recent studies demonstrate that KMT2 genes are frequently mutated in many types of human cancers. Moreover, other components of the KMT2 complexes have been reported to contribute to oncogenesis. This review summarizes the recent advances in our knowledge of the role of KMT2 complexes in cell transformation. In addition, it discusses the therapeutic targeting of different components of the KMT2 complexes.
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Park, Ki-Eun, Christine M. Johnson, and Ryan A. Cabot. "IVMBIX-01294, an inhibitor of the histone methyltransferase EHMT2, disrupts histone H3 lysine 9 (H3K9) dimethylation in the cleavage-stage porcine embryo." Reproduction, Fertility and Development 24, no. 6 (2012): 813. http://dx.doi.org/10.1071/rd11205.
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Global patterns of histone methylation are remodelled during cleavage development. Of the five histone methyltransferases known to mediate methylation of the lysine 9 residue of histone H3 (H3K9), euchromatic histone-lysine N-methyltransferase 2 (EHMT2; also known as G9a) has been shown to be a primary mediator of H3K9 dimethylation; BIX-01294 has been shown to be a specific inhibitor of EHMT2. The objective of the present study was to determine the effect of BIX-01294 treatment on global H3K9 dimethylation in porcine embryos. We hypothesised that inhibition of EHMT2 by BIX-01294 would result in reduced levels of H3K9 dimethylation and compromised embryo development. Our results showed that incubation in 5 µM BIX-01294 markedly reduced global levels of H3K9 dimethylation at the pronuclear, 2-cell and 4-cell stages of development and resulted in developmental arrest before blastocyst formation. Although transient exposure of embryos to BIX-01294 did not alter in vitro development, embryos transiently exposed to BIX-01294 did not establish pregnancy. These data demonstrate that BIX-01294 is a potent inhibitor of H3K9 dimethylation and that transient alterations in global histone modifications can have profound effects on embryo developmental potential.
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Dai, Shaobo, Matthew V. Holt, John R. Horton, Clayton B. Woodcock, Anamika Patel, Xing Zhang, Nicolas L. Young, Alex W. Wilkinson, and Xiaodong Cheng. "Characterization of SETD3 methyltransferase–mediated protein methionine methylation." Journal of Biological Chemistry 295, no. 32 (June 5, 2020): 10901–10. http://dx.doi.org/10.1074/jbc.ra120.014072.
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Most characterized protein methylation events encompass arginine and lysine N-methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methionine substitutions at positions 27 and 36 of histone H3.3. In these instances, the methionine substitution localizes to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain–containing 3 (SETD3) is a protein (i.e. actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating S-methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr312 and Trp273, as well as the hydrocarbon side chain of Ile310. Our results suggest that placing methionine properly in the active site—within close proximity to and in line with the incoming methyl group of SAM—would allow some SET domain proteins to selectively methylate methionine in proteins.
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Gauthier, Nancy, Mireille Caron, Liliana Pedro, Mathieu Arcand, Julie Blouin, Anne Labonté, Claire Normand, et al. "Development of Homogeneous Nonradioactive Methyltransferase and Demethylase Assays Targeting Histone H3 Lysine 4." Journal of Biomolecular Screening 17, no. 1 (September 21, 2011): 49–58. http://dx.doi.org/10.1177/1087057111416659.
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Histone posttranslational modifications are among the epigenetic mechanisms that modulate chromatin structure and gene transcription. Histone methylation and demethylation are dynamic processes controlled respectively by histone methyltransferases (HMTs) and demethylases (HDMs). Several HMTs and HDMs have been implicated in cancer, inflammation, and diabetes, making them attractive targets for drug therapy. Hence, the discovery of small-molecule modulators for these two enzyme classes has drawn significant attention from the pharmaceutical industry. Herein, the authors describe the development and optimization of homogeneous LANCE Ultra and AlphaLISA antibody-based assays for measuring the catalytic activity of two epigenetic enzymes acting on lysine 4 of histone H3: SET7/9 methyltransferase and LSD1 demethylase. Both the SET7/9 and LSD1 assays were designed as signal-increase assays using biotinylated peptides derived from the N-terminus of histone H3. In addition, the SET7/9 assay was demonstrated using full-length histone H3 protein as substrate in the AlphaLISA format. Optimized assays in 384-well plates are robust (Z′ factors ≥0.7) and sensitive, requiring only nanomolar concentrations of enzyme and substrate. All assays allowed profiling of known SET7/9 and LSD1 inhibitors. The results demonstrate that the optimized LANCE Ultra and AlphaLISA assay formats provide a relevant biochemical screening approach toward the identification of small-molecule inhibitors of HMTs and HDMs that could lead to novel epigenetic therapies.
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Li, Yong, Yousef I. Hassan, Hideaki Moriyama, and Janos Zempleni. "Holocarboxylase synthetase interacts physically with euchromatic histone-lysine N-methyltransferase, linking histone biotinylation with methylation events." Journal of Nutritional Biochemistry 24, no. 8 (August 2013): 1446–52. http://dx.doi.org/10.1016/j.jnutbio.2012.12.003.
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Golding, Michael C., Matthew Snyder, Gayle L. Williamson, Kylee J. Veazey, Michael Peoples, Jane H. Pryor, Mark E. Westhusin, and Charles R. Long. "Histone-lysine N-methyltransferase SETDB1 is required for development of the bovine blastocyst." Theriogenology 84, no. 8 (November 2015): 1411–22. http://dx.doi.org/10.1016/j.theriogenology.2015.07.028.
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Duan, Baojun, Jun Bai, Jian Qiu, Jianhua Wang, Cong Tong, Xiaofei Wang, Jiyu Miao, et al. "Histone-lysine N-methyltransferase SETD7 is a potential serum biomarker for colorectal cancer patients." EBioMedicine 37 (November 2018): 134–43. http://dx.doi.org/10.1016/j.ebiom.2018.10.036.
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Min, Wenjian, Zeng Hou, Fang Zhang, Shengnan Xie, Kai Yuan, Haojie Dong, Liping Wang, et al. "Computational discovery and biological evaluation of novel inhibitors targeting histone-lysine N-methyltransferase SET7." Bioorganic & Medicinal Chemistry 28, no. 7 (April 2020): 115372. http://dx.doi.org/10.1016/j.bmc.2020.115372.
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Linscott, Joshua A., Kanishk Kapilashrami, Zhen Wang, Chamara Senevirathne, Ian R. Bothwell, Gil Blum, and Minkui Luo. "Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8." Proceedings of the National Academy of Sciences 113, no. 52 (December 9, 2016): E8369—E8378. http://dx.doi.org/10.1073/pnas.1609032114.
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Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)–PKMT–substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (SN2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactorS-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic13C KIE of 1.04, an inverse intrinsic α-secondary CD3KIE of 0.90, and a small but statistically significant inverse CD3BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 transition state with the C-N and C-S distances of 2.35–2.40 Å and 2.00–2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analogSe-adenosyl-l-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.
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Sharma, Monal, Chhaya Dhiman, Poonam Dangi, and Shailja Singh. "Designing synthetic drugs against Plasmodium falciparum: a computational study of histone-lysine N-methyltransferase (PfHKMT)." Systems and Synthetic Biology 8, no. 2 (April 8, 2014): 155–60. http://dx.doi.org/10.1007/s11693-014-9144-8.
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Kerchner, Keshia M., Tung-Chung Mou, Yizhi Sun, Domniţa-Valeria Rusnac, Stephen R. Sprang, and Klára Briknarová. "The structure of the cysteine-rich region from human histone-lysine N-methyltransferase EHMT2 (G9a)." Journal of Structural Biology: X 5 (2021): 100050. http://dx.doi.org/10.1016/j.yjsbx.2021.100050.
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Kim, Haeun, Seo Yoon Choi, Jinyeong Lim, Anders M. Lindroth, and Yoon Jung Park. "EHMT2 Inhibition Induces Cell Death in Human Non-Small Cell Lung Cancer by Altering the Cholesterol Biosynthesis Pathway." International Journal of Molecular Sciences 21, no. 3 (February 3, 2020): 1002. http://dx.doi.org/10.3390/ijms21031002.
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Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer. Besides genetic and environmental factors, epigenetic alterations contribute to the tumorigenesis of NSCLC. Epigenetic changes are considered key drivers of cancer initiation and progression, and altered expression and activity of epigenetic modifiers reshape the epigenetic landscape in cancer cells. Euchromatic histone-lysine N-methyltransferase 2 (EHMT2) is a histone methyltransferase and catalyzes mono- and di-methylation at histone H3 lysine 9 (H3K9me1 and H3K9me2, respectively), leading to gene silencing. EHMT2 overexpression has been reported in various types of cancer, including ovarian cancer and neuroblastoma, in relation to cell proliferation and metastasis. However, its role in NSCLC is not fully understood. In this study, we showed that EHMT2 gene expression was higher in NSCLC than normal lung tissue based on publicly available data. Inhibition of EHMT2 by BIX01294 (BIX) reduced cell viability of NSCLC cell lines via induction of autophagy. Through RNA sequencing analysis, we found that EHMT2 inhibition significantly affected the cholesterol biosynthesis pathway. BIX treatment directly induced the expression of SREBF2, which is a master regulator of cholesterol biosynthesis, by lowering H3K9me1 and H3K9me2 at the promoter. Treatment of a cholesterol biosynthesis inhibitor, 25-hydroxycholesterol (25-HC), partially recovered BIX-induced cell death by attenuating autophagy. Our data demonstrated that EHMT2 inhibition effectively induced cell death in NSCLC cells through altering cholesterol metabolism-dependent autophagy.
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Cattaneo, Francesca, Alexander Zakharov, and Giuseppina Nucifora. "EVI1 Is a Transcriptional Repressor Able To Recruit SUV39H1 Histone Methylation Activity." Blood 110, no. 11 (November 16, 2007): 1235. http://dx.doi.org/10.1182/blood.v110.11.1235.1235.
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Abstract The Evi1 (Ecotropic viral integration site 1) gene is located on chromosome 3q26 and encodes a highly conserved nuclear protein that belongs to the Kruppel family of transcription factors, containing two Cys2-His2 repeat zinc finger motifs domains. EVI1 is not expressed in normal hematopoietic tissues, but can be found overexpressed in acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) cells as a consequence of chromosome 3 rearrangements. In vitro and in vivo studies have shown that a number of biological properties can be attributed to the EVI1 protein that might contribute to leukemogenesis: deregulation of cell proliferation, inhibition of TGF-ß signaling pathway, and suppression of stress induced apoptosis. We and others reported that EVI1 acts as a transcriptional repressor through its interaction with a subset of co-repressor proteins. More recently, we showed that EVI1 directly interacts with RUNX1 and GATA1 transcription factors. In both cases this interaction alters the ability of the transcription factors to bind their recognition sites on the DNA, leading to the deregulation of their target genes and to the impairment of the normal hematopoietic development. Interestingly, EVI1-mediated transcriptional silencing in association with HDAC proteins can be only partially rescued by HDAC inhibitors, suggesting that additional chromosomal modification might occur for gene silencing. Methylation by histone methyltransferase proteins (HMTs) is one type of these modifications, and when it occurs on certain residues can lead both to repression or activation of gene expression. The SUV39H1 (Suppressor of Variegation 3–9 Homolog 1) gene encodes a histone 3 specific methyltransferase. SUV39H1 selectively methylates lysine 9 of the amino terminus of the histone 3. Tri-methylation of histone H3 is important for recruiting heterochromatin protein 1 (HP1), thereby regulating gene expression, chromatin packaging and heterochromatin formation. Here we investigate the hypothesis that histone methylation may contribute to the transcriptional repressive potential of EVI1. We found that EVI1 physically interacts with the histone methyltransferase SUV39H1. The interaction between the two proteins requires the N-terminus of EVI1 where the proximal zinc finger domain is located; in particular, out of the seven motifs that compose this domain, the distal three motifs seem to be responsible for the binding between the two proteins. By pull-down assay with in vitro translated EVI1 this interaction appears to be direct. Futhermore, using in vitro histone methylation assay we show that EVI1 forms a complex with SUV39H1 that is able to methylate a recombinant H3, suggesting that the EVI1-SUV39H1 interaction does not affect the histone methyltransferase activity of SUV39H1. We suggest here that EVI1 interacts with SUV39H1 and that this binding could have a role in the epigenetic remodellig of the chromatin. According to this model, it is possible that EVI1 recruits histone methylation activity to selected genomic loci to silence tumor suppressor genes and repress differentiation factors. Because lysine methylation by SUV39H1 cannot take place on an already acetylated lysine, deacetylation activity associated with EVI1, through its interaction with HDACs, becomes a necessary preceeding step to SUV39H1 mediated histone methylation.
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Marques, F. "Tazemetostat. Histone-lysine N-methyltransferase EZH2 inhibitor, Treatment of solid tumors, Treatment of B-cell lymphomas." Drugs of the Future 41, no. 10 (2016): 595. http://dx.doi.org/10.1358/dof.2016.041.10.2533859.
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Li, Qian, Min-Di He, Lin Mao, Xue Wang, Yu-Lin Jiang, Min Li, Yong-Hui Lu, Zheng-Ping Yu, and Zhou Zhou. "Nicotinamide N-Methyltransferase Suppression Participates in Nickel-Induced Histone H3 Lysine9 Dimethylation in BEAS-2B Cells." Cellular Physiology and Biochemistry 41, no. 5 (2017): 2016–26. http://dx.doi.org/10.1159/000475432.
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Background: Nickel compounds are well-established human carcinogens with weak mutagenic activity. Histone methylation has been proposed to play an important role in nickel-induced carcinogenesis. Nicotinamide N-methyltransferase (NNMT) decreases histone methylation in several cancer cells by altering the cellular ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). However, the role of NNMT in nickel-induced histone methylation remains unclear. Methods: BEAS-2B cells were exposed to different concentrations of nickel chloride (NiCl2) for 72 h or 200 μM NiCl2 for different time periods. Histone H3 on lysine 9 (H3K9) mono-, di-, and trimethylation and NNMT protein levels were measured by western blot analysis. Expressions of NNMT mRNA and the H3k9me2-associated genes, mitogen-activated protein kinase 3 (MAP2K3) and dickkopf1 (DKK1), were determined by qPCR analysis. The cellular ratio of nicotinamide adenine dinucleotide (NAD+) to reduced NAD (NADH) and SAM/SAH ratio were determined. Results: Exposure of BEAS-2B cells to nickel increased H3K9 dimethylation (H3K9me2), suppressed the expressions of H3K9me2-associated genes (MAP2K3 and DKK1), and induced NNMT repression at both the protein and mRNA levels. Furthermore, over-expression of NNMT inhibited nickel-induced H3K9me2 and altered the cellular SAM/SAH ratio. Additionally, the NADH oxidant phenazine methosulfate (PMS) not only reversed the nickel-induced reduction in NAD+/NADH but also inhibited the increase in H3K9me2. Conclusions: These findings indicate that the repression of NNMT may underlie nickel-induced H3K9 dimethylation by altering the cellular SAM/SAH ratio.
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Cao, Ziyang, Wei Wu, Haiting Wei, Wei Zhang, Yan Huang, and Zhengwei Dong. "Downregulation of histone‑lysine N‑methyltransferase EZH2 inhibits cell viability and enhances chemosensitivity in lung cancer cells." Oncology Letters 21, no. 1 (November 11, 2020): 1. http://dx.doi.org/10.3892/ol.2020.12287.
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Yuan, Qinghua, Xiang Xie, Zhenyan Fu, Xiang Ma, Yining Yang, Ding Huang, Fen Liu, Chuanfang Dai, and Yitong Ma. "Association of the histone-lysine N-methyltransferase MLL5 gene with coronary artery disease in Chinese Han people." Meta Gene 2 (December 2014): 514–24. http://dx.doi.org/10.1016/j.mgene.2014.06.001.
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Chin, Hang Gyeong, Pierre-Olivier Esteve, Cristian Ruse, Jiyoung Lee, Scott E. Schaus, Sriharsa Pradhan, and Ulla Hansen. "The microtubule-associated histone methyltransferase SET8, facilitated by transcription factor LSF, methylates α-tubulin." Journal of Biological Chemistry 295, no. 14 (February 28, 2020): 4748–59. http://dx.doi.org/10.1074/jbc.ra119.010951.
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Microtubules are cytoskeletal structures critical for mitosis, cell motility, and protein and organelle transport and are a validated target for anticancer drugs. However, how tubulins are regulated and recruited to support these distinct cellular processes is incompletely understood. Posttranslational modifications of tubulins are proposed to regulate microtubule function and dynamics. Although many of these modifications have been investigated, only one prior study reports tubulin methylation and an enzyme responsible for this methylation. Here we used in vitro radiolabeling, MS, and immunoblotting approaches to monitor protein methylation and immunoprecipitation, immunofluorescence, and pulldown approaches to measure protein–protein interactions. We demonstrate that N-lysine methyltransferase 5A (KMT5A or SET8/PR-Set7), which methylates lysine 20 in histone H4, bound α-tubulin and methylated it at a specific lysine residue, Lys311. Furthermore, late SV40 factor (LSF)/CP2, a known transcription factor, bound both α-tubulin and SET8 and enhanced SET8-mediated α-tubulin methylation in vitro. In addition, we found that the ability of LSF to facilitate this methylation is countered by factor quinolinone inhibitor 1 (FQI1), a specific small-molecule inhibitor of LSF. These findings suggest the general model that microtubule-associated proteins, including transcription factors, recruit or stimulate protein-modifying enzymes to target tubulins. Moreover, our results point to dual functions for SET8 and LSF not only in chromatin regulation but also in cytoskeletal modification.
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Tie, Feng, Carl A. Stratton, Rebeccah L. Kurzhals, and Peter J. Harte. "The N Terminus of Drosophila ESC Binds Directly to Histone H3 and Is Required for E(Z)-Dependent Trimethylation of H3 Lysine 27." Molecular and Cellular Biology 27, no. 6 (January 8, 2007): 2014–26. http://dx.doi.org/10.1128/mcb.01822-06.
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ABSTRACT Polycomb group proteins mediate heritable transcriptional silencing and function through multiprotein complexes that methylate and ubiquitinate histones. The 600-kDa E(Z)/ESC complex, also known as Polycomb repressive complex 2 (PRC2), specifically methylates histone H3 lysine 27 (H3 K27) through the intrinsic histone methyltransferase (HMTase) activity of the E(Z) SET domain. By itself, E(Z) exhibits no detectable HMTase activity and requires ESC for methylation of H3 K27. The molecular basis for this requirement is unknown. ESC binds directly, via its C-terminal WD repeats (β-propeller domain), to E(Z). Here, we show that the N-terminal region of ESC that precedes its β-propeller domain interacts directly with histone H3, thereby physically linking E(Z) to its substrate. We show that when expressed in stable S2 cell lines, an N-terminally truncated ESC (FLAG-ESC61-425), like full-length ESC, is incorporated into complexes with E(Z) and binds to a Ubx Polycomb response element in a chromatin immunoprecipitation assay. However, incorporation of this N-terminally truncated ESC into E(Z) complexes prevents trimethylation of histone H3 by E(Z). We also show that a closely related Drosophila melanogaster paralog of ESC, ESC-like (ESCL), and the mammalian homolog of ESC, EED, also interact with histone H3 via their N termini, indicating that the interaction of ESC with histone H3 is evolutionarily conserved, reflecting its functional importance. Our data suggest that one of the roles of ESC (and ESCL and EED) in PRC2 complexes is to enable E(Z) to utilize histone H3 as a substrate by physically linking enzyme and substrate.
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Chang, Y. L., H. Y. Chen, K. B. Chen, K. C. Chen, K. L. Chang, P. C. Chang, T. T. Chang, and Y. C. Chen. "Investigation of the inhibitors of histone-lysine N-methyltransferase SETD2 for acute lymphoblastic leukaemia from traditional Chinese medicine." SAR and QSAR in Environmental Research 27, no. 7 (May 24, 2016): 589–608. http://dx.doi.org/10.1080/1062936x.2016.1186112.
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Strelow, John M., Min Xiao, Rachel N. Cavitt, Nathan C. Fite, Brandon J. Margolis, and Kyu-Jin Park. "The Use of Nucleosome Substrates Improves Binding of SAM Analogs to SETD8." Journal of Biomolecular Screening 21, no. 8 (July 10, 2016): 786–94. http://dx.doi.org/10.1177/1087057116656596.
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SETD8 is the methyltransferase responsible for monomethylation of lysine at position 20 of the N-terminus of histone H4 (H4K20). This activity has been implicated in both DNA damage and cell cycle progression. Existing biochemical assays have utilized truncated enzymes containing the SET domain of SETD8 and peptide substrates. In this report, we present the development of a mechanistically balanced biochemical assay using full-length SETD8 and a recombinant nucleosome substrate. This improves the binding of SAM, SAH, and sinefungin by up to 10,000-fold. A small collection of inhibitors structurally related to SAM were screened and 40 compounds were identified that only inhibit SETD8 when a nucleosome substrate is used.
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Yin, Jie, Jianmei W. Leavenworth, Yang Li, Qi Luo, Huafeng Xie, Xinhua Liu, Shan Huang, et al. "Ezh2 regulates differentiation and function of natural killer cells through histone methyltransferase activity." Proceedings of the National Academy of Sciences 112, no. 52 (December 14, 2015): 15988–93. http://dx.doi.org/10.1073/pnas.1521740112.
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Changes of histone modification status at critical lineage-specifying gene loci in multipotent precursors can influence cell fate commitment. The contribution of these epigenetic mechanisms to natural killer (NK) cell lineage determination from common lymphoid precursors is not understood. Here we investigate the impact of histone methylation repressive marks (H3 Lys27 trimethylation; H3K27me3) on early NK cell differentiation. We demonstrate that selective loss of the histone-lysine N-methyltransferase Ezh2 (enhancer of zeste homolog 2) or inhibition of its enzymatic activity with small molecules unexpectedly increased generation of the IL-15 receptor (IL-15R) CD122+ NK precursors and mature NK progeny from both mouse and human hematopoietic stem and progenitor cells. Mechanistic studies revealed that enhanced NK cell expansion and cytotoxicity against tumor cells were associated with up-regulation of CD122 and the C-type lectin receptor NKG2D. Moreover, NKG2D deficiency diminished the positive effects of Ezh2 inhibitors on NK cell commitment. Identification of the contribution of Ezh2 to NK lineage specification and function reveals an epigenetic-based mechanism that regulates NK cell development and provides insight into the clinical application of Ezh2 inhibitors in NK-based cancer immunotherapies.
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Youdell, Michael L., Kelby O. Kizer, Elena Kisseleva-Romanova, Stephen M. Fuchs, Eris Duro, Brian D. Strahl, and Jane Mellor. "Roles for Ctk1 and Spt6 in Regulating the Different Methylation States of Histone H3 Lysine 36." Molecular and Cellular Biology 28, no. 16 (June 9, 2008): 4915–26. http://dx.doi.org/10.1128/mcb.00001-08.
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ABSTRACT Set2 (KMT3)-dependent methylation (me) of histone H3 at lysine 36 (H3K36) promotes deacetylation of transcribed chromatin and represses cryptic promoters within genes. Although Set2 is the only methyltransferase (KMTase) for H3K36 in yeast, it is not known if Set2 is regulated or whether the different methylation states at H3K36 are functionally distinct. Here we show that the N-terminal 261 residues of Set2 (Set21-261), containing the SET KMTase domain, are sufficient for H3K36me2, histone deacetylation, and repression of cryptic promoters at STE11. Set2-catalyzed H3K36me2 does not require either Ctk1-dependent phosphorylation of RNA polymerase II (RNAPII) or the presence of the phospho-C-terminal domain (CTD) interaction (SRI) domain of Set2. This finding is consistent with a known correlation between H3K36me2 and whether a gene is on or off, but not the level of activity of a gene. By contrast, H3K36me3 requires Spt6, proline 38 on histone H3 (H3P38), the CTD of RNAPII, Ctk1, and the C-terminal SRI domain of Set2. We suggest that the C-terminal region of Set2, in conjunction with the phosphorylated CTD of RNAPII, influences the KMTase activity to promote H3K36me3 during transcription elongation.
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Xu, Junyao, Chuanchao He, Jianlong Zhang, and Jie Wang. "Association of high histone H3K4 trimethylation level and prognosis of patients with low-TNM-stage hepatocellular carcinoma." Journal of Clinical Oncology 30, no. 4_suppl (February 1, 2012): 171. http://dx.doi.org/10.1200/jco.2012.30.4_suppl.171.
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171 Background: Along with genetic events, tumor associated epigenetic alterations including DNA methylation and post-translational histone modifications are important determinants in the initiation and progression of hepatocellular cancer (HCC) and represent promising biomarkers and therapeutic targets. Locus-specific trimethylation of histone H3 lysine 4 (H3K4me) is a well-known modification linked to the enhanced transcriptional expression of many genes activated in HCC. However its expression and association with prognosis of HCC patients remain unclear. Our aim was to assess the cellular expression pattern of H3K4me3 in HCC and its association with clinicopathologic variables and outcome. Methods: Expression of H3K4me3 and the histone methyltransferase SMYD3 was studied by western blotting and immunohistochemistry in human HCC cell lines and tumor tissue micmicroarray, which is from a well–characterized series of HCC patients(n=168). Tissue staining were assessed using blinded semiquantitative scoring. The optimal cut-point of H3K4me3 expression for prognosis was determined by the X-tile program. The prognostic significance was evaluated using Kaplan-Meier survival estimates and log-rank tests. Tumor tissue micmicroarray from another independent HCC patients cohort(n=147) was used for validation studies. Results: Expression of H3K4me3 and the histone methyltransferase SMYD3 were enhanced in HCC cell lines.In clinical tumor specimens, enhanced expression of H3K4me3 was correlated with reduced overall survival (P < 0.0001), especially in early-stage HCC patients(TNM I/II).Furthermore,both univariate and multivariate analysis revealed that H3K4me3 level was a significant and independent predictor of poor survival (hazard ratio,3.592; 95% CI, 2.302-5.605). In addition, H3K4m3 expression was positively correlated with SMYD3 expression in both testing and validation cohorts (P<0.0001). Conclusions: H3K4me3 level defines previously unrecognized subsets of HCC patients with distinct epigenetic phenotype and clinical outcome, thus can be a novel predictor for poor prognosis of HCC patients, especially within TNM I/IIstage.
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Borikar, Sneha, Vivek Philip, Lauren Kuffler, and Jennifer J. Trowbridge. "Lysine Methyltransferase Kmt5a Restricts Myeloid-Biased Output of Lymphoid-Primed Multipotent Progenitors." Blood 128, no. 22 (December 2, 2016): 1487. http://dx.doi.org/10.1182/blood.v128.22.1487.1487.
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Abstract Distinct, lineage-biased subsets of multipotent progenitor cells (MPP) dynamically respond to the demands of the hematopoietic system to replenish mature hematopoietic cells as needed. It currently remains unclear as to whether distinct epigenetic mechanisms regulate lineage-specific expansion and differentiation from MPPs. Focusing on lymphoid-primed multipotent progenitor cells (LMPP/MPP4), we performed a lentiviral shRNA screen of 15 epigenetic factors, selected based on differential expression between myeloid-restricted and lymphoid-restricted progenitors. Following a 48 hour infection with lentiviral shRNA constructs or a non-targeting control, the lineage potential of lymphoid-primed multipotent progenitors was interrogated by myeloid and lymphoid colony forming unit (CFU) assays. From this screen, knockdown of the lysine methyltransferase Kmt5a most dramatically altered lineage output from lymphoid-primed multipotent progenitors through an expansion of myeloid lineage colonies without altering lymphoid colony production. To confirm target specificity, two independent shRNA hairpins targeting distinct locations of the Kmt5a transcript demonstrated that knockdown of Kmt5a (97.1% and 99.5% versus non-targeting control shRNA) increased macrophage colony production by 1.94 and 1.95 fold, respectively (P < 0.01 and P < 0.05, n = 3). Preliminary single cell culture experiments support that the enhanced myeloid lineage output from lymphoid-primed multipotent progenitors occurs at the single-cell level through increased cloning efficiency of myeloid-biased cells. Our results suggest that Kmt5a functions to restrict myeloid lineage output from lymphoid-primed multipotent progenitors. Mechanistically, KMT5A is responsible for monomethylation of histone H4K20 and the methylation of non-histone proteins (ex. p53K376). Our ongoing work aims to distinguish between these histone and non-histone targets to determine the precise mechanisms restricting myeloid lineage output from lymphoid-primed multipotent progenitors. This work has direct implications for a better understanding of the molecular drivers of transient myeloid lineage reprogramming of lymphoid-primed multipotent progenitors during hematopoietic regeneration, age associated myeloid lineage skewing of hematopoiesis, and myeloid malignancies. Disclosures No relevant conflicts of interest to declare.
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Klocko, Andrew D., Calvin A. Summers, Marissa L. Glover, Robert Parrish, William K. Storck, Kevin J. McNaught, Nicole D. Moss, et al. "Selection and Characterization of Mutants Defective in DNA Methylation in Neurospora crassa." Genetics 216, no. 3 (September 1, 2020): 671–88. http://dx.doi.org/10.1534/genetics.120.303471.
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DNA methylation, a prototypical epigenetic modification implicated in gene silencing, occurs in many eukaryotes and plays a significant role in the etiology of diseases such as cancer. The filamentous fungus Neurospora crassa places DNA methylation at regions of constitutive heterochromatin such as in centromeres and in other A:T-rich regions of the genome, but this modification is dispensable for normal growth and development. This and other features render N. crassa an excellent model to genetically dissect elements of the DNA methylation pathway. We implemented a forward genetic selection on a massive scale, utilizing two engineered antibiotic-resistance genes silenced by DNA methylation, to isolate mutants defective in methylation (dim). Hundreds of potential mutants were characterized, yielding a rich collection of informative alleles of 11 genes important for DNA methylation, most of which were already reported. In parallel, we characterized the pairwise interactions in nuclei of the DCDC, the only histone H3 lysine 9 methyltransferase complex in Neurospora, including those between the DIM-5 catalytic subunit and other complex members. We also dissected the N- and C-termini of the key protein DIM-7, required for DIM-5 histone methyltransferase localization and activation. Lastly, we identified two alleles of a novel gene, dim-10 – a homolog of Clr5 in Schizosaccharomyces pombe – that is not essential for DNA methylation, but is necessary for repression of the antibiotic-resistance genes used in the selection, which suggests that both DIM-10 and DNA methylation promote silencing of constitutive heterochromatin.
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Lin, Yan, Xu Cheng, Peter Sutovsky, De Wu, Lian-Qiang Che, Zheng-Feng Fang, Sheng-Yu Xu, Bo Ren, and Hong-Jun Dong. "Effect of intra-uterine growth restriction on long-term fertility in boars." Reproduction, Fertility and Development 29, no. 2 (2017): 374. http://dx.doi.org/10.1071/rd15130.
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The present study assessed the effect of birthweight on reproductive performance, including a possible mechanism, in male pigs. Ten newborn male piglets, including five normal birthweight (NBW) piglets and five intra-uterine growth restricted (IUGR) piglets, were used in the study. All piglets were weaned on Day 28 and fed the same diet during the experiment (10 months). Average daily weight gain, feed intake and the feed conversion ratio were higher in NBW than IUGR piglets. Similarly, testis volume and the number of Leydig and Sertoli cells in the distal portion of the testes were higher in NBW than IUGR piglets (P < 0.05). Semen volume (P < 0.05) and the total number of spermatozoa per ejaculate (P = 0.08) were lower in IUGR boars. Testosterone concentrations on Day 141 and prostaglandin E2 concentrations on Days 82 and 141 were higher in IUGR than NBW boars (P < 0.05). The malondialdehyde content of seminal plasma was higher in IUGR boars, whereas sperm glutathione peroxidase activity was lower in IUGR versus NBW boars (P < 0.05). Expression of DNA methyltransferase (Dnmt) genes Dnmt1, Dnmt3a, histone-lysine N-methyltransferase (Suv39h2), and lysine (K)-specific demethylase Kdm4a was upregulated in testes from IUGR boars. These findings suggest that growth restriction affects sperm production via reproductive organ development and epigenetic regulation.
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Braig, Melanie, Christoph Loddenkemper, Antoine H. F. M. Peters, Harald Stein, Bernd Dorken, Thomas Jenuwein, and Clemens A. Schmitt. "Impaired Histone H3 Lysine 9 Methylation Accelerates Ras-Driven Lymphomagenesis by Disabling Cellular Senescence." Blood 104, no. 11 (November 16, 2004): 346. http://dx.doi.org/10.1182/blood.v104.11.346.346.
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Abstract As a barrier against malignant transformation, checkpoint-mediated failsafe mechanisms such as apoptosis or cellular senescence may be activated in response to mitogenic oncogenes. Acute induction of oncogenic Ras has been shown to provoke a senescence-like cell-cycle arrest that involves the retinoblastoma (Rb) pathway. Recently, Rb-mediated formation of heterochromatin was identified as a critical feature of cellular senescence. Since methylation of histone H3 lysine 9 by the Rb-bound histone methyltransferase Suv39h1 contributes to the transcriptional repression of growth promoting genes, we asked whether inactivation of Suv39h1 may disable cellular senescence as a suppressor mechanism against malignant transformation by oncogenic Ras. To investigate the role of Suv39h1 in Ras-driven lymphomagenesis, Eμ-N-ras-transgenic mice were crossbred to mice harboring targeted deletions in the Suv39h1 locus, as well as to p53 knockout mice. Ras-transgenic mice with no additional defined genetic lesion - hereafter referred to as controls - developed a terminal condition mostly due to a neoplastic histiocytic infiltration of the liver with a median latency of about 250 days. In stark contrast, ras-transgenic mice lacking Suv39h1, or carrying heterozygous defects in the Suv39h1 or p53 locus, respectively, entered a final disease stage significantly earlier, i.e. at a median age of 50 to 100 days, due to aggressive T cell lymphomas that were virtually absent or only sporadically found in the control group. Importantly, similar to p53+/− mice which typically lost the remaining wild-type allele in lymphoma cells, Suv39h1 heterozygous mice produced tumors that invariably lacked Suv39h1 expression, rendering these lymphomas in fact Suv39h1 null. Suv39h1 null cells propagated as primary cultures grew readily in an exponential fashion, surpassed only by the growth potential of p53 null cells. In short-term cytotoxicity assays, the anticancer agent adriamycin efficiently killed both control and Suv39h1 null lymphoma cells, whereas p53 null cells were resistant, indicating no overt apoptotic defect in Suv39h1 null cells. While non-neoplastic ras-transgenic Suv39h1+/+ cells exhibited heterochromatin foci reminiscent of cellular senescence, this phenotype was completely lost in Suv39h1 null lymphoma cells. In the presence of Bcl2 - introduced to block apoptotic cell death - adriamycin-treated control cells entered cellular senescence, whereas Suv39h1 null cells failed to display a senescent phenotype in response to therapy, demonstrating that stress-inducible senescence is a Suv39h1-dependent program. The data unveil the fundamental role of cellular senescence as a tumor suppressor principle by establishing a novel link between Ras-driven transformation and deregulated histone modification in vivo. Impaired cellular senescence due to inactivation of the histone H3 lysine 9 methyltransferase Suv39h1 promotes malignant transformation by oncogenic Ras, leading to a dramatic acceleration of Ras-driven lymphoma formation. Moreover, the observation that a full-blown malignancy may arise as a consequence of disabled senescence but without an apparent apoptotic defect has important therapeutic implications.
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Byun, Woong Sub, Gyu Ho Lee, Hyeung-geun Park, and Sang Kook Lee. "Inhibition of DOT1L by Half-Selenopsammaplin A Analogs Suppresses Tumor Growth and EMT-Mediated Metastasis in Triple-Negative Breast Cancer." Pharmaceuticals 14, no. 1 (December 28, 2020): 18. http://dx.doi.org/10.3390/ph14010018.
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Due to a lack of hormone receptors, current treatment strategies for triple-negative breast cancer (TNBC) are limited with frequent disease recurrence and metastasis. Recent findings have suggested that aberrant methylation of histone H3 lysine 79 residue (H3K79me) by the histone methyltransferase disruptor of telomeric silencing 1-like (DOT1L) is a potential therapeutic target for TNBC clinical management. Therefore, we developed DOT1L inhibitors as potential antitumor agents against TNBC cells. We reveal that a synthetic half-selenopsammaplin A analog 9l (subsequently known as 9l) exhibited inhibitory activity against DOT1L-mediated H3K79 methylation, and showed antitumor activity in TNBC cells. The analog 9l also significantly inhibited TNBC invasion and migration via the modulation of epithelial-mesenchymal transition (EMT) markers, including N-cadherin and vimentin downregulation and E-cadherin upregulation. In an MDA-MB-231/Luc-implanted orthotopic mouse metastasis model, treatment with 9l effectively inhibited tumor growth and lung metastasis via DOT1L regulatory activity and EMT processes. Taken together, these findings highlight the potential of 9l as a novel therapeutic candidate for treating metastatic TNBC via DOT1L modulation.
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Johnson, C. M., and R. A. Cabot. "4 THE HISTONE METHYLTRANSFERASE Suv39h2 ADOPTS A NUCLEAR LOCALIZATION DURING CLEAVAGE DEVELOPMENT IN PARTHENOGENETIC PORCINE EMBRYOS." Reproduction, Fertility and Development 21, no. 1 (2009): 102. http://dx.doi.org/10.1071/rdv21n1ab4.
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Successful cleavage development of mammalian embryos requires precise activation and repression of transcription. Covalent modifications to histone proteins, such as methylation and acetylation, play a key role in transcriptional regulation. In particular, dimethylation of the lysine 9 residue of histone protein H3 (H3K9) results in gene silencing and heterochromatin formation. Our lab has previously shown that transcripts encoding the five histone methyltransferases known to methylate H3K9 (Suv39h1, Suv39h2, ESET, G9a, and EHMT1) are present in different amounts during oocyte maturation and cleavage development. Specifically, Suv39h2 is in the greatest abundance in GV and metaphase II stage oocytes and is also present throughout cleavage development. The aim of this study was to determine the localization of Suv39h2 protein in the GV-stage oocyte and pronuclear, 2 cell, and 4 cell stage parthenogenetic porcine embryos. We hypothesized that Suv39h2 protein would localize to the nucleus based on its high transcript abundance throughout cleavage development. To test this hypothesis, we performed a microinjection experiment in which mRNA encoding a porcine Suv39h2-GFP fusion protein was injected into metaphase II porcine oocytes. Porcine oocytes were matured in a defined medium (TCM-199 supplemented with 0.1% PVA, 0.069 mg mL–1 cysteine, 10 ng mL–1 EGF, 0.5 IU mL–1 LH and FSH) for 42 to 44 h at 39°C in 5% CO2, then denuded of cumulus cells just before microinjection. Two separate treatment groups were microinjected intracytoplasmically with 1 μg μL–1 GFP or Suv39h2-GFP mRNA, respectively. Microinjection was performed using a FemtoJet microinjector (Eppendorf, Hamburg, Germany). The treatment groups and non-injected controls were electroactivated independently and cultured in PZM medium supplemented with 3 mg mL–1 BSA for 12 (pronuclear), 24 (2 cell), or 48 (4 cell) hours at 39°C in 5% CO2, depending on desired stage of development. Before visualization under UV light, embryos were stained with Hoechst 33342 for 15 minutes. Oocytes and embryos were analyzed for GFP expression at the GV, pronuclear, 2 and 4 cell stages of development using epifluorescence microscopy. Two to four biological replicates were performed for each stage of embryo development. We found that Suv39h2-GFP protein showed nuclear localization in most GV-stage oocytes (n = 11/14) and pronuclear (n = 17/17), 2-cell (n = 34/36), and 4-cell (n = 9/9) stage embryos. Chi square analysis revealed this pattern to be different from that observed in embryos injected with GFP mRNA, where GFP did not display nuclear localization at any stage of development (n = 12; P < 0.05). These results indicate that Suv39h2 is localized in the nucleus of oocytes and cleaved embryos, which suggests that this histone methyltransferase plays an important role in methylating H3K9.
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Banumathy, Gowrishankar, Neeta Somaiah, Rugang Zhang, Yong Tang, Jason Hoffmann, Mark Andrake, Hugo Ceulemans, David Schultz, Ronen Marmorstein, and Peter D. Adams. "Human UBN1 Is an Ortholog of Yeast Hpc2p and Has an Essential Role in the HIRA/ASF1a Chromatin-Remodeling Pathway in Senescent Cells." Molecular and Cellular Biology 29, no. 3 (November 24, 2008): 758–70. http://dx.doi.org/10.1128/mcb.01047-08.
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ABSTRACT Cellular senescence is an irreversible proliferation arrest, tumor suppression process and likely contributor to tissue aging. Senescence is often characterized by domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), which repress expression of proliferation-promoting genes. Given its likely contribution to tumor suppression and tissue aging, it is essential to identify all components of the SAHF assembly pathway. Formation of SAHF in human cells is driven by a complex of histone chaperones, namely, HIRA and ASF1a. In yeast, the complex orthologous to HIRA/ASF1a contains two additional proteins, Hpc2p and Hir3p. Using a sophisticated approach to search for remote orthologs conserved in multiple species through evolution, we identified the HIRA-associated proteins, UBN1 and UBN2, as candidate human orthologs of Hpc2p. We show that the Hpc2-related domain of UBN1, UBN2, and Hpc2p is an evolutionarily conserved HIRA/Hir-binding domain, which directly interacts with the N-terminal WD repeats of HIRA/Hir. UBN1 binds to proliferation-promoting genes that are repressed by SAHF and associates with histone methyltransferase activity that methylates lysine 9 of histone H3, a site that is methylated in SAHF. UBN1 is indispensable for formation of SAHF. We conclude that UBN1 is an ortholog of yeast Hpc2p and a novel regulator of senescence.
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Wang, Jingyuan, Joanne Xiu, Yasmine Baca, Richard M. Goldberg, Philip Agop Philip, Andreas Seeber, Francesca Battaglin, et al. "Molecular landscape of gastric cancer (GC) harboring mutations of histone methyltransferases." Journal of Clinical Oncology 38, no. 4_suppl (February 1, 2020): 418. http://dx.doi.org/10.1200/jco.2020.38.4_suppl.418.
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418 Background: Alteration of histone modifications participating in transcription and genomic instability, has been recognized as an important role in tumorigenesis. Aberrant expression of histone-lysine N-methyltransferase 2 ( KMT2) family, which methylate histone H3 on lysine 4, is significantly correlated with poor survival in GC. Understanding how gene mutations of KMT2 family interact to affect cancer progression could lead to new treatment strategies. Methods: A total of 1,245 GC were analyzed using next-generation sequencing (NGS) and immunohistochemistry (IHC; Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous mutations, and MSI status was evaluated by a combination of IHC, fragment analysis and NGS. PD-L1 status was analyzed by IHC (SP142). Gene fusions were detected by Archer (N = 59) or whole-transcriptome sequencing (N = 129). Results: The overall mutation rate of genes in KMT2 family was 10.6% ( KMT2A: 1.7 %, KMT2C: 4.7%, KMT2D: 7.1%). Overall, the mutation rates were significantly higher in KMT2-mutated (MT) GC than KMT2-wild type (WT) GC, except for TP53 (43% vs 63%, p < .0001). Interestingly, among the genes with significant higher mutation rates in KMT2-MT GC, 28% (21/76) of them were related to DNA damage repair (including BRCA1/ 2, RAD50) and 33% (25/76) of them were related to chromatin remodeling (including ARID1A/ 2, SMARCA4). Overexpression of HER2, amplifications of KRAS, CDK6 and HER2 were significant lower, while PCM1 and BCL3 amplifications were significant higher in KMT2-MT, compared to KMT2-WT GC ( p < .05). Significantly higher prevalence of TMB-high ( > 17mut/MB) (49% vs 3%), MSI-H (53% vs 2%), and PD-L1 overexpression (20% vs 7%) were present in KMT2-MT GC, compared to KMT2-WT GC ( p < .001). The rates of fusions involving ARHGAP26 (19% vs 3%, p < .01)and RELA (29% vs 0%, p < .0001) were significantly higher in KMT2-MT than those in KMT2-WT GC. Conclusions: This is the largest study to investigate the distinct genomic landscape between KMT2-MT and WT GC. Our data indicates that KMT2-MT GC patients could potentially benefit from agents targeting DNA damage repair and immunotherapy, which warrants further in-vitro and in-vivo investigation.
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Amjadi, Mohammad, Tooba Hallaj, and Niko Hildebrandt. "A sensitive homogeneous enzyme assay for euchromatic histone-lysine-N-methyltransferase 2 (G9a) based on terbium-to-quantum dot time-resolved FRET." BioImpacts 11, no. 3 (July 8, 2020): 173–79. http://dx.doi.org/10.34172/bi.2021.23.
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Introduction: Histone modifying enzymes include several classes of enzymes that are responsible for various post-translational modifications of histones such as methylation and acetylation. They are important epigenetic factors, which may involve several diseases and so their assay, as well as screening of their inhibitors, are of great importance. Herein, a bioassay based on terbium-to-quantum dot (Tb-to-QD) time-resolved Förster resonance energy transfer (TR-FRET) was developed for monitoring the activity of G9a, the euchromatic histone-lysine N-methyltransferase 2. Overexpression of G9a has been reported in some cancers such as ovarian carcinoma, lung cancer, multiple myeloma and brain cancer. Thus, inhibition of this enzyme is important for therapeutic purposes. Methods: In this assay, a biotinylated peptide was used as a G9a substrate in conjugation with streptavidin-coated ZnS/CdSe QD as FRET acceptor, and an anti-mark antibody labeled with Tb as a donor. Time-resolved fluorescence was used for measuring FRET ratios. Results: We examined three QDs, with emission wavelengths of 605, 655 and 705 nm, as FRET acceptors and investigated FRET efficiency between the Tb complex and each of them. Since the maximum FRET efficiency was obtained for Tb to QD705 (more than 50%), this pair was exploited for designing the enzyme assay. We showed that the method has excellent sensitivity and selectivity for the determination of G9a at concentrations as low as 20 pM. Furthermore, the designed assay was applied for screening of an enzyme inhibitor, S-(5’-Adenosyl)-L-homocysteine (SAH). Conclusion: It was shown that Tb-to-QD FRET is an outstanding platform for developing a homogenous assay for the G9a enzyme and its inhibitors. The obtained results confirmed that this assay was quite sensitive and could be used in the field of inhibitor screening.
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ZHANG, Wenzheng, Yoshihide HAYASHIZAKI, and Bruce C. KONE. "Structure and regulation of the mDot1 gene, a mouse histone H3 methyltransferase." Biochemical Journal 377, no. 3 (February 1, 2004): 641–51. http://dx.doi.org/10.1042/bj20030839.
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Recently, a new class of histone methyltransferases that plays an indirect role in chromatin silencing by targeting a conserved lysine residue in the nucleosome core was described, namely the Dot1 (disruptor of telomeric silencing) family [Feng, Wang, Ng, Erdjument-Bromage, Tempst, Struhl and Zhang (2002) Curr. Biol. 12, 1052–1058; van Leeuwen, Gafken and Gottschling (2002) Cell (Cambridge, Mass.) 109, 745–756; Ng, Feng, Wang, Erdjument-Bromage, Tempst, Zhang and Struhl (2002) Genes Dev. 16, 1518–1527]. In the present study, we report the isolation, genomic organization and in vivo expression of a mouse Dot1 homologue (mDot1). Expressed sequence tag analysis identified five mDot1 mRNAs (mDot1a–mDot1e) derived from alternative splicing. mDot1a and mDot1b encode 1540 and 1114 amino acids respectively, whereas mDot1c–mDot1e are incomplete at the 5´-end. mDot1a is closest to its human counterpart (hDot1L), sharing 84% amino acid identity. mDot1b is truncated at its N- and C-termini and contains an internal deletion. The five mDot1 isoforms are encoded by 28 exons on chromosome 10qC1, with exons 24 and 28 further divided into two and four sections respectively. Alternative splicing occurs in exons 3, 4, 12, 24, 27 and 28. Northern-blot analysis with probes corresponding to the methyltransferase domain or the mDot1a-coding region detected 7.6 and 9.5 kb transcripts in multiple tissues, but only the 7.6 kb transcript was evident in mIMCD3-collecting duct cells. Transfection of mDot1a–EGFP constructs (where EGFP stands for enhanced green fluorescent protein) into human embryonic kidney (HEK)-293T or mIMCD3 cells increased the methylation of H3-K79 but not H3-K4, -K9 or -K36. Furthermore, DMSO induced mDot1 gene expression and methylation specifically at H3-K79 in mIMCD3 cells in a time- and dose-dependent manner. Collectively, these results add new members to the Dot1 family and show that mDot1 is involved in a DMSO-mediated signal-transduction pathway in collecting duct cells.
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Wen, Xue, Yao Xiong, Huimin Liu, Ting Geng, Ling Jin, Ming Zhang, Ling Ma, and Yuanzhen Zhang. "Decreased mixed lineage leukemia 1 is involved in endometriosis-related infertility." Journal of Molecular Endocrinology 66, no. 1 (January 2021): 45–57. http://dx.doi.org/10.1530/jme-20-0193.
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The aberrant histone methylation patterns contribute to the pathogenesis of endometriosis (EM). Mixed lineage leukemia 1 (MLL1), a histone methyltransferase, is crucial for gene expression by catalyzing the trimethylation of histone 3 lysine 4 (H3K4me3) in gene promoter. This study aimed to explore whether MLL1 is involved in EM-related infertility. The expressions of MLL1 and H3K4me3 were analyzed in the eutopic endometria from EM women with infertility (n = 22) and the normal endometria from EM-free women (n = 22). Mouse EM model was established. The MLL1 and H3K4me3 expression patterns in mice endometria of early pregnancy were also investigated. Immortalized human endometrial stromal cells (iESCs) were cultured and underwent in vitro decidualization. The chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) was performed to find the target gene of MLL1 during decidual process. Results showed that both MLL1 and H3K4me3 decreased in the eutopic endometrium from EM patients compared to that in the normal endometrium. During early pregnancy and the decidual process, MLL1 and H3K4me3 were significantly upregulated in stromal cells. ChIP-seq and ChIP-qPCR found that the cytochrome c oxidase subunit 4I 2 (COX4I2) was directly targeted by MLL1. The dominance of COX4I2-containing enzyme induced the expression of hypoxia-inducible factor-2α (HIF-2α), whose expression in the peri-implantation endometrium is essential for embryo implantation. Further results showed that MLL1 was directly regulated by progesterone (P4) – P4 receptors (PRs). Our study proved that MLL1 was involved in EM-related infertility, which may provide a novel approach to treat the nonreceptive endometrium in EM patients.
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Huang, Gang, Xinghui Zhao, Lan Wang, Shannon Elf, Hao Xu, Xinyang Zhao, Goro Sashida, et al. "The ability of MLL to bind RUNX1 and methylate H3K4 at PU.1 regulatory regions is impaired by MDS/AML-associated RUNX1/AML1 mutations." Blood 118, no. 25 (December 15, 2011): 6544–52. http://dx.doi.org/10.1182/blood-2010-11-317909.
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Abstract The mixed-lineage leukemia (MLL) H3K4 methyltransferase protein, and the heterodimeric RUNX1/CBFβ transcription factor complex, are critical for definitive and adult hematopoiesis, and both are frequently targeted in human acute leukemia. We identified a physical and functional interaction between RUNX1 (AML1) and MLL and show that both are required to maintain the histone lysine 4 trimethyl mark (H3K4me3) at 2 critical regulatory regions of the AML1 target gene PU.1. Similar to CBFβ, we show that MLL binds to AML1 abrogating its proteasome-dependent degradation. Furthermore, a subset of previously uncharacterized frame-shift and missense mutations at the N terminus of AML1, found in MDS and AML patients, impairs its interaction with MLL, resulting in loss of the H3K4me3 mark within PU.1 regulatory regions, and decreased PU.1 expression. The interaction between MLL and AML1 provides a mechanism for the sequence-specific binding of MLL to DNA, and identifies RUNX1 target genes as potential effectors of MLL function.
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Byun, Woong Sub, Won Kyung Kim, Ji-seong Yoon, Dnyandev B. Jarhad, Lak Shin Jeong, and Sang Kook Lee. "Antiproliferative and Antimigration Activities of Fluoro-Neplanocin A via Inhibition of Histone H3 Methylation in Triple-Negative Breast Cancer." Biomolecules 10, no. 4 (March 31, 2020): 530. http://dx.doi.org/10.3390/biom10040530.
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Triple-negative breast cancer (TNBC) is among the most aggressive and potentially metastatic malignancies. Most affected patients have poor clinical outcomes due to the lack of specific molecular targets on tumor cells. The upregulated expression of disruptor of telomeric silencing 1-like (DOT1L), a histone methyltransferase specific for the histone H3 lysine 79 residue (H3K79), is strongly correlated with TNBC cell aggressiveness. Therefore, DOT1L is considered a potential molecular target in TNBC. Fluoro-neplanocin A (F-NepA), an inhibitor of S-adenosylhomocysteine hydrolase, exhibited potent antiproliferative activity against various types of cancer cells, including breast cancers. However, the molecular mechanism underlying the anticancer activity of F-NepA in TNBC cells remains to be elucidated. We determined that F-NepA exhibited a higher growth-inhibitory activity against TNBC cells relative to non-TNBC breast cancer and normal breast epithelial cells. Moreover, F-NepA effectively downregulated the level of H3K79me2 in MDA-MB-231 TNBC cells by inhibiting DOT1L activity. F-NepA also significantly inhibited TNBC cell migration and invasion. These activities of F-NepA might be associated with the upregulation of E-cadherin and downregulation of N-cadherin and Vimentin in TNBC cells. Taken together, these data highlight F-NepA as a strong potential candidate for the targeted treatment of high-DOT1L-expressing TNBC.
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Revenko, Alexey S., Ekaterina V. Kalashnikova, Abigael T. Gemo, June X. Zou, and Hong-Wu Chen. "Chromatin Loading of E2F-MLL Complex by Cancer-Associated Coregulator ANCCA via Reading a Specific Histone Mark." Molecular and Cellular Biology 30, no. 22 (September 20, 2010): 5260–72. http://dx.doi.org/10.1128/mcb.00484-10.
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ABSTRACT Histone modifications are regarded as the carrier of epigenetic memory through cell divisions. How the marks facilitate cell cycle-dependent gene expression is poorly understood. The evolutionarily conserved AAA ATPase ANCCA (AAA nuclear coregulator cancer-associated protein)/ATAD2 was identified as a direct target of oncogene AIB1/ACTR/SRC-3 and a transcriptional coregulator for estrogen and androgen receptors and is strongly implicated in tumorigenesis. We report here that ANCCA directly interacts with E2F1 to E2F3 and that its N terminus interacts with both the N and C termini of E2F1. ANCCA preferentially associates via its bromodomain with H3 acetylated at lysine 14 (H3K14ac) and is required for key cell cycle gene expression and cancer cell proliferation. ANCCA associates with chromosomes at late mitosis, and its occupancy at E2F targets peaks at the G1-to-S transition. Strikingly, ANCCA is required for recruitment of specific E2Fs to their targets and chromatin assembly of the host cell factor 1 (HCF-1)-MLL histone methyltransferase complex. ANCCA depletion results in a marked decrease of the gene activation-linked H3K4me3 mark. Bromodomain mutations disable ANCCA function as an E2F coactivator and its ability to promote cancer cell proliferation, while ANCCA overexpression in tumors correlates with tumor growth. Together, these results suggest that ANCCA acts as a pioneer factor in E2F-dependent gene activation and that a novel mechanism involving ANCCA bromodomain may contribute to cancer cell proliferation.
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O'Carroll, Dónal, Harry Scherthan, Antoine H. F. M. Peters, Susanne Opravil, Andrew R. Haynes, Götz Laible, Stephen Rea, et al. "Isolation and Characterization ofSuv39h2, a Second Histone H3 Methyltransferase Gene That Displays Testis-Specific Expression." Molecular and Cellular Biology 20, no. 24 (December 15, 2000): 9423–33. http://dx.doi.org/10.1128/mcb.20.24.9423-9433.2000.
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ABSTRACT Higher-order chromatin has been implicated in epigenetic gene control and in the functional organization of chromosomes. We have recently discovered mouse (Suv39h1) and human (SUV39H1) histone H3 lysine 9-selective methyltransferases (Suv39h HMTases) and shown that they modulate chromatin dynamics in somatic cells. We describe here the isolation, chromosomal assignment, and characterization of a second murine gene, Suv39h2. Like Suv39h1,Suv39h2 encodes an H3 HMTase that shares 59% identity with Suv39h1 but which differs by the presence of a highly basic N terminus. Using fluorescent in situ hybridization and haplotype analysis, theSuv39h2 locus was mapped to the subcentromeric region of mouse chromosome 2, whereas the Suv39h1 locus resides at the tip of the mouse X chromosome. Notably, although bothSuv39h loci display overlapping expression profiles during mouse embryogenesis, Suv39h2 transcripts remain specifically expressed in adult testes. Immunolocalization of Suv39h2 protein during spermatogenesis indicates enriched distribution at the heterochromatin from the leptotene to the round spermatid stage. Moreover, Suv39h2 specifically accumulates with chromatin of the sex chromosomes (XY body) which undergo transcriptional silencing during the first meiotic prophase. These data are consistent with redundant enzymatic roles for Suv39h1 and Suv39h2 during mouse development and suggest an additional function of the Suv39h2 HMTase in organizing meiotic heterochromatin that may even impart an epigenetic imprint to the male germ line.
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Moody, James D., Shiri Levy, Julie Mathieu, Yalan Xing, Woojin Kim, Cheng Dong, Wolfram Tempel, et al. "First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor." Proceedings of the National Academy of Sciences 114, no. 38 (September 1, 2017): 10125–30. http://dx.doi.org/10.1073/pnas.1706907114.
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The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein–protein interactions in living cells and organisms. Here, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.
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Snyder, M. D., J. H. Pryor, M. D. Peoples, G. L. Williamson, M. C. Golding, M. E. Westhusin, and C. R. Long. "122 SUPPRESSION OF EPIGENETIC MODIFIERS ALTERS THE BOVINE EMBRYONIC DEVELOPMENTAL PROGRAM DURING IN VITRO CULTURE." Reproduction, Fertility and Development 26, no. 1 (2014): 175. http://dx.doi.org/10.1071/rdv26n1ab122.
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During early bovine embryogenesis, the regular establishment of DNA methylation and histone modification patterns is essential for proper gene expression and continuation of embryonic development. Epigenome patterns established during this period, if improperly maintained, can lead to developmental anomalies and may partially explain the lower pregnancy rates of in vitro-produced embryos. We hypothesised that the suppression of translation of the genes euchromatic histone-lysine N-methyltransferase 2 (EHMT2), DNA methyltransferase 3A (DNMT3A), absent, small, or homeotic-like (ASH2L), and SET domain, bifurcated 1 (SETDB1) would provide insightful information on the importance of these genes during early embryonic development in an in vitro setting. In order to define the roles of these genes, small interfering RNA (siRNA) targeting the gene of interest were synthesised and target verified in bovine cell culture using quantitative real-time RT-PCR (RT-qPCR). We acquired matured bovine oocytes from commercial suppliers, followed by IVF by standard laboratory procedures. Eighteen hours post IVF, cumulus cells were removed and zygotes separated into 3 different treatment groups: non-injected controls (CNTL), non-targeting siRNA injected controls (siNULL), and injection with siRNA targeting the gene of interest (si “gene target”). Each siRNA was mixed with a green fluorescent dextran at a concentration of 20 μM and ~100 pL injected cytoplasmically. The green fluorescent dextran was used to give visual confirmation that zygotes were indeed injected. Post-injection, fluorescent embryos were separated and cultured in Bovine Evolve (Zentih Biotech) medium supplemented with 4 mg mL–1 of BSA (Probumin, Millipore). Cleavage rates were monitored on Day 2, and only cleaved embryos were cultured further. On Day 8 post-IVF, embryos were morphologically examined and numbers of blastocysts recorded. Mean development rates between siNULL and targeting siRNA were compared using a t-test statistic. Over the course of these experiments the mean blastocyst rate for CNTL zygotes was 34.5% ± 2.6 s.e.m. (n = 1647). None of the zygotes injected with siEHMT2 (n = 1184) or siSETDB1 (n = 361) reached the blastocyst stage and these rates differed from the siNULL rate (21.0% ± 2.5 s.e.m., n = 1587; P < 0.05). Morphologically, embryos from both groups developed to the morula stage before they exhibited fragmentation. Injection of siDNMT3A also resulted in significant loss of viability at the 8-cell stage and few zygotes injected (n = 1057) developed to blastocyst (2.1% ± 0.5 s.e.m.; P < 0.001). Inhibiting gene expression of ASH2L showed little variation in blastocyst rate from our siNULL embryos (31.3% ± 2.0 s.e.m., n = 466 v. 34.8% ± 1.9 s.e.m., n = 418, respectively, P > 0.2). It is unknown at this time if inhibition of ASH2L translation will have effects later in development. Ongoing experiments analysing DNA methylation and histone modifications through immunocytochemistry and global gene expression via RT-qPCR will further explore the establishment and maintenance of these genes in the embryonic epigenome.
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Akishina, A. A., Yu E. Vorontsova, R. O. Cherezov, E. E. Kuvaeva, O. B. Simonova, and B. A. Kuzin. "Epigenetic modulation of transcription of human aryl-hydrocarboxylic receptor target genes in the Drosophila melanogaster transgenic line." Biomics 12, no. 4 (2020): 504–9. http://dx.doi.org/10.31301/2221-6197.bmcs.2020-44.
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Aryl hydrocarbon receptor (AHR), is an important ligand-dependent transcription factor, which retained its structural and functional features during evolution. AHR target genes play a key role in detoxification, in the regulation of the development and maintenance of eukaryotic homeostasis. The high conservatism of human AHR allowed us to study its functions in vivo using transgenic Drosophila melanogaster lines with an inducible human AHR gene. It is believed that Drosophila AHR homolog isn't activated by exogenous ligands, so human AHR can be tissue-specific activated in the body of a transgenic fly. This allows us to analyze the transcription level of AHR target genes in various organs and tissues. Unexpectedly we found that exogenous AHR ligands can increase as well as decrease the transcription levels of the AHR target genes, including genes that control proliferation, motility, polarization, and programmed cell death. We hypothesized that the absence of the expected increase in the transcription of some AHR target genes was caused by its epigenetic silencing. This hypothesis was confirmed in the experiments using histone lysine-N-methyltransferase inhibitors and with Drosophila null mutation of the methyltransferase Pc gene. As a result a new epigenetic mechanism of modulation of human aryl hydrocarbon receptor target genes transcription was discovered. Since exogenous AHR ligands and small molecule inhibitors of epigenetic modifiers are often used as pharmaceutical anticancer drugs, our findings may have significant implications in designing new combinations of therapeutic treatments for oncological diseases.
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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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Huang, Yanfang, Xiaohong Jiang, Miao Yu, Rongfu Huang, Jianfeng Yao, Ming Li, Fangfang Zheng, and Xiaoyu Yang. "Beneficial effects of diazepin-quinazolin-amine derivative (BIX-01294) on preimplantation development and molecular characteristics of cloned mouse embryos." Reproduction, Fertility and Development 29, no. 6 (2017): 1260. http://dx.doi.org/10.1071/rd15463.
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Somatic cell nuclear transfer is frequently associated with abnormal epigenetic modifications that may lead to the developmental failure of cloned embryos. BIX-01294 (a diazepine–quinazoline–amine derivative) is a specific inhibitor of the histone methyltransferase G9a. The aim of the present study was to investigate the effects of BIX-01294 on development, dimethylation of histone H3 at lysine 9 (H3K9), DNA methylation and the expression of imprinted genes in cloned mouse preimplantation embryos. There were no significant differences in blastocyst rates of cloned embryos treated with or without 0.1 μM BIX-01294. Relative to clone embryos treated without 0.1 μM BIX-01294, exposure of embryos to BIX-01294 decreased histone H3K9 dimethylation and DNA methylation in cloned embryos to levels that were similar to those of in vivo-fertilised embryos at the 2-cell and blastocyst stages. Cloned embryos had lower expression of octamer-binding transcription factor 4 (Oct4) and small nuclear ribonucleoprotein N (Snrpn), but higher expression of imprinted maternally expressed transcript (non-protein coding) (H19) and growth factor receptor-bound protein 10 (Grb10) compared with in vivo-fertilised counterparts. The addition of 0.1 μM BIX-01294 to the activation and culture medium resulted in lower H19 expression and higher cyclin dependent kinase inhibitor 1C (Cdkn1c) and delta-like 1 homolog (Dlk1) expression, but had no effect on the expression of Oct4, Snrpn and Grb10. The loss of methylation at the Grb10 cytosine–phosphorous–guanine (CpG) islands in cloned embryos was partially corrected by BIX-01294. These results indicate that BIX-01294 treatment of cloned embryos has beneficial effects in terms of correcting abnormal epigenetic modifications, but not on preimplantation development.
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Pareek, C. S., J. Michno, R. Smoczynski, J. Tyburski, M. Gołębiewski, K. Piechocki, M. Średzińska, et al. "Identification of predicted genes expressed differentially in pituitary gland tissue of young growing bulls revealed by cDNA-AFLP technique." Czech Journal of Animal Science 58, No. 4 (April 3, 2013): 147–58. http://dx.doi.org/10.17221/6709-cjas.
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Differentially expressed transcript derived fragments (TDFs) of bovine pituitary gland tissue at different developmental ages of Limousine and Hereford bulls were identified by cDNA-AFLP technique. Study revealed comparatively higher differentially expressed transcripts in 6-month Limousine bulls and 12-month Hereford bulls. The BLASTn/p analysis identified 3 and 21 predicted genes which gave significant e-values for Limousine and Hereford respectively, in assembled Bos taurus genome. The identified predicted genes expressed in bovine pituitary gland showed their mapped positions on bovine chromosomes: BTA2, 3, 5, 8, 9, 11, 12, 15–23, 26, and 28, respectively. Results based on TDF annotation identified 10 sequences that have BLAST hits to known annotated bovine genes and 14 sequences to unannotated contig regions in the latest gene Ensembl database Btau_4.0. Two breed specific predicted target genes were validated by qRT-PCR. Within and between breeds, qRT-PCR results revealed highly significant differences in the expression levels of bovine euchromatic histone-lysine N-methyltransferase 1 (EHMT1) and NCK adaptor protein 2 (NCK2) predicted genes. Obtained results conclude that cDNA-AFLP is a reliable technique for studying within breed age dependent gene expression patterns.
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Nowak-Imialek, M., C. Wrenzycki, D. Herrmann, I. Lagutina, A. Lucas-Hahn, E. Lemme, G. Lazzari, C. Galli, K. G. Hadeler, and H. Niemann. "258 MESSENGER RNA EXPRESSION PATTERNS OF HISTONE MODIFICATION GENES IN BOVINE EMBRYOS DERIVED FROM DIFFERENT ORIGINS." Reproduction, Fertility and Development 18, no. 2 (2006): 236. http://dx.doi.org/10.1071/rdv18n2ab258.
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Epigenetic modifications of the genome, such as covalent modifications of histones, are crucial for transcriptional regulation during development. The N-terminal tails of the histones are subject to post-translational modifications, including acetylation, deacetylation and methylation. histone acetylation loosens chromatin packing and correlates with transcriptional activation. The enzymes Histone acetyltransferases (HATs) transfer acetyl moieties to the lysine residues of histones H2A, H2B, H3, and H4. Histone acetylation is a reversible process which is catalyzed by the histone deacetylase (HDAC) and results in transcriptional repression. Histone methyltransferase (HMT) is responsible for the methylation of arginine in histones 3 and 4, playing an important role in transcriptional activation of genes. In contrast, the H3 Lys 9 methylation is associated with a transcriptionally repressive heterochromatin. The objective of the present study was to determine the effects of different origins of embryos on the relative abundance of transcripts for the histone acetyltransferase 1 (HAT1), histone deacetylase 2 (HDAC2), histone metyltransferases (SUV39H1 and G9A), and heterochromatin protein 1 (HP1). Messenger RNA expression profiles of these genes were investigated in bovine oocytes and pre-implantation embryos up to the blastocyt stage produced either in vitro by two different culture systems, i.e. SOF+BSA or TCM+SERUM, by somatic cloning using adult male and female fibroblasts, parthenogenetic activation, and androgenetic construction, or in vivo, employing semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Significant differences are described below. HAT1, SUV39H1, G9A, and HP1 mRNA transcripts decreased in enucleated oocytes, compared with immature oocytes. The relative abundance of HAT1 and SUV39H1 transcripts was significantly increased in NT-derived zygotes produced from adult female fibroblasts, compared to their in vitro fertilized and parthenogenetic counterparts. No differences were found in the relative abundances of gene transcripts at the 8-16-cell stage, except for parthenogenetic embryos in which SUV39H1 transcripts were significantly higher than in all other 8-16 cell groups. The relative abundance of SUV39H1, G9A, and HP1 transcripts were significantly higher in NT-derived blastocysts derived from adult male fibroblasts than in their in vivo-generated counterparts. HP1 and G9A transcript levels were significantly increased in NT-derived blastocysts derived from male fibroblasts compared to NT-derived embryos produced from female fibroblasts. The results show that the in vitro environment and the nuclear transfer protocol affect mRNA expression patterns of histone modification genes in pre-implantation bovine embryos.
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Laurie, Herviou, Fanny Izard, Elke De Bruyne, Eva Desmedt, Anqi Ma, Jian Jin, Karin Vanderkerken, Eric Julien, and Jerome Moreaux. "SET8 Is a Potential Therapeutic Target in MM." Blood 128, no. 22 (December 2, 2016): 4435. http://dx.doi.org/10.1182/blood.v128.22.4435.4435.
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Abstract Epigenetic regulation mechanisms - such as histone marks, DNA methylation and miRNA - are often misregulated in cancers and are associated with tumorigenesis and drug resistance. Multiple Myeloma (MM) is a malignant plasma cell disease that accumulates within the bone marrow. Epigenetic modifications in MM are associated not only with cancer development and progression, but also with resistance to chemotherapy. This epigenetic plasticity can be targeted with epidrugs, nowadays used in treatment of several cancers. We recently identified a significant overexpression of the lysine histone methyltransferase SETD8 in MM cells (HMCLs; N=40) compared with normal plasma cells (N=5) (P<0.001). SETD8 (also known as SET8, PR-Set7, KMT5A) is the sole enzyme responsible for the monomethylation of histone H4 at lysine 20 (H4K20me1) which has been linked to chromatin compaction and cell-cycle regulation. In addition, SETD8 induces the methylation of non-histone proteins, such as the replication factor PCNA, the tumor suppressor P53 and its stabilizing protein Numb. While SETD8-mediated methylation of P53 and Numb inhibits apoptosis, PCNA methylation upon SETD8 enhances the interaction with the Flap endonuclease FEN1 and promotes cancer cell proliferation. SETD8 is also implicated in DNA damage response, helping 53BP1 recruitment at DNA double-strand breaks. Consistent with this, overexpression of SETD8 is found in various types of cancer and has been directly implicated in breast cancer invasiveness and metastasis. A role of SETD8 in development of MM has however never been described. We found that high SETD8 expression is associated with a poor prognosis in 2 independent cohorts of newly diagnosed patients (UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N=158). Specific SETD8 inhibition with UNC-0379 inhibitor, causing its degradation and H4K20me1 depletion, leads to significant growth inhibition of HMCLs (N=10) and the murine cell lines 5T33MM and 5TGM1. MM cells treated with UNC-0379 presented a G0/G1 cell cycle arrest after 24h of treatment, followed by apoptosis 48h later. To confirm that SETD8 inhibition is as efficient on primary MM cells from patients, primary MM cells (N=8) were co-cultured with their bone marrow microenvironment and recombinant IL-6 and treated for 4 days with UNC-0379. Interestingly, treatment of MM patient samples with UNC-0379 reduces the percentage of myeloma cells (65%; P<0.005) without significantly affecting the non-myeloma cells, suggesting a specific addiction of primary myeloma cells to SETD8 activity. Melphalan is an alkylating agent commonly used in MM treatment. As SETD8 is known to be involved in the DNA damage response, we investigated the effect of its combination with Melphalan on HMCLs. Results show that this particular drug combination strongly enhances double strand breaks in HMCLs monitored using 53BP1 foci formation and gH2AX detection. This result emphasizes a potential role of SETD8 in DNA repair in MM cells. Furthermore, GSEA analysis of patients with high SETD8 expression highlighted a significant enrichment of genes involved in DNA repair, MYC-MAX targets and MAPK pathway. Our study is the first to demonstrate the importance of SETD8 for MM cells survival and suggest that SETD8 inhibition represent a promising strategy to improve conventional treatment of MM with DNA damaging agents. Disclosures No relevant conflicts of interest to declare.
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Muntean, Andrew G., Jiaying Tan, Venkatesha Basrur, Kojo S. J. Elenitoba-Johnson, and Jay Hess. "The PAF Complex Synergizes with MLL Fusion Proteins at .Hox Loci to Promote Leukemogenesis." Blood 114, no. 22 (November 20, 2009): 1277. http://dx.doi.org/10.1182/blood.v114.22.1277.1277.
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Abstract Abstract 1277 Poster Board I-299 Mixed lineage leukemia (MLL) is a histone H3 lysine 4 methyltransferase that is required to maintain a normal hematopoietic stem cell compartment. MLL functions to maintain expression of HOX genes as well as the HOX co-factor MEIS1, which play significant roles in regulating hematopoiesis. MLL is involved in chromosomal translocations with up to sixty different partners in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). HOXA9 and MEIS1, are directly regulated by MLL fusion proteins and are crucial for MLL fusion protein mediated transformation. The deregulated expression of target genes in AML is dependent on specific protein-protein interactions and functional domains of MLL. For example, the tumor suppressor Menin bridges LEDGF to the extreme N-terminus of MLL and both of these interactions are necessary for transformation. Furthermore, a DNA methyltransferase homology region (CxxC domain) of MLL is essential for binding to non-methylated CpG islands and MLL-fusion protein oncogenesis. We have found that sequences downstream of the CxxC domain, termed the RD2 region, that interact with the Polymerase Associated Factor (PAF) complex are also required for MLL fusion protein mediated transformation. The PAF complex interacts with RNA polymerase II and is required for H2B mono-ubiquitination and subsequent histone H3K4 and H3K79 methylation. Together the PAF complex has been shown to be involved in transcriptional initiation, elongation and termination. Interaction of MLL with the PAF complex is mediated through direct contacts with two subunits: Ctr9 and PAF1. The PAF complex synergizes with MLL-AF9 to augment transcriptional activation of the Hoxa9 promoter. Furthermore, MLL fusion proteins recruit high levels of the PAF complex to the Hoxa9 promoter. Importantly, deletions of the MLL RD2 region that abolish interactions with the PAF complex eliminate MLL-AF9 mediated transformation of mouse bone marrow cells. Transcription of PAF components is dramatically downregulated during differentiation of hematopoietic cells, consistent with recent data showing a requirement for the PAF complex to maintain an embryonic stem cell phenotype. Knock down and transplantation experiments are underway to further define how the PAF complex regulates normal MLL function and cooperates with MLL fusion proteins to promote leukemogenesis. Disclosures No relevant conflicts of interest to declare.
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Tan, Hwei Ling, Yi Bing Zeng, and Ee Sin Chen. "N-Terminus Does Not Govern Protein Turnover of Schizosaccharomyces pombe CENP-A." International Journal of Molecular Sciences 21, no. 17 (August 26, 2020): 6175. http://dx.doi.org/10.3390/ijms21176175.
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Centromere integrity underlies an essential framework for precise chromosome segregation and epigenetic inheritance. Although centromeric DNA sequences vary among different organisms, all eukaryotic centromeres comprise a centromere-specific histone H3 variant, centromeric protein A (CENP-A), on which other centromeric proteins assemble into the kinetochore complex. This complex connects chromosomes to mitotic spindle microtubules to ensure accurate partitioning of the genome into daughter cells. Overexpression of CENP-A is associated with many cancers and is correlated with its mistargeting, forming extra-centromeric kinetochore structures. The mislocalization of CENP-A can be counteracted by proteolysis. The amino (N)-terminal domain (NTD) of CENP-A has been implicated in this regulation and shown to be dependent on the proline residues within this domain in Saccharomyces cerevisiae CENP-A, Cse4. We recently identified a proline-rich GRANT motif in the NTD of Schizosaccharomyces pombe CENP-A (SpCENP-A) that regulates the centromeric targeting of CENP-A via binding to the CENP-A chaperone Sim3. Here, we investigated whether the NTD is required to confer SpCENP-A turnover (i.e., counter stability) using various truncation mutants of SpCENP-A. We show that sequential truncation of the NTD did not improve the stability of the protein, indicating that the NTD of SpCENP-A does not drive turnover of the protein. Instead, we reproduced previous observations that heterochromatin integrity is important for SpCENP-A stability, and showed that this occurs in an NTD-independent manner. Cells bearing the null mutant of the histone H3 lysine 9 methyltransferase Clr4 (Δclr4), which have compromised constitutive heterochromatin integrity, showed reductions in the proportion of SpCENP-A in the chromatin-containing insoluble fraction of the cell extract, suggesting that heterochromatin may promote SpCENP-A chromatin incorporation. Thus, a disruption in heterochromatin may result in the delocalization of SpCENP-A from chromatin, thus exposing it to protein turnover. Taken together, we show that the NTD is not required to confer SpCENP-A protein turnover.