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Chen, Xin, Mark Hiller, Yasemin Sancak, and Margaret T. Fuller. "Tissue-Specific TAFs Counteract Polycomb to Turn on Terminal Differentiation." Science 310, no. 5749 (November 3, 2005): 869–72. http://dx.doi.org/10.1126/science.1118101.
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Polycomb transcriptional silencing machinery is implicated in the maintenance of precursor fates, but how this repression is reversed to allow cell differentiation is unknown. Here we show that testis-specific TAF (TBP-associated factor) homologs required for terminal differentiation of male germ cells may activate target gene expression in part by counteracting repression by Polycomb. Chromatin immunoprecipitation revealed that testis TAFs bind to target promoters, reduce Polycomb binding, and promote local accumulation of H3K4me3, a mark of Trithorax action. Testis TAFs also promoted relocalization of Polycomb Repression Complex 1 components to the nucleolus in spermatocytes, implicating subnuclear architecture in the regulation of terminal differentiation.
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Chiacchiera, Fulvio, and Diego Pasini. "Control of adult intestinal identity by the Polycomb repressive machinery." Cell Cycle 16, no. 3 (November 28, 2016): 243–44. http://dx.doi.org/10.1080/15384101.2016.1252582.
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Kaundal, Babita, Anup K. Srivastava, Mohammed Nadim Sardoiwala, Surajit Karmakar, and Subhasree Roy Choudhury. "A NIR-responsive indocyanine green-genistein nanoformulation to control the polycomb epigenetic machinery for the efficient combinatorial photo/chemotherapy of glioblastoma." Nanoscale Advances 1, no. 6 (2019): 2188–207. http://dx.doi.org/10.1039/c9na00212j.
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Kuehner and Yao. "The Dynamic Partnership of Polycomb and Trithorax in Brain Development and Diseases." Epigenomes 3, no. 3 (August 21, 2019): 17. http://dx.doi.org/10.3390/epigenomes3030017.
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Epigenetic mechanisms, including DNA and histone modifications, are pivotal for normal brain development and functions by modulating spatial and temporal gene expression. Dysregulation of the epigenetic machinery can serve as a causal role in numerous brain disorders. Proper mammalian brain development and functions depend on the precise expression of neuronal-specific genes, transcription factors and epigenetic modifications. Antagonistic polycomb and trithorax proteins form multimeric complexes and play important roles in these processes by epigenetically controlling gene repression or activation through various molecular mechanisms. Aberrant expression or disruption of either protein group can contribute to neurodegenerative diseases. This review focus on the current progress of Polycomb and Trithorax complexes in brain development and disease, and provides a future outlook of the field.
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Flora, Pooja, Gil Dalal, Idan Cohen, and Elena Ezhkova. "Polycomb Repressive Complex(es) and Their Role in Adult Stem Cells." Genes 12, no. 10 (September 24, 2021): 1485. http://dx.doi.org/10.3390/genes12101485.
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Populations of resident stem cells (SCs) are responsible for maintaining, repairing, and regenerating adult tissues. In addition to having the capacity to generate all the differentiated cell types of the tissue, adult SCs undergo long periods of quiescence within the niche to maintain themselves. The process of SC renewal and differentiation is tightly regulated for proper tissue regeneration throughout an organisms’ lifetime. Epigenetic regulators, such as the polycomb group (PcG) of proteins have been implicated in modulating gene expression in adult SCs to maintain homeostatic and regenerative balances in adult tissues. In this review, we summarize the recent findings that elucidate the composition and function of the polycomb repressive complex machinery and highlight their role in diverse adult stem cell compartments.
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Cruz-Becerra, Grisel, Mandy Juárez, Viviana Valadez-Graham, and Mario Zurita. "Analysis of Drosophila p8 and p52 mutants reveals distinct roles for the maintenance of TFIIH stability and male germ cell differentiation." Open Biology 6, no. 10 (October 2016): 160222. http://dx.doi.org/10.1098/rsob.160222.
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Eukaryotic gene expression is activated by factors that interact within complex machinery to initiate transcription. An important component of this machinery is the DNA repair/transcription factor TFIIH. Mutations in TFIIH result in three human syndromes: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Transcription and DNA repair defects have been linked to some clinical features of these syndromes. However, how mutations in TFIIH affect specific developmental programmes, allowing organisms to develop with particular phenotypes, is not well understood. Here, we show that mutations in the p52 and p8 subunits of TFIIH have a moderate effect on the gene expression programme in the Drosophila testis, causing germ cell differentiation arrest in meiosis, but no Polycomb enrichment at the promoter of the affected differentiation genes, supporting recent data that disagree with the current Polycomb-mediated repression model for regulating gene expression in the testis. Moreover, we found that TFIIH stability is not compromised in p8 subunit-depleted testes that show transcriptional defects, highlighting the role of p8 in transcription. Therefore, this study reveals how defects in TFIIH affect a specific cell differentiation programme and contributes to understanding the specific syndrome manifestations in TFIIH-afflicted patients.
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Breiling, Achim, Edgar Bonte, Simona Ferrari, Peter B. Becker, and Renato Paro. "The Drosophila Polycomb Protein Interacts with Nucleosomal Core Particles In Vitro via Its Repression Domain." Molecular and Cellular Biology 19, no. 12 (December 1, 1999): 8451–60. http://dx.doi.org/10.1128/mcb.19.12.8451.
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ABSTRACT The proteins of the Polycomb group (PcG) are required for maintaining regulator genes, such as the homeotic selectors, stably and heritably repressed in appropriate developmental domains. It has been suggested that PcG proteins silence genes by creating higher-order chromatin structures at their chromosomal targets, thus preventing the interaction of components of the transcriptional machinery with theircis-regulatory elements. An unresolved issue is how higher order-structures are anchored at the chromatin base, the nucleosomal fiber. Here we show a direct biochemical interaction of a PcG protein—the Polycomb (PC) protein—with nucleosomal core particles in vitro. The main nucleosome-binding domain coincides with a region in the C-terminal part of PC previously identified as the repression domain. Our results suggest that PC, by binding to the core particle, recruits other PcG proteins to chromatin. This interaction could provide a key step in the establishment or regulation of higher-order chromatin structures.
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Luo, Xi, Kelly Schoch, Sharayu V. Jangam, Venkata Hemanjani Bhavana, Hillary K. Graves, Sujay Kansagra, Joan M. Jasien, et al. "Rare deleterious de novo missense variants in Rnf2/Ring2 are associated with a neurodevelopmental disorder with unique clinical features." Human Molecular Genetics 30, no. 14 (April 16, 2021): 1283–92. http://dx.doi.org/10.1093/hmg/ddab110.
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Abstract The Polycomb group (PcG) gene RNF2 (RING2) encodes a catalytic subunit of the Polycomb repressive complex 1 (PRC1), an evolutionarily conserved machinery that post-translationally modifies chromatin to maintain epigenetic transcriptional repressive states of target genes including Hox genes. Here, we describe two individuals, each with rare de novo missense variants in RNF2. Their phenotypes include intrauterine growth retardation, severe intellectual disabilities, behavioral problems, seizures, feeding difficulties and dysmorphic features. Population genomics data suggest that RNF2 is highly constrained for loss-of-function (LoF) and missense variants, and both p.R70H and p.S82R variants have not been reported to date. Structural analyses of the two alleles indicate that these changes likely impact the interaction between RNF2 and BMI1, another PRC1 subunit or its substrate Histone H2A, respectively. Finally, we provide functional data in Drosophila that these two missense variants behave as LoF alleles in vivo. The evidence provide support for deleterious alleles in RNF2 being associated with a new and recognizable genetic disorder. This tentative gene-disease association in addition to the 12 previously identified disorders caused by PcG genes attests to the importance of these chromatin regulators in Mendelian disorders.
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Leicher, Rachel, Eva J. Ge, Xingcheng Lin, Matthew J. Reynolds, Wenjun Xie, Thomas Walz, Bin Zhang, Tom W. Muir, and Shixin Liu. "Single-molecule and in silico dissection of the interaction between Polycomb repressive complex 2 and chromatin." Proceedings of the National Academy of Sciences 117, no. 48 (November 18, 2020): 30465–75. http://dx.doi.org/10.1073/pnas.2003395117.
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Polycomb repressive complex 2 (PRC2) installs and spreads repressive histone methylation marks on eukaryotic chromosomes. Because of the key roles that PRC2 plays in development and disease, how this epigenetic machinery interacts with DNA and nucleosomes is of major interest. Nonetheless, the mechanism by which PRC2 engages with native-like chromatin remains incompletely understood. In this work, we employ single-molecule force spectroscopy and molecular dynamics simulations to dissect the behavior of PRC2 on polynucleosome arrays. Our results reveal an unexpectedly diverse repertoire of PRC2 binding configurations on chromatin. Besides reproducing known binding modes in which PRC2 interacts with bare DNA, mononucleosomes, and adjacent nucleosome pairs, our data also provide direct evidence that PRC2 can bridge pairs of distal nucleosomes. In particular, the “1–3” bridging mode, in which PRC2 engages two nucleosomes separated by one spacer nucleosome, is a preferred low-energy configuration. Moreover, we show that the distribution and stability of different PRC2–chromatin interaction modes are modulated by accessory subunits, oncogenic histone mutations, and the methylation state of chromatin. Overall, these findings have implications for the mechanism by which PRC2 spreads histone modifications and compacts chromatin. The experimental and computational platforms developed here provide a framework for understanding the molecular basis of epigenetic maintenance mediated by Polycomb-group proteins.
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Rouleau, M., D. McDonald, P. Gagné, M. E. Ouellet, A. Droit, J. M. Hunter, S. Dutertre, C. Prigent, M. J. Hendzel, and G. G. Poirier. "PARP-3 associates with polycomb group bodies and with components of the DNA damage repair machinery." Journal of Cellular Biochemistry 100, no. 2 (February 1, 2007): 385–401. http://dx.doi.org/10.1002/jcb.21051.
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Lee, Patrick C., Phuong Le, Keegan Korthauer, Jingwei Cheng, John Doench, James A. DeCaprio, Derin B. Keskin, and Catherine J. Wu. "Identifying regulators of reversible MHC I loss in Merkel cell carcinoma through genome-scale screens." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 243.18. http://dx.doi.org/10.4049/jimmunol.204.supp.243.18.
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Abstract Loss of surface MHC I is an important mechanism by which cancer cells evade immune surveillance, and it can correlate with worse prognosis and resistance to immunotherapy. MHC I surface expression can be affected by genomic or transcriptional alterations to HLA-A/B/C, β2M, or the class I antigen processing machinery. In the case of transcriptional loss, we hypothesized that certain cancers might employ specific epigenetic programs to enforce downregulation of the MHC I pathway. Thus, we sought to identify novel MHC I regulators in MHC I-low cancers. In particular, we focused on Merkel cell carcinoma (MCC), a rare but aggressive neuroendocrine skin cancer that is caused by the Merkel cell polyomavirus in 80% of cases. Notably, MHC I downregulation is prevalent in MCC. We first generated and characterized a series of patient-derived MCC cell lines, in which MHC I surface expression was low but inducible with IFN-γ. We then conducted genome-scale CRISPR-KO and open reading frame (ORF) gain-of-function screens in one of our virus-positive MCC lines, using FACS to select for perturbations that upregulated surface MHC I. One of the top hits from these screens was PRC1.1, a non-canonical Polycomb repressive complex. Polycomb complexes are known to mediate gene silencing through chromatin modification and play an important role in development and cancer. These studies suggest a possible role for PRC1.1 in suppressing MHC I in MCC.
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Infante, Teresa, Francesco P. Mancini, Alessandro Lanza, Andrea Soricelli, Filomena de Nigris, and Claudio Napoli. "Polycomb YY1 is a critical interface between epigenetic code and miRNA machinery after exposure to hypoxia in malignancy." Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1853, no. 5 (May 2015): 975–86. http://dx.doi.org/10.1016/j.bbamcr.2015.01.009.
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Hanly, David J., Manel Esteller, and María Berdasco. "Interplay between long non-coding RNAs and epigenetic machinery: emerging targets in cancer?" Philosophical Transactions of the Royal Society B: Biological Sciences 373, no. 1748 (April 23, 2018): 20170074. http://dx.doi.org/10.1098/rstb.2017.0074.
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Of the diverse array of putative molecular and biological functions assigned to long non-coding RNAs (lncRNAs), one attractive perspective in epigenetic research has been the hypothesis that lncRNAs directly interact with the proteins involved in the modulation of chromatin conformation. Indeed, epigenetic modifiers are among the most frequent protein partners of lncRNAs that have been identified to date, of which histone methyltransferases and protein members of the Polycomb Repressive Complex PRC2 have received considerable attention. This review is focused on how lncRNAs interface with epigenetic factors to shape the outcomes of crucial biological processes such as regulation of gene transcription, modulation of nuclear architecture, X inactivation in females and pre-mRNA splicing. Because of our increasing knowledge of their role in development and cellular differentiation, more research is beginning to be done into the deregulation of lncRNAs in human disorders. Focusing on cancer, we describe some key examples of disease-focused lncRNA studies. This knowledge has significantly contributed to our ever-improving understanding of how lncRNAs interact with epigenetic factors of human disease, and has also provided a plethora of much-needed novel prognostic biomarker candidates or potential therapeutic targets. Finally, current limitations and perspectives on lncRNA research are discussed here. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.
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Garrick, David, Marco De Gobbi, Vasiliki Samara, Michelle Rugless, Michelle Holland, Helena Ayyub, Karen Lower, et al. "The role of the polycomb complex in silencing α-globin gene expression in nonerythroid cells." Blood 112, no. 9 (November 1, 2008): 3889–99. http://dx.doi.org/10.1182/blood-2008-06-161901.
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Although much is known about globin gene activation in erythroid cells, relatively little is known about how these genes are silenced in nonerythroid tissues. Here we show that the human α- and β-globin genes are silenced by fundamentally different mechanisms. The α-genes, which are surrounded by widely expressed genes in a gene dense region of the genome, are silenced very early in development via recruitment of the Polycomb (PcG) complex. By contrast, the β-globin genes, which lie in a relatively gene-poor chromosomal region, are not bound by this complex in nonerythroid cells. The PcG complex seems to be recruited to the α-cluster by sequences within the CpG islands associated with their promoters; the β-globin promoters do not lie within such islands. Chromatin associated with the α-globin cluster is modified by histone methylation (H3K27me3), and silencing in vivo is mediated by the localized activity of histone deacetylases (HDACs). The repressive (PcG/HDAC) machinery is removed as hematopoietic progenitors differentiate to form erythroid cells. The α- and β-globin genes thus illustrate important, contrasting mechanisms by which cell-specific hematopoietic genes (and tissue-specific genes in general) may be silenced.
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Birve, Anna, Aditya K. Sengupta, Dirk Beuchle, Jan Larsson, James A. Kennison, Åsa Rasmuson-Lestander, and Jürg Müller. "Su(z)12, a novelDrosophilaPolycomb group gene that is conserved in vertebrates and plants." Development 128, no. 17 (September 1, 2001): 3371–79. http://dx.doi.org/10.1242/dev.128.17.3371.
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In both Drosophila and vertebrates, spatially restricted expression of HOX genes is controlled by the Polycomb group (PcG) repressors. Here we characterize a novel Drosophila PcG gene, Suppressor of zeste 12 (Su(z)12). Su(z)12 mutants exhibit very strong homeotic transformations and Su(z)12 function is required throughout development to maintain the repressed state of HOX genes. Unlike most other PcG mutations, Su(z)12 mutations are strong suppressors of position-effect variegation (PEV), suggesting that Su(z)12 also functions in heterochromatin-mediated repression. Furthermore, Su(z)12 function is required for germ cell development. The Su(z)12 protein is highly conserved in vertebrates and is related to the Arabidopsis proteins EMF2, FIS2 and VRN2. Notably, EMF2 is a repressor of floral homeotic genes. These results suggest that at least some of the regulatory machinery that controls homeotic gene expression is conserved between animals and plants.
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Shen, Qingwen, Yisheng Lin, Yingbo Li, and Guifeng Wang. "Dynamics of H3K27me3 Modification on Plant Adaptation to Environmental Cues." Plants 10, no. 6 (June 8, 2021): 1165. http://dx.doi.org/10.3390/plants10061165.
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Given their sessile nature, plants have evolved sophisticated regulatory networks to confer developmental plasticity for adaptation to fluctuating environments. Epigenetic codes, like tri-methylation of histone H3 on Lys27 (H3K27me3), are evidenced to account for this evolutionary benefit. Polycomb repressive complex 2 (PRC2) and PRC1 implement and maintain the H3K27me3-mediated gene repression in most eukaryotic cells. Plants take advantage of this epigenetic machinery to reprogram gene expression in development and environmental adaption. Recent studies have uncovered a number of new players involved in the establishment, erasure, and regulation of H3K27me3 mark in plants, particularly highlighting new roles in plants’ responses to environmental cues. Here, we review current knowledge on PRC2-H3K27me3 dynamics occurring during plant growth and development, including its writers, erasers, and readers, as well as targeting mechanisms, and summarize the emerging roles of H3K27me3 mark in plant adaptation to environmental stresses.
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King, Ian F. G., Nicole J. Francis, and Robert E. Kingston. "Native and Recombinant Polycomb Group Complexes Establish a Selective Block to Template Accessibility To Repress Transcription In Vitro." Molecular and Cellular Biology 22, no. 22 (November 15, 2002): 7919–28. http://dx.doi.org/10.1128/mcb.22.22.7919-7928.2002.
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ABSTRACT Polycomb group (PcG) proteins are responsible for stable repression of homeotic gene expression during Drosophila melanogaster development. They are thought to stabilize chromatin structure to prevent transcription, though how they do this is unknown. We have established an in vitro system in which the PcG complex PRC1 and a recombinant PRC1 core complex (PCC) containing only PcG proteins are able to repress transcription by both RNA polymerase II and by T7 RNA polymerase. We find that assembly of the template into nucleosomes enhances repression by PRC1 and PCC. The subunit Psc is able to inhibit transcription on its own. PRC1- and PCC-repressed templates remain accessible to Gal4-VP16 binding, and incubation of the template with HeLa nuclear extract before the addition of PCC eliminates PCC repression. These results suggest that PcG proteins do not merely prohibit all transcription machinery from binding the template but instead likely inhibit specific steps in the transcription reaction.
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Tagore, Mohita, Michael J. McAndrew, Alison Gjidoda, and Monique Floer. "The Lineage-Specific Transcription Factor PU.1 Prevents Polycomb-Mediated Heterochromatin Formation at Macrophage-Specific Genes." Molecular and Cellular Biology 35, no. 15 (May 26, 2015): 2610–25. http://dx.doi.org/10.1128/mcb.00027-15.
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Lineage-specific transcription factors (TFs) are important determinants of cellular identity, but their exact mode of action has remained unclear. Here we show using a macrophage differentiation system that the lineage-specific TF PU.1 keeps macrophage-specific genes accessible during differentiation by preventing Polycomb repressive complex 2 (PRC2) binding to transcriptional regulatory elements. We demonstrate that the distal enhancer of a gene becomes bound by PRC2 as cells differentiate in the absence of PU.1 binding and that the gene is wrapped into heterochromatin, which is characterized by increased nucleosome occupancy and H3K27 trimethylation. This renders the gene inaccessible to the transcriptional machinery and prevents induction of the gene in response to an external signal in mature cells. In contrast, if PU.1 is bound at the transcriptional regulatory region of a gene during differentiation, PRC2 is not recruited, nucleosome occupancy is kept low, and the gene can be induced in mature macrophages. Similar results were obtained at the enhancers of other macrophage-specific genes that fail to bind PU.1 as an estrogen receptor fusion (PUER) in this system. These results show that one role of PU.1 is to exclude PRC2 and to prevent heterochromatin formation at macrophage-specific genes.
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Tan, Jiaying, and Jay L. Hess. "CBX8, a Polycomb-Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis." Blood 116, no. 21 (November 19, 2010): 4174. http://dx.doi.org/10.1182/blood.v116.21.4174.4174.
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Abstract Abstract 4174 Trithorax and Polycomb-group (Trx-G and Pc-G) proteins are antagonistic regulators of homeobox-containing (Hox) gene expression that play a major role in regulation of hematopoiesis and leukemogenesis. Mixed lineage leukemia (MLL), a mammalian Trx-G protein, is a histone methyltransferase crucial for embryonic development and hematopoiesis that is commonly altered by translocation in acute leukemia. Recent evidence suggests that transformation by MLL fusion proteins is dependent on multiple interaction complexes, including the polymerase associated factor complex (PAFc) and the elongation activating protein complex (EAPc) or a closely related AF4 family/ENL family/P-TEFb complex (AEPc). CBX8 is a human PcG protein, functioning as a transcription repressor in the polycomb repressive complex 1 (PRC1). Previous studies have shown that CBX8 also interacts with the EAPc components AF9 and ENL; however, its role in leukemogenesis is unknown. To elucidate the significance of this interaction between these two proteins thought to have antagonistic function, we generated a large series of point mutations in AF9 and identified two amino acids that are essential for CBX8 interaction but preserve the interaction with other EAP components. Mutation of the two sites reduced the transcriptional activation of the MLL-AF9 target promoters by nearly 50% and completely inhibits the ability of MLL-AF9 to immortalize bone marrow (BM) as assessed by methylcellulose replating assays. This finding suggests that CBX8 interaction is essential for MLL-AF9-induced leukemogenesis. Several lines of evidence further support this finding. First, CBX8 knockdown by siRNAs decreased MLL-AF9-induced transcriptional activation by approximately 50%. Second, the ability of MLL-AF9 to transform primary BM was markedly reduced by retroviral shCbx8 transduction. Notably, this inhibitory effect is specific for MLL-AF9 because the BM transformation ability of E2A-HLF was unaffected by Cbx8 suppression. Third, Cbx8 suppression by shCbx8 in MLL-AF9 and MLL-ENL, but not E2A-HLF transformed AML cell lines, significantly inhibited the expression of MLL-dependent target genes, as well as cell growth and colony forming ability. Fourth, inducing CBX8 knockdown in human leukemia cell lines expressing MLL-AF9 led to a marked decrease in the localization of basic transcription machinery at the Hoxa9 locus and a corresponding reduction in Hoxa9 transcription. Importantly, the observed effects of CBX8 on MLL-rearranged leukemia cells are PRC1-independent: no effects on MLL target gene expression, cell growth, or BM transformation ability were observed by suppressing other core components of PRC1. Taken together, our results indicate that CBX8, independent of its transcription repression role in PRC1, interacts with and synergizes with MLL fusion proteins to promote leukemogenesis. Defining the interaction sites between AF9/ENL and CBX8 and the dependence of other AML subtypes and normal hematopoiesis on CBX8 will be important for the further development of agents that target this mechanism in MLL-rearranged and potentially other AML subtypes. Disclosures: No relevant conflicts of interest to declare.
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Gillespie, Robert F., and Lorraine J. Gudas. "Retinoic Acid Receptor Isotype Specificity in F9 Teratocarcinoma Stem Cells Results from the Differential Recruitment of Coregulators to Retinoic Acid Response Elements." Journal of Biological Chemistry 282, no. 46 (September 17, 2007): 33421–34. http://dx.doi.org/10.1074/jbc.m704845200.
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The retinoic acid receptor (RAR) α, β2, and γ isotypes each regulate specific subsets of target genes in F9 teratocarcinoma stem cells. We used chromatin immunoprecipitation assays to monitor the association of RARγ, retinoic X receptor (RXR) α, and coregulators with the RARβ2, Hoxa1, and Cyp26A1 retinoic acid response elements (RAREs) in F9 wild type and RARα, -β2, and -γ null cells. Additionally we quantitatively monitored expression of the corresponding mRNAs. We demonstrated that the association of RARγ and/or RXRα with a RARE was not sufficient for retinoic acid (RA)-mediated transcription of the corresponding target gene. However, the ability of RARγ and/or RXRα to recruit pCIP (AIB1/ACTR/RAC-3/TRAM-1/SRC-3) and p300 to a RARE did correlate with RA-associated transcription of target mRNAs. Therefore, the specific functions of the RAR isotypes do not manifest at the level of their DNA binding but rather from a differential ability to recruit specific components of the transcriptional machinery. We also demonstrated that RA-mediated displacement of the polycomb group protein SUZ12 from a RARE was inhibited in the absence of RARγ. Thus, transcriptional components of the RAR signaling pathway are specifically required for displacement of SUZ12 from RAREs during RA-mediated differentiation of F9 cells.
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Xu, Jian, Zhen Shao, Dan Li, Huafeng Xie, Woojin Kim, Jialiang Huang, Luca Pinello, Kimberly Glass, Guo-Cheng Yuan, and Stuart H. Orkin. "Developmental Control of Polycomb Subunit Composition Mediates a Switch to Non-Canonical Functions during Hematopoiesis." Blood 124, no. 21 (December 6, 2014): 241. http://dx.doi.org/10.1182/blood.v124.21.241.241.
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Abstract The epigenetic machinery plays crucial roles in hematopoiesis, and its deregulation drives the pathogenesis of blood disorders. Polycomb Repressive Complex 2 (PRC2) is a major class of epigenetic repressor that catalyzes the di/tri-methylation of histone H3 lysine 27 (or H3K27me2/3). The canonical PRC2 complex consists of EED, SUZ12, and the histone methyltransferase EZH2. The functions of PRC2 in hematopoiesis remain elusive due in large to the existence of two highly related enzymatic subunits EZH1 and EZH2. While amplification or overexpression of PRC2 proteins is common in many cancers, inactivating mutation in PRC2 is frequently found in hematopoietic malignancies, indicating that PRC2 can be oncogenic or tumor suppressive in different cellular contexts. In light of recent efforts to therapeutically target EZH2 enzyme activities or canonical EZH2-PRC2 functions in various hematopoietic malignancies, it will be critical to fully assess the context-specific activity of this epigenetic complex in normal and malignant developmental processes. The molecular mechanisms by which PRC2 regulates normal and neoplastic hematopoiesis is unclear, as are the non-redundant effects of canonical versus non-canonical PRC2 functions, which are mediated by EZH1 or EZH2 independent of H3K27me2/3. In this study, we demonstrate that the PRC2 enzymatic subunits EZH1 and EZH2 undergo an expression switch during hematopoiesis. EZH2 is highly expressed in primary human CD34+ hematopoietic stem/progenitor (HSPC) cells and progressively downregulated during erythroid and lymphoid specification, whereas EZH1 is significantly upregulated during differentiation. We next examined the in vivo stoichiometry of the PRC2 complexes by quantitative proteomics and revealed the existence of an EZH1-SUZ12 sub-complex lacking EED subunit in human erythroid cells. Through genome scale chromatin occupancy (by ChIP-seq) and transcriptional profiling (by RNA-seq) analyses, we provide evidence that EZH1 together with SUZ12 form a non-canonical PRC2 complex, occupy active chromatin domains marked by H3K4me3 and H3K27me1, and positively regulate gene expression. Furthermore, loss of EZH2 expression leads to global repositioning of EZH1 chromatin occupancy to EZH2 targets, and EZH1 complements EZH2 loss within canonical PRC2 target genes. To elucidate the regulatory networks underlying the developmental control of EZH1/2 switch, we profiled the histone modifications and chromatin accessibility surrounding the EZH1 gene in both CD34+ HSPCs and committed erythroid cells. We identified and characterized an erythroid-selective enhancer element that is indispensable for the transcriptional activation of EZH1. Loss of function analysis using CRISPR/cas9-mediated enhancer deletion results in markedly decrease in EZH1 expression in human erythroid cells. Moreover, a switch from GATA2 to GATA1 expression controls the developmental EZH1/2 switch by differential association with distinct EZH1 enhancers during erythroid differentiation. Thus, the lineage- and developmental stage-specific regulation of PRC2 subunit composition leads to a switch from canonical silencing to non-canonical PRC2 functions. Our study also establishes a molecular link between the switch of master lineage regulators and developmental control of PRC2 composition, providing a means to coordinate linage-specific transcription and accompanying changes in the epigenetic landscape during blood stem cell specification. Disclosures No relevant conflicts of interest to declare.
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Price, Colles, Ping Chen, Shenglai Li, Zejuan Li, Yuanyuan Li, Xi Jiang, Hao Huang, et al. "Polycomb Group Member Rybp Is a Functional Tumor Suppressor Repressed By Mir-9 in MLL-Rearranged AML." Blood 124, no. 21 (December 6, 2014): 871. http://dx.doi.org/10.1182/blood.v124.21.871.871.
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Abstract MicroRNAs (miRNAs), are small non-coding RNA molecules known to be important regulators of cancer biology. Notably, we and others have shown that miRNAs play important roles in Acute Myeloid Leukemia (AML), a heterogeneous malignancies with multiple chromosomal and molecular abnormalities. Patients with chromosomal rearrangements involving mixed lineage leukemia (MLL), the mammalian homology of trithorax gene, are associated with poor survival. Previously, we have found that MLL-rearranged AML drives aberrant expression of several miRNAs, most notably microRNA-9 (miR-9). Expression of miR-9 with MLL-AF9, a common MLL-translocation, was sufficient to promote transformation normal hematopoietic progenitor cells in vitro and leukemogenesis in vivo. We previously found that miR-9 reduces expression of several genes but we did not know which genes were critical tumor suppressors. We found that the polycomb group member RING1- and YY1-Bindin Protein (RYBP) was consistently inhibited upon miR-9 expression. To assess the regulation of RYBP we used publically available data from the Cancer Genome Atlas (TCGA) and looked at genome-wide Illumina 450K methylation data. We did not find a strong correlation with methylation and RYBP expression, suggesting that expression of RYBP is likely not regulated by the DNA methylation machinery in patients. Upon looking at copy number alterations we found that a small population of AML patients contained either homozygous or heterozygous loss of RYBP, suggesting a potential role of RYBP in leukemia pathogenesis. To assess the role of RYBP we did a series of in vitro experiments. We found that expression of RYBP was sufficient to attenuate colony-forming growth driven by MLL- AF9. Furthermore, RYBP expression was able to reduce proliferation, increase apoptosis, and significantly reduce immature cell population. To determine the role of RYBP expression in vivo, we transplanted lethally irradiated mice with progenitors retrovirally transduced with MLL-AF9 compared to MLL-AF9 and RYBP. We found that expression of RYBP was sufficient to reduce leukemia burden in vivo as well as induce differentiation as shown by flow cytometry and histological analysis. Thus, this demonstrates that RYBP is a functional tumor suppressor in MLL-rearranged AML. In conclusion, we have demonstrated that chromosomal rearrangements involving MLL, the mammalian homology of trithorax, downregulates a member of the polycomb complex through upregulation of miR-9. Disclosures No relevant conflicts of interest to declare.
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Schnerch, Angelique, Jung Bok Lee, Monica Graham, Borhane Guezguez, and Mickie Bhatia. "Human Embryonic Stem Cell-Derived Hematopoietic Cells Maintain Core Epigenetic Machinery of the Polycomb Group/Trithorax Group Complexes Distinctly from Functional Adult Hematopoietic Stem Cells." Stem Cells and Development 22, no. 1 (January 2013): 73–89. http://dx.doi.org/10.1089/scd.2012.0204.
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Akkouche, Abdou, Sara Moodad, Rita Hleihel, Hala Skayneh, Séverine Chambeyron, Hiba El Hajj, and Ali Bazarbachi. "In vivo antagonistic role of the Human T-Cell Leukemia Virus Type 1 regulatory proteins Tax and HBZ." PLOS Pathogens 17, no. 1 (January 20, 2021): e1009219. http://dx.doi.org/10.1371/journal.ppat.1009219.
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Adult T cell leukemia (ATL) is an aggressive malignancy secondary to chronic infection by the human T-cell leukemia virus type 1 (HTLV-1) infection. Two viral proteins, Tax and HBZ, play central roles in ATL leukemogenesis. Tax expression transforms T cells in vitro and induces ATL-like disease in mice. Tax also induces a rough eye phenotype and increases hemocyte count in Drosophila melanogaster, indicative of transformation. Among multiple functions, Tax modulates the expression of the enhancer of zeste homolog 2 (EZH2), a methyltransferase of the Polycomb Repressive Complex 2 (PRC2), leading to H3K27me3-dependent reprogramming of around half of cellular genes. HBZ is a negative regulator of Tax-mediated viral transcription. HBZ effects on epigenetic signatures are underexplored. Here, we established an hbz transgenic fly model, and demonstrated that, unlike Tax, which induces NF-κB activation and enhanced PRC2 activity creating an activation loop, HBZ neither induces transformation nor NF-κB activation in vivo. However, overexpression of Tax or HBZ increases the PRC2 activity and both proteins directly interact with PRC2 complex core components. Importantly, overexpression of HBZ in tax transgenic flies prevents Tax-induced NF-κB or PRC2 activation and totally rescues Tax-induced transformation and senescence. Our results establish the in vivo antagonistic effect of HBZ on Tax-induced transformation and cellular effects. This study helps understanding long-term HTLV-1 persistence and cellular transformation and opens perspectives for new therapeutic strategies targeting the epigenetic machinery in ATL.
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Gurvich, Nadia, Francesca Voza, Silvia Menendez, and Stephen Nimer. "Loss of L3MBTL1, a Candidate 20q12 Tumor Suppressor Gene, Leads to DNA Damage." Blood 114, no. 22 (November 20, 2009): 1974. http://dx.doi.org/10.1182/blood.v114.22.1974.1974.
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Abstract Abstract 1974 Poster Board I-997 The L3MBTL1 gene is located on the long arm of chromosome 20 (q12), a region commonly lost in several myeloid malignancies, including myeloproliferative diseases (MPD), myeloprodysplastic disorders (MDS), and acute myeloid leukemias (AML). MDS and AML frequently have complex cytogenetic profiles, and are thought to arise due to accumulation of several cooperating mutations. We have previously reported that L3MBTL1 is highly expressed in human hematopoietic progenitor cells. L3MBTL1 is a homolog of Drosophila polycomb L3MBTL tumor suppressor protein. Thus, L3MBTL1 is a candidate gene in 20q12 myeloid disorders. We have depleted L3MBTL1 by RNAi in several human cell lines, and find that loss of L3MBTL1 leads to a decrease of cells in the S phase of the cell cycle and accumulation in G2/M phase. Cells with depleted L3MBTL1 have an increase of spontaneous DNA breaks, as evidenced by accumulation of γH2A.X foci and comet assay. The presence of DNA breaks leads to activation of DNA damage response, as the L3MBTL1 knockdown cells have increased levels of phosphoChk1 (Ser317 and Ser345), phosphoChk2 (Thr68), phosphoATM (Ser1981), p21 and p53. The DNA damage in the L3MBTL1-depleted cells activates DNA repair machinery, as shown by the increase in Rad51 levels. We propose that the spontaneous DNA damage caused by depletion of L3MBTL1 could contribute to the development of 20q12 myeloid malignancies. Disclosures: No relevant conflicts of interest to declare.
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Jiao, Lianying, Murtada Shubbar, Xin Yang, Qi Zhang, Siming Chen, Qiong Wu, Zhe Chen, Josep Rizo, and Xin Liu. "A partially disordered region connects gene repression and activation functions of EZH2." Proceedings of the National Academy of Sciences 117, no. 29 (July 6, 2020): 16992–7002. http://dx.doi.org/10.1073/pnas.1914866117.
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Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), which minimally requires two other subunits, EED and SUZ12, for enzymatic activity. EZH2 has been traditionally known to mediate histone H3K27 trimethylation, a hallmark of silent chromatin. Emerging evidence indicates that EZH2 also activates gene expression in cancer cells in a context distinct from canonical PRC2. The molecular mechanism underlying the functional conversion of EZH2 from a gene repressor to an activator is unclear. Here, we show that EZH2 harbors a hidden, partially disordered transactivation domain (TAD) capable of interacting with components of active transcription machinery, mimicking archetypal acidic activators. The EZH2 TAD comprises the SRM (Stimulation-Responsive Motif) and SANT1 (SWI3, ADA2, N-CoR, and TFIIIB 1) regions that are normally involved in H3K27 methylation. The crystal structure of an EZH2−EED binary complex indicates that the EZH2 TAD mediates protein oligomerization in a noncanonical PRC2 context and is entirely sequestered. The EZH2 TAD can be unlocked by cancer-specific EZH2 phosphorylation events to undergo structural transitions that may enable subsequent transcriptional coactivator binding. The EZH2 TAD directly interacts with the transcriptional coactivator and histone acetyltransferase p300 and activates gene expression in a p300-dependent manner in cells. The corresponding TAD may also account for the gene activation function of EZH1, the paralog of EZH2. Distinct kinase signaling pathways that are known to abnormally convert EZH2 into a gene activator in cancer cells can now be understood in a common structural context of the EZH2 TAD.
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Matilla, Angel J. "Exploring Breakthroughs in Three Traits Belonging to Seed Life." Plants 11, no. 4 (February 11, 2022): 490. http://dx.doi.org/10.3390/plants11040490.
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Based on prior knowledge and with the support of new methodology, solid progress in the understanding of seed life has taken place over the few last years. This update reflects recent advances in three key traits of seed life (i.e., preharvest sprouting, genomic imprinting, and stored-mRNA). The first breakthrough refers to cloning of the mitogen-activated protein kinase-kinase 3 (MKK3) gene in barley and wheat. MKK3, in cooperation with ABA signaling, controls seed dormancy. This advance has been determinant in producing improved varieties that are resistant to preharvest sprouting. The second advance concerns to uniparental gene expression (i.e., imprinting). Genomic imprinting primarily occurs in the endosperm. Although great advances have taken place in the last decade, there is still a long way to go to complete the puzzle regarding the role of genomic imprinting in seed development. This trait is probably one of the most important epigenetic facets of developing endosperm. An example of imprinting regulation is polycomb repressive complex 2 (PRC2). The mechanism of PRC2 recruitment to target endosperm with specific genes is, at present, robustly studied. Further progress in the knowledge of recruitment of PRC2 epigenetic machinery is considered in this review. The third breakthrough referred to in this update involves stored mRNA. The role of the population of this mRNA in germination is far from known. Its relations to seed aging, processing bodies (P bodies), and RNA binding proteins (RBPs), and how the stored mRNA is targeted to monosomes, are aspects considered here. Perhaps this third trait is the one that will require greater experimental dedication in the future. In order to make progress, herein are included some questions that are needed to be answered.
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Ntziachristos, Panagiotis, Aristotelis Tsirigos, Grant Welstead, Thomas Trimarchi, Linda Holmfeldt, Takashi Satoh, Elisabeth M. Paietta, et al. "An Oncogene-Regulated Epigenetic Switch in T Cell Acute Lymphoblastic Leukemia." Blood 124, no. 21 (December 6, 2014): 56. http://dx.doi.org/10.1182/blood.v124.21.56.56.
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Abstract Although the cure rate for acute lymphoblastic leukemia (ALL), a frequent pediatric leukemia, has improved dramatically, the overall prognosis remains dismal due to frequent disease relapse and the absence of non-cytotoxic targeted therapy options. Up to 25% of children fail frontline therapy and in these cases prognosis is dismal and the cure rate is approximate 20%. Main current therapies are based on intensive induction chemotherapy that is most frequently coupled to intrathecal chemotherapy alone or with cranial irradiation for central nervous system prophylaxis, which has severe short and long-term side effects. Thus, the ultimate and most critical aim for developing new treatments in different types of leukemia is to block the effects of specific cancer-inducing oncogenes. Others and we have previously shown that T cell ALL (T-ALL) is characterized by activating mutations in the NOTCH signaling pathway. It is currently unclear how key transcription factors in T-ALL such as NOTCH1 recruit the epigenetic machinery and bring together different chromosomal domain, in order to carry out the oncogenic transformation program. We generated evidence that NOTCH1 oncogenic action leads to important epigenetic changes through antagonizing the polycomb repressive complex 2 (PRC2) and leads to loss of the repressive mark histone 3 lysine 27 di/tri-methylation (H3K27me2/3). Moreover, we identified inactivating mutations of the polycomb repressive complex 2 (PRC2), the “writer” of Histone 3 lysine 27 methylation, in primary samples from human patients revealing a tumor suppressor role for the complex in T-ALL. Further extending our work on the H3K27me3 mark, we showed the oncogenic role for the Jumonji d3 (JMJD3) demethylase. Functionally, genomic ablation of the JMJD3 modulator as well as targeting with a specific chemical inhibitor, GSKJ4, generated by GlaxoSmithKline, leads to apoptosis and cell cycle arrest of T-ALL lines and primary cells. Genetic ablation of JMJD3 leads to slower initiation of the disease with significantly improved survival rates of the mice. Surprisingly, UTX acts as a tumor suppressor in the context of the same disease, as part of different transcriptional complexes, and we found that it is genetically inactivated in T-ALL patients. In light of recent developments on novel epigenetic inhibitors against JMJD3, these findings pave the way to specific pharmacological targeting of T cell leukemia. Based on this activity of Notch1 oncogene on epigenetic marks we further hypothesized that the switch from physiological to oncogenic activity might be mediated by changes in enhancer-promoter interaction networks forming chromosomal domains. A substantial percentage of these interactions are likely to be specific for the malignant state, and their disruption with epigenetic pharmacological inhibitors would not potentially affect healthy tissues. Studies in our laboratory show for the first time in leukemia that NOTCH1 chromatin binding sites are associated with enhancer-promoter interactions at oncogenic loci, using up-to-date chromosome conformation capture technology. We hereby show the importance of these interactions for oncogenic gene expression and pharmacological targeting of leukemic cells. These findings lend further rationale to the use of epigenetic drugs for targeted treatment of T cell leukemia. Disclosures Kruidenier: GlaxoSmithKline: Employment. Prinjha:GlaxoSmithKline: Employment.
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Ariës, Ingrid, Triona Ni Chonghaile, Salmaan Karim, Mina Jacob, Kristen E. Stevenson, Donna S. Neuberg, Meenakshi Devidas, et al. "PRC2 Mutations Induce Resistance to Conventional Chemotherapy By Inhibiting Mitochondrial Apoptosis in T-Cell Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 604. http://dx.doi.org/10.1182/blood.v128.22.604.604.
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Abstract Although contemporary combination chemotherapy can cure a substantial fraction of patients with T-cell acute lymphoblastic leukemia (T-ALL), front-line therapy fails in 15-20% of children and 50-70% of adults, and these patients have a poor prognosis. Strikingly, half of treatment failure events in childhood T-ALL are induction failure, suggesting pre-existing resistance to chemotherapeutics with distinct molecular targets. The molecular basis for induction failure remains poorly understood. Recent work has shown that mitochondrial apoptosis resistance is a cellular phenotype that predicts chemotherapy failure in some tumor types. However, the molecular mechanisms responsible for the striking variability in chemotherapy response among different patients with seemingly identical tumors remain largely unknown. Using a technique known as BH3 profiling, we analyzed mitochondrial apoptosis sensitivity or resistance in pre-treatment clinical specimens from a cohort of 47 children and adolescents treated on the COG AALL0434 or DFCI 05001 clinical trials. We found that mitochondrial apoptosis resistance was strongly associated with a poor response to induction chemotherapy (P = 0.008), as well as inferior 5-year event-free survival (65% vs 88%; P = 0.036 by log-rank test). Apoptosis resistance was weakly associated with the early T-cell precursor (ETP) immunophenotype (P = 0.08), but univariate and multivariable Cox regression analysis including both revealed that apoptosis resistance predicts clinical outcome more strongly than ETP status. To identify molecular lesions underlying mitochondrial apoptosis resistance, we applied targeted exome sequencing and array CGH to this cohort. We found that loss-of-function mutations in genes encoding core components of the polycomb repressive complex 2 (PRC2), including EZH2, EED or SUZ12, are associated with resistance to mitochondrial apoptosis (P = 0.015). PRC2 is a chromatin-modifying complex best known for its role in transcriptional repression. The PRC2 complex has been implicated as a tumor suppressor in T-ALL, but whether PRC2 plays a direct role in chemotherapy response is unknown. To test whether PRC2 regulates mitochondrial apoptosis in human T-ALL, we performed shRNA knockdown of the PRC2 core components EZH2, EED or SUZ12 in human T-ALL cell lines. Knockdown of each of these genes significantly induced mitochondrial apoptosis resistance, as assessed by BH3-profiling. This effect was dependent on the lysine methyltransferase activity of the PRC2 complex, because the effect of EZH2 knock-down was rescued by expression of wild-type EZH2, but not a point mutant that is methyltransferase-defective (P < 0.001). PRC2 knockdown also induced significant resistance to apoptosis induction (assessed using caspase 3/7 activation or annexin V/PI staining) in response to various chemotherapeutics with distinct molecular targets, including vincristine, dexamethasone, asparaginase, methotrexate, mercaptopurine, nelarabine, cytarabine and etoposide. To test whether PRC2 regulates mitochondrial apoptosis during normal T-cell development, we took advantage of mice heterozygous for a floxed Ezh2 or Eed allele, and induced deletion of one allele in hematopoietic cells using Mx-Cre activation by pIpC. Controls were Ezh2 and Eed wild-type mice with Mx-Cre activation. BH3 profiling analysis revealed that loss of one Ezh2 or Eed allele is sufficient to induce apoptosis resistance in non-transformed double-negative thymocytes (P = 0.003 for Ezh2 and P = 0.008 for Eed), suggesting that chemotherapy resistance can develop prior to oncogenic transformation. To define the transcriptional consequences of PRC2 inhibition in T-ALL, we performed RNA sequencing of T-ALL cells infected with shRNAs targeting EZH2, EED or SUZ12 (2 independent hairpins for each gene), or two control shRNAs. RNA sequencing analysis revealed a number of candidate transcriptional targets linking PRC2 to the mitochondrial apoptotic machinery, which are currently being investigated using functional genetics and small molecule inhibitors. Collectively, these data implicate polycomb repressive complex 2 function as a key determinant of chemotherapy response in childhood T-ALL. Defining the mechanism linking PRC2 to the mitochondria will provide a rational target for therapeutic intervention. Disclosures Teachey: Novartis: Research Funding. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding.
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Yamagishi, Makoto, Harutaka Katano, Tsunekazu Hishima, Yasunori Ota, Seiji Okada, and Toshiki Watanabe. "Epigenetically Programmed Defenseless Signaling in Malignant Lymphoma." Blood 126, no. 23 (December 3, 2015): 1230. http://dx.doi.org/10.1182/blood.v126.23.1230.1230.
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Abstract B-cell receptor (BCR) signaling is a major source of gene expression signature important for B cell survival, functions, and development. The integrated signaling amplitude should be equilibrated; when chronically activated by genetic perturbations or other mechanisms, BCR signaling has been accepted as a stem in the pathogenesis of malignant lymphoma/leukemia. The promiscuous signaling network without appropriate alleviation is an essential property of cancers. Diffuse large B-cell lymphoma (DLBCL) with aggressive phenotypes often associates with BCR signaling activation and epigenetic abnormalities, both of which are well correlated with lymphoma subtypes and disease progression. However, the relationship of the two fundamental characteristics is unknown. It is also still unclear the mechanism by which the expression of functionally important genes is continuously deregulated. microRNAs (miRNAs) are an emerging class of intrinsic buffering molecules that have diverse functions in mainly post-transcriptional regulation through miRNA-RISC (miRISC) formation. Using optimized RISC-capture assay, we revealed that multiple BCR signaling factors were persistently regulated by miRNA in human B cells. Clinical samples from newly diagnosed patients with DLBCL (n=83) showed epigenetic loss of an essential miRNA set (miR-200c, miR-203, miR-31). Conventional screening and RISC profiling identified multiple targets (CD79B, SYK, PKCbII, PLCg1, IKKb, NIK, MYD88, PI3K class I (a/b/d/g), RasGRP3); the miRNA-orchestrated overlapping interactome suggested multiple regulatory windows in signaling pathways, which implied their compensatory roles. The shared components within the interconnected signaling pathways were under constant surveillance by the miRNAs. We demonstrated that simultaneous depletion of the key miRNAs enhanced translation of the multiple targets and caused seamless signaling crosstalk among canonical/noncanonical NF-kB pathways, PI3K-Akt-mTOR and Ras-Erk cascades, and downstream of BCR and BAFF receptors. In addition to the miRNA loss, mTOR activation was also positively correlated with signaling amplitude, offering the possibility of selective therapy targeting the translation machinery. The defenseless signaling crosstalk appeared to be a prerequisite for lymphoma development because the essential miRNA set were functionally lost in all tested lymphoma samples. Surprisingly, a common epigenetic mechanism was identified. Polycomb-mediated H3K27me3 accumulation and reciprocal H3K4me3 loss were frequently observed at the miRNAs loci in primary DLBCL samples. We found that lymphoma-associated deregulation (mutation and/or expression change) of EZH2 and MLL2 cooperated to cause the sustained signaling activities of NF-kB, Ras-Erk, PI3K-Akt and mTOR pathways through miRNA silencing en masse. Of note, the perturbation of histone-modifying polycomb and trithorax groups resulted from variations of pooled miRNAs and signaling integrity. The identified coherent circuit may be a source of pathological robustness and phenotypic convergence. Most malignant B cell clones have some kind of genetic lesions. In particular, recurrent variations have been identified within signaling cascades, where miRNA-formed cooperative gene interference (CGI) could buffer the signaling noise. We re-established some experimental models that mimicked the signaling cascade observed in clinically established malignant clones. We experimentally confirmed the buffering roles of the key miRNAs against genetic mutation/alteration of CARD11, A20, MYD88 and PTEN. Reciprocally, coordinated reduction of the functional miRNAs promoted biological processes caused by genetic perturbations. It is therefore conceivable that systematic redundancy of miRNA is inherently required for robustness against genetic alterations that are acquired in lymphoma evolution. We illustrate an example of a biological masterplan comprised of signaling pathways, compensatory actions of multi-layered miRNAs, translation regulation and epigenetic mechanisms. Malignant cell opts for epigenetic reprogramming to stabilize mutation-driven processes and to surmount the demarcation threshold of transformation. Reversing the plastic characteristics of epigenetic alterations is expected in future clinical settings. Disclosures Yamagishi: Daiichi Sankyo Co., Ltd.: Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.
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Papaemmanuil, Elli. "Somatic Mutations in Myelodysplastic Syndrome." Blood 124, no. 21 (December 6, 2014): SCI—22—SCI—22. http://dx.doi.org/10.1182/blood.v124.21.sci-22.sci-22.
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Abstract Myelodysplastic syndromes (MDS) are clonal stem cell neoplasms affecting patients usually over 60 years old that typically present into the clinic with common symptoms including cytopenias, recurrent infections, bleeding and bruising. Approximately 20-30% of MDS patients progress to acute myeloid leukemia (AML) and are associated with inferior survival1. Diagnosis of MDS relies on findings from peripheral blood counts, examination of bone marrow morphology and evaluation of cytogenetic profiles for chromosomal aberrations. Using the WHO 2008 criteria, the proportion of blasts in the bone marrow, the number of cell lineages affected and the presence of del(5q) are collectively evaluated to classify patients into one of the five MDS categories [refractory anemia, refractory anemia with ring sideroblasts, refractory cytopenia multilineage dysplasia, refractory anemia with excess blasts, MDS with del(5q)]. The International Prognostication Scoring System (IPSS & IPSS-R) is the most widely used prognostic system in MDS. IPSS utilizes morphological variables to assign patients into low, intermediate or high-risk groups2. Accurate classification into one of these prognostic categories is critical as it determines selection of therapy regimes. Recent systematic profiling screens of MDS genomes have unraveled a complex network of cellular pathways that are causally implicated in MDS pathogenesis. Mutations have now been characterized in a number of key components of the spliceosome machinery (SF3B1, SRSF2, U2AF1, U2AF2, ZRSR2), regulators of DNA methylation (DNMT3A, IDH1, IDH2, TET2), chromatin modification (ASXL1, EZH2), transcription (EVI1, RUNX1, GATA2), signal transduction (NRAS, JAK2, KRAS, CBL) and cell cycle control (TP53)3-9. Collectively, more than 40 genes are significantly mutated in MDS; these mutations account for nearly 90% of MDS patients. The majority of patients present with two or more oncogenic mutations at diagnosis, and significant patterns of gene-gene interactions and mutual exclusivity have been reported10,11. Systematic integration of mutation data with large and well-annotated clinical datasets offers an unprecedented opportunity to decipher both the diagnostic as well as prognostic potential of these mutations as clinical biomarkers. However, the underlying genetic heterogeneity imposes significant challenges and important considerations that need to be accounted for when interpreting observed correlations between genotype, morphology and patient outcome. To unravel the interlocking genetic heterogeneity in MDS, Bejar et al., Papaemmanuil et al., and Haferlach et al. have studied the prevalence of acquired gene mutations in MDS and closely related chronic myeloid neoplasms in ~ 2100 MDS patients with well-annotated diagnostic and clinical outcome variables10-12. Univariate analysis has identified more than 10 genes to be significantly correlated with clinical outcome, including SF3B1, SRSF2, ASXL1, RUNX1, TP53, BCOR, RUNX1, EZH2, IDH2, ZRSR2, U2AF1 and CUX1. The total number of oncogenic mutations identified in each patient is selected as one of the most significant genetic predictors of outcome. Mutations in gene components of the spliceosome machinery are observed in approximately 50% of MDS patients, identifying pre-mRNA splicing as the most frequently altered biological process in MDS. Additionally, clonal relationship analysis of these mutations identifies that mutations in splicing genes occur early, followed by mutations in preferred partner genes, and mutations in different genes of the spliceosome machinery are associated with distinct morphological classification groups. The present talk will provide an overview of our current understanding of the underlying molecular mechanisms that underpin MDS biology. It will also evaluate how the genetic architecture of MDS can be incorporated in developing reliable and informative patient classification as well as outcome prediction models that can support clinical decision making in the future. References: 1. Tefferi A, Vardiman JW. Myelodysplastic syndromes. N Engl J Med. 2009;361(19):1872-1885. 2. Greenberg PL, Tuechler H, Schanz J, et al. Revised International Prognostic Scoring System (IPSS-R) for myelodysplastic syndromes. Blood. 2012. 3. Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 2011;478(7367):64-69. 4. Graubert TA, Shen D, Ding L, et al. Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes. Nat Genet. 2012;44(1):53-57. 5. Ernst T, Chase AJ, Score J, et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet. 2010;42(8):722-726. 6. Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. N Engl J Med. 2010;363(25):2424-2433. 7. Mardis ER, Ding L, Dooling DJ, et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med. 2009;361(11):1058-1066. 8. Gelsi-Boyer V, Trouplin V, Adelaide J, et al. Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia. Br J Haematol. 2009;145(6):788-800. 9. Shih AH, Levine RL. Molecular biology of myelodysplastic syndromes. Semin Oncol. 2011;38(5):613-620. 10. Haferlach T, Nagata Y, Grossmann V, et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia. 2014;28(2):241-247. 11. Papaemmanuil E, Gerstung M, Malcovati L, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood. 2013;122(22):3616-3627; quiz 3699. 12. Bejar R, Stevenson K, Abdel-Wahab O, et al. Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med. 2011;364(26):2496-2506. Disclosures No relevant conflicts of interest to declare.
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Chung, Jihyun, Vrajesh Karkhanis, Sif Said, and Robert A. Baiocchi. "Protein Arginine Methyltransferase 5 Regulates WNT/β-Catenin Target Gene Expression in at Multiple Levels." Blood 128, no. 22 (December 2, 2016): 4106. http://dx.doi.org/10.1182/blood.v128.22.4106.4106.
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Abstract Introduction: It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promotes lymphomagenesis. Over-expression of the type II protein arginine methyltransferase (PRMT5) enzyme has been associated with increased cell proliferation and survival in both solid and blood cancers. We have reported that PRMT5 is essential for EBV-driven B cell transformation and that it regulates the EZH2, EED and SUZ12, components of the Polycomb-repressive complex 2 (PRC2) complex via transcriptional silencing of RBL2and hyper-phosphorylation of RB1 in aggressive lymphomas. Here we report a mechanistic assessment of how PRMT5 over-expression supports the WNT/β-CATENIN pathway at multiple levels and drives oncogenic β-CATENIN target genes in lymphoma. Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells was achieved by infecting with sh-PRMT5 lentivirus (or sh-GFP control), a selective small molecule PRMT5 inhibitor (Alinari et al, Blood 2015, CMP5) or CRISPR/CAS9 PRMT5 deletion. Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP-PCR assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by MTS proliferation assay and FACS analysis. WIF1 protein detection in cell culture media was performed by ELISA. Results: PRMT5 regulated WNT/β-CATENIN signaling by direct transcriptional repression of AXIN2 and WIF1, two proteins that negatively regulate this pathway. PRMT5 inhibition with shRNA, CRISPR/CAS9 deletion, or CMP5 led to restored expression of AXIN2 and WIF1 transcript and protein that was associated with transcriptional repression of WNT/β-CATENIN target genes CYCLIN D1, MYC, and SURVIVIN. With PRMT5 inhibition, we observed differential enhanced recruitment of co-repressors LSD and HDAC2 and loss of associated epigenetic marks H3K4Me3 and H3K9Me (associated with the LSD demethylase) and H3K9Ac and H3K14Ac (associated with HDAC2) at each b-CATENIN target promoter. PRMT5 inhibition was found to reduce recruitment of co-activators CBP and MLL1 and respective epigenetic mark H3K4me3. The de-repression of AXIN2 and WIF1 was associated with loss of phospho-AKT (S450, S473) and down-stream survival pathways. Reduction in phospho-AKT was attributed to physical association between AXIN2 and the down-stream activity of secreted WIF1 in media of lymphoma cells treated with PRMT5 inhibition or CRISPR/CAS9 PRMT5 deletion. Furthermore, reduction of phospho-AKT prevented dimerization of MLL1 leading to dissociation of the BCL9-Pygopus TCF1/b-CATENIN transcriptional activation complex at MYC, CYCLIND1, and SURVIVIN promoters. Our observations show that PRMT5 is an important epigenetic regulator that governs the expression WNT/β-CATENIN-driven oncogenes c-MYC, CYCLIND D1 and SURVIVIN. PRMT5 inhibition restores regulation at several levels that converge on AKT signaling; (i) AXIN2-AKT interaction and (ii) WIF1 inhibition of WNT signaling. The restored regulation occurs via modulation of the downstream transcriptional machinery that is supported by constitutive AKT activity. This data identifies a novel pathway to interfere with WNT/β-CATENIN signaling and validates the driver role orchestrated by PRMT5 in lymphoma. Disclosures Baiocchi: Essanex: Research Funding.
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Lee, Miyoung, Aleksandra Filipovic, and Curtis J. Henry. "Combinatorial Inhibition of Galectin-9 and CHK1 Represent a Novel Treatment Strategy for T-Cell Acute Lymphoblastic Leukemia." Blood 138, Supplement 1 (November 5, 2021): 4400. http://dx.doi.org/10.1182/blood-2021-154404.
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Abstract Due to improvements in treatment strategies, the five-year event-free survival rate for pediatric patients with acute lymphoblastic leukemia (ALL) is 90%. However, patients with relapse and refractory disease fare much worse with 5-year overall survival rates of less than 50% in patients receiving chimeric antigen receptor T-cell therapy and fewer than 20% of patients surviving after receiving hematopoietic stem cell transplantation. These dismal outcomes for patients with relapse and refractory disease highlight the need for novel treatment regimens when current therapeutic options are exhausted. T-cell acute lymphoblastic leukemia (T-ALL) accounts for around 15% and 25% of ALL cases in pediatric and adult populations, respectively. This disease is driven by various molecular changes including alterations in the epigenome due, in part, to deregulated epigenetic machinery such as the polycomb repressive complex 2 (PRC2). Despite this observation, and ongoing clinical trials determining the utility of epigenetic drugs for treating various hematological malignancies, the role of the epigenome in T-ALL pathogenesis and the efficacy of epigenetic modifying drugs as treatments for this disease is heavily understudied. Galectins are members of s-type lectins which promote diverse biological processes including adhesion, signaling, and immunosuppression. Galectin-9 (Gal-9) is an emerging therapeutic target for solid cancers and hematological malignancies given that its presence is associated with poor outcomes for multiple cancers. In unpublished studies, we have found that Gal-9 is expressed on the surface of multiple human ALL subtypes with the highest basal surface expression found on T-ALL cells. To determine how this lectin impacts the function of human T-ALL cells, we treated leukemia cells with immunoglobulin control (Ig Ctrl) or anti-Gal-9 antibody (αGal-9Ab) and assessed the impact of treatment on cell cycle progression, DNA damage, and apoptosis. We used two αGal-9Ab clones for these experiments, a commercially available antibody and LYT-200 (a proprietary antibody in Phase I clinical trials for solid tumors from PureTech Health). Treatment with the commercially available antibody, but not Ctrl Ig, increased histone 3 trimethylation (H3K2me 3/H3K4me 3) with accompanying decreases in EZH2 and RING1A protein expression in human T-ALL cell lines. Antibody-induced epigenetic changes also promoted cell cycle progression (G2M transition), DNA damage, and extensive apoptosis (>90%) in multiple human T-ALL cell lines (n>6). Importantly, LYT-200 single-agent treatment also induced cell death in human T-ALL cells, demonstrating that blocking multiple epitopes on Gal-9 is sufficient to induce T-ALL cytotoxicity. These results highlight a previously unreported role for Galectin-9 in the epigenetic regulation and survival of human T-ALL cells. Given our observations that epigenome stability is critical for the survival of human T-ALL cells, we next sought to determine if the combination of αGal-9Ab treatment and epigenetic modifying drugs would further enhance the cytotoxicity of human T-ALL cells. We tested the combination of αGal-9Ab treatment and multiple drugs targeting either histone acetylation, methylation, or phosphorylation. Of these, we found that combining αGal-9Ab and GDC-0575 (a CHK1 inhibitor) resulted in extensive DNA damage and cytotoxicity (>98%). Mechanistically, we found αGal-9Ab treatment induces DNA damage in multiple human T-ALL lines, which leads to CHK1 activation. Given that GDC-0575 inhibits CHK1 activity, and CHK1 is a master regulator of the DNA damage response, we predict that the enhanced cytotoxicity of human T-ALL cells treated with the combination therapy results from the inability to effectively repair DNA damage induced by αGal-9Ab treatment. Our findings describe a previously unrecognized role for Gal-9 in T-ALL pathogenesis and demonstrates the cytotoxic effects αGal-9Ab treatment (including LYT-200) in preclinical models of human T-ALL. Disclosures Lee: PureTech Health: Research Funding. Filipovic: PureTech Health: Research Funding. Henry: PureTech Health: Research Funding.
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Sahasrabuddhe, Anagh A., Xiaofei Chen, Thirunavukkarasu Velusamy, Fuzon Chung, Megan S. Lim, and Kojo S. J. Elenitoba-Johnson. "A Novel Non-Canonical Phosphodegron Regulates EZH2 Proteasomal Degradation and H3K27 Trimethylation Activity in Hematopoietic Malignancies." Blood 124, no. 21 (December 6, 2014): 1678. http://dx.doi.org/10.1182/blood.v124.21.1678.1678.
Full textAbstract:
Abstract Introduction: Enhancer of zeste homologue 2 (EZH2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2) which trimethylates histone H3 (H3K27) to mediate gene repression. EZH2 is aberrantly activated and overexpressed in several hematologic malignancies and is associated with aggressive clinical behavior. In particular, recurrent mutations targeting Y641 of EZH2 are common in germinal center derived lymphomas. Critically, the cellular machinery and mechanisms that regulate EZH2 by post-translational modification are not well understood. We have previously shown that β-transducin repeat containing protein (βTrCP) is the substrate specific adaptor for ubiquitin mediated degradation of EZH2, however the degron motif by which βTrCP recognizes EZH2 is unknown. In order to better understand the post-translational regulation of EZH2, we sought to identify the non-canonical degron motif that regulates EZH2 stability. Methods: βTrCP recognizes a phosphorylated consensus degron motif DpSG(X)2-5pS in its substrates. To investigate the post-translational modifications regulating the half-life of EZH2 protein we generated a series of truncation and site directed mutants of βTrCP including deletion of 7th WD repeat domain and mutation of R474A. The afore mentioned βTrCP mutants were assessed for their interaction with EZH2. Having established the non-canonical degron (DpS601KNVpS605) in EZH2, to further characterize the role of this degron in EZH2-βTrCP interaction we generated several EZH2 mutants including site-directed deletion of putative degron motif (DSKNVS) or alanine substitution mutations of critical serine residues S601A and S605A alone or in combination. Using wild-type as well as EZH2 mutants we investigated their ability to interact with βTrCP and consequent lysine (K48) linked polyubiquitination on EZH2 using co-immunoprecipitation and western blotting techniques. Further we characterized the impact of the identified degron in EZH2 stability using cycloheximide chase mediated protein turnover analysis of wild type and EZH2 degron deletion and serine (S601A/S605A) substitution mutants. Moreover, we investigated the impact of increased stability of EZH2 on its H3K27 trimethylation activity using wild type and degron deletion and serine substitution mutants by western blot analysis. We also investigated the impact of increased stability of EZH2 via degron modification by analyzing its downstream target p21. Results: Extensive interaction between substrates and R474 and Y488 residues in the 7th WD repeat domain of βTrCP is critical for stable binding and this is mediated via the conserved degron motif. In co-immunoprecipitation experiments, the mutation of R474A or deletion of 7th WD repeat domain in βTrCP abrogated the EZH2-βTrCP interaction. Further, the deletion of the identified degron DSKNVS in EZH2 or substitution of critical serine residues residing within this degron abrogated EZH2-βTrCP interaction and consequent lysine K48-linked polyubiquitination and proteasomal degradation. Half-life measurements by cycloheximide chase experiments demonstrated that the degron deletion or substitution mutations exhibit increased protein stability as compared to wild type EZH2. Motif analysis revealed that the novel EZH2 degron harbors the GSK3β recognition and phosphorylation motif (DpSKNVpS) involved in the phosphorylation of several known βTrCP substrates. Therefore, we examined the involvement of GSK3β in EZH2-βTrCP interaction. Pharmacologic inhibition of GSK3β compromised EZH2- βTrCP interaction in a dose dependent manner. Further, deletion of the degron or alanine substitution mutation of the critical serine residues (S601A and S605A) within the degron increased H3K27 trimethylation activity of EZH2 and consequently increased its repression on its downstream target p21. Conclusion: In the present study, we identify the presence of a novel non-canonical degron and GSK3β-mediated phosphorylation of the site that regulates EZH2 stability and activity. Our studies demonstrate that βTrCP/GSK3β axis plays an important role in controlling H3K27 trimethylation activity by targeting EZH2 for degradation. We propose that this newly identified mechanism might help in designing novel therapeutic approaches for clinical management of hematologic malignancies driven by aberrant activity of EZH2. Disclosures No relevant conflicts of interest to declare.
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Shinde, Sneha, Azim M. Mohamedali, and Ghulam Mufti. "Mutation and Expression Analysis of Jumonji Genes in Myelodysplastic Syndrome & Acute Myeloid Leukaemia." Blood 124, no. 21 (December 6, 2014): 3555. http://dx.doi.org/10.1182/blood.v124.21.3555.3555.
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Abstract Introduction Discovery of cytogenetic abnormalities together with specific aberrations in RNA splicing, cell signalling, translational regulation and tumour suppressor genes are increasingly been applied for the prognostic stratification well as understanding the pathobiology of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML).Epigenetic regulation of transcription has attracted particular attention, because of the efficacy of DNA methyltransferase and Histone deacetylase inhibitor therapies for the treatment of both MDS and AML. The Jumonji (JMJ) family of histone demethylases are epigenetic regulators that demethylate lysine residues of histones in a site and methylation state specific context. Given the importance of these in the transcriptional machinery of myeloid progenitors, we examined the role of seventeen Jumonji genes; JARID1A-1D, JARID2, JMJD3, UTX, JMJD4, JMJD2A-2D, JMJD1A-1C, JHJDM1A-1B in the bone marrow samples from patients with MDS/AML. Majority of the genes tested are involved in the demethylation of H3K4, H3K9, H3K27 or H3K36 histone tails while others associate with the polycomb repressive complex (PRC), abnormalities of which are well documented in both MDS/AML. Methods SNP6 array karyotyping was carried out in 124 MDS patients [M/F ratio = 1:1.7, median age= 69 yrs, WHO subtypes: RCMD 21%, RAEB I/II 50 %, AML 1%, sAML/tMDS 16 %, CMML 6.5 %, MDS/MPD 5.5 %], focusing exclusively on the Jumonji genes loci to identify copy number variations [CNVs] (deletion/ gain) & Uniparental disomy. The patients with SNP6 aberrations in the Jumonji genes were examined by 454 DNA parallel sequencing and Real-time PCR for mutational analysis and alterations in gene expression respectively. The sequencing depth was 350-450 reads / amplicon. Results Of the 124 cases, 28 patients (22.5%) were identified with a deletion, gain or UPD at 15/17 Jumonji gene loci while two genes showed no SNP6 abnormalities. Of the 22.5 % patients, the highest frequencies of deletions were found in JMJD1B (chr 5q31.2) [41 %], JARID2 [10.7 %] and JMJD3, JMJD2D, JMJD1A [7.14 %] each. On the other hand, the highest frequency of gain was observed in JMJD2C [10.7 %], followed by JMJD2A & UTX [7.14 %] each. Interestingly, only three genes showed both deletion and amplification in different patients in our cohort (JARID2, JMJD4, and JMJD2D) while the rest were either deleted or amplified. Compared to CNVs (69 %), only twelve patients (44 %) carried UPD [telomeric, size: majority were >20Mb]. JMJD2D and JMJD4 had the highest frequency (14.3 %) while JMJD3 had [10.7 %] of UPDs. 454 DNA parallel sequencing of the fifteen Jumonji genes in the 28 patients with SNP6 abnormalities revealed no mutations. To elucidate changes in gene expression as a result of CNVs; two patients with CN gain at the UTX locus showed 5 fold increase (p value < 0.0001) in the expression of this gene while two patients with deletion at the JARID1A locus showed knock down in the expression levels as compared to patients with normal SNP6 profile (n =20). Conclusion In summary, 22.5 % of high-risk MDS patients show SNP6 aberrations at the Jumonji gene loci. No mutations were associated with the SNP6 abnormalities even with a read depth of 350-450 reads/ amplicon. However, alteration in the expression of JARIDA and UTX was consistent with the CNVs detected on SNP6 which might have direct consequence on the methylation status of the genome or may assist as yet unidentified targets in the pathogenesis of MDS. Table 1: Table 1:. Gain, deletion and UPD in the Jumonji genes. Numbers in brackets indicate size of the aberration in Mb Figure 1 (a) Five-fold increase in UTX expression in two patients, one with trisomy chr: Xp and second with CN=4 (b) Knock down of JARID1A expression in two patients with CN=1 in compared to twenty control patients with normal SNP6 profile at the UTX and JARID1A loci. cDNA from K562 cell line was used as a positive technical control in all experiments. Figure 1. (a) Five-fold increase in UTX expression in two patients, one with trisomy chr: Xp and second with CN=4 (b) Knock down of JARID1A expression in two patients with CN=1 in compared to twenty control patients with normal SNP6 profile at the UTX and JARID1A loci. cDNA from K562 cell line was used as a positive technical control in all experiments. Disclosures Shinde: Celgene: Research Funding.
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van Dijk, Anneke D., Fieke W. Hoff, Yihua Qiu, Mary Figueroa, Joya Chandra, Elias Jabbour, Eveline S. de Bont, and Steven M. Kornblau. "Trimethylated H3K27, and Di- and Trimethylated H3K4 Proteomic Profiling Distinguishes Acute Lymphoid Leukemia (ALL) from Acute Myeloid Leukemia (AML) and Associates with Overall Survival and Tyrosine Kinase Inhibitor Sensitivity in Adult ALL." Blood 134, Supplement_1 (November 13, 2019): 1460. http://dx.doi.org/10.1182/blood-2019-124960.
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Background: Lineage-specific gene transcription signatures between AML and ALL are recognized, but post-translational phenotype-specific protein expression profiles remain undefined. We hypothesized that functional proteomic patterns vary between AML and ALL and that the activity state of cells correlates with response to therapy within subgroups, complementing cytogenetic and molecular data. Methods: Reverse phase protein arrays (RPPA) were generated using bone marrow (BM) and peripheral blood (PB) samples from newly diagnosed B-ALL (n=114), T-ALL (n=14), and AML (n=241) adult patients admitted at the MD Anderson Cancer Center. RPPA allowed simultaneous expression measurement of 229 highly validated protein antibodies including 3 Histone 3 (H3) post-translational methylation regulatory modifications; H3K4Me2, H3K4Me3 and H3K27Me3. Results: Unsupervised clustering of histone modification protein expressions distinguished AML from ALL in freshly prepared lysates from BM (n=241) and PB (n=127) as well as when BM and PB samples were combined (fig. 1A). The ALL-enriched cluster was dominated by high H3K27Me3. Elevated H3K27Me3 levels were found in the BM derived leukemic blasts compared to PB blasts in ALL (P < 0.001), but not AML (P = 0.35). Trimethylation of the repressive mark H3K27 is catalyzed by the polycomb group protein Ezh2. Oncogenic gain-of-functions of Ezh2 are seen in patients with lymphoid malignancies and others have shown that mutated Ezh2 increased H3K27Me3 in B-cells which associated with tumorigenesis. H3K27Me3 and Ezh2 antibody expressions were highly correlated in another RPPA of ALL and AML we created (R2=0.49, P < 0.001). Profiling of methylation marks using unsupervised clustering in ALL divided patients in 2 clusters that correlated with survival (fig. 1B-C, P = 0.02). Cluster 1 (C1) with higher H3K27Me3, H3K4Me2 and H3K4Me3 was associated with better outcome. In ALL, Ph+ historically associated with poor prognosis but outcomes have improved substantially with the use of tyrosine kinase inhibitors (TKI). In our cohort, 11/26 Ph+ ALL patients were treated with TKIs and it is notable that sensitivity to TKIs correlated with cluster membership; all C1 patients (high degree of methylation) were alive after 7 years of follow-up in contrast to none of the TKI-treated Ph+ ALL patients in cluster 2 (C2, low degree of methylation) (fig. 1D, P = 0.01). Recently, TKI resistance in Ph+ ALL has been proposed to associate with smoking due to altered DNA methylation patterns caused by chemical components of cigarette smoke. Retrospectively, we identified that 2 of 11 TKI treated patients were smokers. Both had membership in C2, were resistant against TKIs and died after 1 year. Thus, 2 out of 3 resistant TKI treated Ph+ ALL were smokers compared to none of the 8 responders. We then aimed to identify proteins that are potentially downregulated by increased expression of the repressive mark H3K27Me3. Pathway enrichment analysis of 59 significant negatively correlated proteins with H3K27Me3 revealed that these are involved in tyrosine kinase activity and resistance, including Jak/STAT and PI3K/Akt signaling pathways. If these pathways are less activated in patients with high H3K27Me3, then this can partially explain the increased sensitivity to TKIs in this subgroup. Clinically, no differences were found in age, BM and PB blast counts between TKI-treated C1 and C2 patients to provide an explanation for the higher death rate in C2. Conclusion: ALL and AML share some pathophysiology and the identification of differences in the functional activity of cells may contribute to a better understanding of the etiology of both diseases. Here we report that high H3K27Me3 protein levels in BM and PB distinguish ALL from AML and are related to TKI sensitivity in Ph+ ALL. Histone methylation status defines a group of Ph+ ALL patients that does not benefit from the addition of TKI therapy. The idea that smoking alters the epigenetic machinery in TKI resistant Ph+ ALL has been proposed and warrants further investigation. Fig. 1 A) Heatmap showing histone methylation levels in BM and PB from AML and ALL patients. B) Heatmap showing histone methylation levels in ALL BM and PB. Unsupervised clustering divided samples into 2 clusters. C) ALL patients in C1 survived longer than patients in C2 (P = 0.02). D) Increased long-term sensitivity for TKI therapy in C1 Ph+ ALL patients compared to C2 (100 vs. 0%, P = 0.01). Figure.1 Disclosures Jabbour: AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding; Adaptive: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Amgen: Consultancy, Research Funding.
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Wang, Zhiquan, Justin C. Boysen, Huihuang Yan, Charla R. Secreto, Sameer A. Parikh, Saad S. Kenderian, Wei Ding, Esteban Braggio, Susan L. Slager, and Neil E. Kay. "Targeting Aberrant Chromatin in Chronic Lymphocytic Leukemia." Blood 136, Supplement 1 (November 5, 2020): 1. http://dx.doi.org/10.1182/blood-2020-140309.
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Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature-appearing malignant lymphocytes (CLL B-cells) in the blood, marrow, lymph nodes, and spleen. Despite improved outcome with the introduction of novel BCR and BCL-2 inhibitors, disease progression is still a therapeutic challenge from either differential responses or acquired drug resistance. Recent studies in CLL reported alterations of the epigenetic landscape as well as mutations of genes encoding key chromatin machineries. These aberrant chromatin structures may provide novel therapeutic targets for CLL. Here, we identify aberrant chromatin features in CLL B-cells as novel therapeutic targets. Methods: Histones were extracted by acid from B cells derived from 10 random selected CLL patients and 10 normal donors and histone modifications were checked by western blot. For ChIP-seq study, published H3K27me3 ChIP-seq data (GSE113336) were downloaded from and analyzed (Control samples, n= 6; CLL samples, n=16). Gene ontology analysis used the Panther Classification System. Cell Survival was determined by CellTiter 96® AQueous Assay (Promega). Results: While most histone modifications do not vary between CLL and controls, H3K27me3 and H3.3S31ph are increased and decreased respectively, albeit variably, in CLL B-cells (Fig1. A and B). Notably, the low level of H3.3S31ph was observed in a subset of samples (7 of 10 CLL samples tested). To further investigate the biology and role of H3K27me3 in CLL, we analyzed its genome-wide distribution by chromatin immunoprecipitation followed by sequencing (ChIP-seq). Our analysis showed that the genes with increased H3K27me3 occupancy were mostly enriched in tumor suppression pathways (e.g., negative regulation of PI3K-Akt pathway) or down-regulated genes in CLL such as genes involved in the pro-apoptotic pathway (FAS) (Fig1. C and D). These results suggested that high enrichment of H3K27me3 may regulate the expression of these genes, contributing to CLL survival. H3K27 methylation, an important suppressive histone modification that is associated with transcription repression, is catalyzed by Polycomb Repressive Complex 2 (PRC2). Therefore, inhibition of Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of PRC2, could be explored as a therapy approach in CLL. However, feedback activation of H3K27 acetylation (H3K27ac) can promote expression of pro-survival genes that confers EZH2 inhibitor (EZH2i) resistance, which limits its use in human malignancy. Thus, the epigenetic determinants that reliably overcome EZH2i resistance or sensitize cells to EZH2 inhibition have yet to be identified. As we observed that the CLL B-cells in a subset of CLL patients have low levels of H3.3S31ph, and a recent study showed the importance of H3.3S31ph for the enzymatic activity of p300 to acetylate H3 at lysine 27(Martire S et al., Nat Genet. 2019), we assessed the role of H3.3S31ph in the process of EZH2 inhibitor-mediated H3K27ac. Our results showed that inhibition of H3.3S31ph by CHK1 inhibitor MK-8776 abolished the activation of H3K27ac by EZH2i in MEC1 cells, which represents the patients who have CLL cells with relatively high level H3.3S31ph. However, we did not see a major increase of H3K27ac and H3.3S31ph in primary CLL B-cells with EZH2 inhibition (Fig. 1E), consistent with the relatively low expression of CHK1 protein in these cells (Fig. 1F). Because our data shows the requirement of H3.3S31ph in H3K27ac activation by EZH2 inhibition, we next tested if H3.3S31ph inhibition could overcome H3K27ac induced EZH2 inhibition resistance. We found that suppression of H3.3S31ph by CHK1 inhibitor MK-8776 sensitizes the CLL-like line MEC1 to EZH1/2 inhibition (Fig. 1 G). We then showed that an EZH2 inhibitor, Valemetostat, reduce the survival of the primary CLL B-cells (Fig. 1 H). These results suggest that the low level of H3.3S31ph may provide a therapeutic opportunity for CLL treatment with EZH inhibition. Conclusion: In summary, we have elucidated how epigenetic features in leukemic CLL B-cells (H3K27me3 and H3.3S31ph), can provide novel treatment targets for CLL (Fig. 1 I). Moreover, this study may provide a proof of concept to develop new treatment strategies based on epigenetic vulnerabilities in other hematological malignancies. Disclosures Parikh: GlaxoSmithKline: Honoraria; MorphoSys: Research Funding; Genentech: Honoraria; Ascentage Pharma: Research Funding; AbbVie: Honoraria, Research Funding; TG Therapeutics: Research Funding; Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Verastem Oncology: Honoraria; Merck: Research Funding. Kenderian:Kite: Research Funding; MorphoSys: Research Funding; Tolero: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; BMS: Research Funding; Gilead: Research Funding; Juno: Research Funding; Lentigen: Research Funding; Mettaforge: Patents & Royalties; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Sunesis: Research Funding. Ding:Beigene: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Research Funding; DTRM: Research Funding; Abbvie: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Kay:Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding.
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Yin, Xiaochang, Francisco J. Romero-Campero, Minqi Yang, Fernando Baile, Yuxin Cao, Jiayue Shu, Lingxiao Luo, et al. "Binding by the Polycomb complex component BMI1 and H2A monoubiquitination shape local and long-range interactions in the Arabidopsis genome." Plant Cell, April 18, 2023. http://dx.doi.org/10.1093/plcell/koad112.
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Abstract Three-dimensional (3D) chromatin organization is highly dynamic during development and seems to play a crucial role in regulating gene expression. Self-interacting domains, commonly called topologically associating domains (TADs) or compartment domains (CDs), have been proposed as the basic structural units of chromatin organization. Surprisingly, although these units have been found in several plant species, they escaped detection in Arabidopsis (Arabidopsis thaliana). Here, we show that the Arabidopsis genome is partitioned into contiguous CDs with different epigenetic features, which are required to maintain appropriate intra-CD and long-range interactions. Consistent with this notion, the histone-modifying Polycomb group machinery is involved in 3D chromatin organization. Yet, while it is clear that Polycomb Repressive Complex 2 (PRC2)-mediated trimethylation of histone H3 on lysine 27 (H3K27me3) helps establish local and long-range chromatin interactions in plants, the implications of PRC1-mediated histone H2A monoubiquitination on lysine 121 (H2AK121ub) are unclear. We found that PRC1, together with PRC2, maintains intra-CD interactions, but it also hinders the formation of H3K4me3-enriched local chromatin loops when acting independently of PRC2. Moreover, the loss of PRC1 or PRC2 activity differentially affects long-range chromatin interactions, and these 3D changes differentially affect gene expression. Our results suggest that H2AK121ub helps prevent the formation of transposable element/H3K27me1-rich long loops and serves as a docking point for H3K27me3 incorporation.
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Wang, Gang, Heng Ye, Xuchao Wang, and Binbin Liu. "Polycomb repressive complex 2 controls cardiac cell fate decision via interacting with RNA: Promiscuously or well-ordered." Frontiers in Genetics 13 (October 14, 2022). http://dx.doi.org/10.3389/fgene.2022.1011228.
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The epigenetic landscape determines cell fate during heart development. Polycomb repressive complex 2 (PRC2) mediates histone methyltransferase activity during cardiac cell differentiation. The PRC2 complex contains the proteins embryonic ectoderm development (EED), suppressor of zeste (SUZ12), the chromatin assembly factor 1 (CAF1) histone-binding proteins RBBP4 and RBBP7, and the histone methyltransferase called enhancer of zeste (EZH2 or EZH1), which incorporates the Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain. Cardiac PRC2-deficient mice display lethal congenital heart malformations. The dynamic process of cardiac cell fate decisions is controlled by PRC2 and the PRC2-mediated epigenetic landscape. Although specific individual long noncoding RNAs (lncRNAs) including Braveheart were widely reported to regulate the recruitments of PRC2 to their specific targets, a promiscuous RNA binding profile by PRC2 was also identified to play an essential role in cardiac cell fate decision. In this review, we focus on RNA-mediated PRC2 recruitment machinery in the process of cardiac cell fate decisions. The roles of individual lncRNAs which recruit PRC2, as well as promiscuous RNA binding by PRC2 in heart development are summarized. Since the binding priority of RNAs with different primary and secondary structures differs in its affinity to PRC2, the competitive relationship between individual lncRNAs binding and promiscuous RNA binding by PRC2 may be important for understanding the machinery by which biding of individual lncRNA and promiscuous RNA by PRC2 coordinately control the well-ordered dynamic cardiac cell lineage differentiation process.
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Fonouni-Farde, Camille, Aurélie Christ, Thomas Blein, María Florencia Legascue, Lucía Ferrero, Michaël Moison, Leandro Lucero, et al. "The Arabidopsis APOLO and human UPAT sequence-unrelated long noncoding RNAs can modulate DNA and histone methylation machineries in plants." Genome Biology 23, no. 1 (August 29, 2022). http://dx.doi.org/10.1186/s13059-022-02750-7.
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Abstract Background RNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. Results Here, we show that in addition to the PRC1-component LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), APOLO interacts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). The APOLO-VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis gene YUCCA2 by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrate that the lncRNA UHRF1 Protein Associated Transcript (UPAT), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology between UPAT and APOLO. In addition, we show that increased levels of APOLO or UPAT hamper VIM1 and LHP1 binding to YUCCA2 promoter and globally alter the Arabidopsis transcriptome in a similar manner. Conclusions Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation through the interaction with VIM1, and indicates that evolutionary unrelated lncRNAs with potentially conserved structures may exert similar functions by interacting with homolog partners.
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Djeghloul, Dounia, Andrew Dimond, Sherry Cheriyamkunnel, Holger Kramer, Bhavik Patel, Karen Brown, Alex Montoya, et al. "Loss of H3K9 trimethylation alters chromosome compaction and transcription factor retention during mitosis." Nature Structural & Molecular Biology, March 20, 2023. http://dx.doi.org/10.1038/s41594-023-00943-7.
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AbstractRecent studies have shown that repressive chromatin machinery, including DNA methyltransferases and polycomb repressor complexes, binds to chromosomes throughout mitosis and their depletion results in increased chromosome size. In the present study, we show that enzymes that catalyze H3K9 methylation, such as Suv39h1, Suv39h2, G9a and Glp, are also retained on mitotic chromosomes. Surprisingly, however, mutants lacking histone 3 lysine 9 trimethylation (H3K9me3) have unusually small and compact mitotic chromosomes associated with increased histone H3 phospho Ser10 (H3S10ph) and H3K27me3 levels. Chromosome size and centromere compaction in these mutants were rescued by providing exogenous first protein lysine methyltransferase Suv39h1 or inhibiting Ezh2 activity. Quantitative proteomic comparisons of native mitotic chromosomes isolated from wild-type versus Suv39h1/Suv39h2 double-null mouse embryonic stem cells revealed that H3K9me3 was essential for the efficient retention of bookmarking factors such as Esrrb. These results highlight an unexpected role for repressive heterochromatin domains in preserving transcription factor binding through mitosis and underscore the importance of H3K9me3 for sustaining chromosome architecture and epigenetic memory during cell division.
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Liu, Xiuli, and Xin Liu. "PRC2, Chromatin Regulation, and Human Disease: Insights From Molecular Structure and Function." Frontiers in Oncology 12 (June 21, 2022). http://dx.doi.org/10.3389/fonc.2022.894585.
Full textAbstract:
Polycomb repressive complex 2 (PRC2) is a multisubunit histone-modifying enzyme complex that mediates methylation of histone H3 lysine 27 (H3K27). Trimethylated H3K27 (H3K27me3) is an epigenetic hallmark of gene silencing. PRC2 plays a crucial role in a plethora of fundamental biological processes, and PRC2 dysregulation has been repeatedly implicated in cancers and developmental disorders. Here, we review the current knowledge on mechanisms of cellular regulation of PRC2 function, particularly regarding H3K27 methylation and chromatin targeting. PRC2-related disease mechanisms are also discussed. The mode of action of PRC2 in gene regulation is summarized, which includes competition between H3K27 methylation and acetylation, crosstalk with transcription machinery, and formation of high-order chromatin structure. Recent progress in the structural biology of PRC2 is highlighted from the aspects of complex assembly, enzyme catalysis, and chromatin recruitment, which together provide valuable insights into PRC2 function in close-to-atomic detail. Future studies on the molecular function and structure of PRC2 in the context of native chromatin and in the presence of other regulators like RNAs will continue to deepen our understanding of the stability and plasticity of developmental transcriptional programs broadly impacted by PRC2.
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Kong, Isabella Y., Stephanie Trezise, Amanda Light, Izabela Todorovski, Gisela Mir Arnau, Sreeja Gadipally, David Yoannidis, et al. "Epigenetic modulators of B cell fate identified through coupled phenotype-transcriptome analysis." Cell Death & Differentiation, July 13, 2022. http://dx.doi.org/10.1038/s41418-022-01037-5.
Full textAbstract:
AbstractHigh-throughput methodologies are the cornerstone of screening approaches to identify novel compounds that regulate immune cell function. To identify novel targeted therapeutics to treat immune disorders and haematological malignancies, there is a need to integrate functional cellular information with the molecular mechanisms that regulate changes in immune cell phenotype. We facilitate this goal by combining quantitative methods for dissecting complex simultaneous cell phenotypic effects with genomic analysis. This combination strategy we term Multiplexed Analysis of Cells sequencing (MAC-seq), a modified version of Digital RNA with perturbation of Genes (DRUGseq). We applied MAC-seq to screen compounds that target the epigenetic machinery of B cells and assess altered humoral immunity by measuring changes in proliferation, survival, differentiation and transcription. This approach revealed that polycomb repressive complex 2 (PRC2) inhibitors promote antibody secreting cell (ASC) differentiation in both murine and human B cells in vitro. This is further validated using T cell-dependent immunization in mice. Functional dissection of downstream effectors of PRC2 using arrayed CRISPR screening uncovered novel regulators of B cell differentiation, including Mybl1, Myof, Gas7 and Atoh8. Together, our findings demonstrate that integrated phenotype-transcriptome analyses can be effectively combined with drug screening approaches to uncover the molecular circuitry that drives lymphocyte fate decisions.
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Becker, Timothy James, Badam Enkhmandakh, and Dashzeveg Bayarsaihan. "Single‐cell RNA analysis of chromodomain‐encoding genes in mesenchymal stromal cells of the mouse dental pulp." Journal of Cellular Biochemistry, May 23, 2024. http://dx.doi.org/10.1002/jcb.30608.
Full textAbstract:
AbstractThe chromodomain helicase DNA‐binding (CHD) and chromobox (CBX) families of proteins play crucial roles in cell fate decisions, differentiation, and cell proliferation in a broad variety of tissues and cell types. CHD proteins are ATP‐dependent epigenetic enzymes actively engaged in transcriptional regulation, DNA replication, and DNA damage repair, whereas CBX proteins are transcriptional repressors mainly involved in the formation of heterochromatin. The pleiotropic effects of CHD and CBX proteins are largely dependent on their versatility to interact with other key components of the epigenetic and transcriptional machinery. Although the function and regulatory modes of CHD and CBX factors are well established in many cell types, little is known about their roles during osteogenic differentiation. A single‐cell RNA‐sequencing analysis of the mouse incisor dental pulp revealed distinct spatiotemporal expression patterns of CHD‐ and CBX‐encoding genes within different clusters of mesenchymal stromal cells (MSCs) representing various stages of osteogenic differentiation. Additionally, genes encoding interaction partners of CHD and CBX proteins, such as subunits of the trithorax‐COMPASS and polycomb chromatin remodeling complexes, exhibited differential co‐expression behaviors within MSC subpopulations. Thus, CHD‐ and CBX‐encoding genes show partially overlapping but distinct expression patterns in MSCs, suggesting their differential roles in osteogenic cell fate decisions.
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Zhang, Pingxian, Chunmei Zhu, Yuke Geng, Yifan Wang, Ying Yang, Qing Liu, Weijun Guo, et al. "Rice and Arabidopsis homologs of yeast CHROMOSOME TRANSMISSION FIDELITY PROTEIN 4 commonly interact with Polycomb complexes but exert divergent regulatory functions." Plant Cell, February 6, 2021. http://dx.doi.org/10.1093/plcell/koab047.
Full textAbstract:
Abstract Both genetic and epigenetic information must be transferred from mother to daughter cells during cell division. The mechanisms through which information about chromatin states and epigenetic marks like histone 3 lysine 27 trimethylation (H3K27me3) are transferred have been characterized in animals; these processes are less well understood in plants. Here, based on characterization of a dwarf rice (Oryza sativa) mutant (dwarf-related wd40 protein 1, drw1) deficient for yeast CTF4 (CHROMOSOME TRANSMISSION FIDELITY PROTEIN 4), we discovered that CTF4 orthologs in plants use common cellular machinery yet accomplish divergent functional outcomes. Specifically, drw1 exhibited no flowering-related phenotypes (as in the putatively orthologous Arabidopsis thaliana eol1 mutant), but displayed cell cycle arrest and DNA damage responses. Mechanistically, we demonstrate that DRW1 sustains normal cell cycle progression by modulating the expression of cell cycle inhibitors KIP-RELATED PROTEIN 1 (KRP1) and KRP5, and show that these effects are mediated by DRW1 binding their promoters and increasing H3K27me3 levels. Thus, although CTF4 orthologs ENHANCER OF LHP1 1 (EOL1) in Arabidopsis and DRW1 in rice are both expressed uniquely in dividing cells, commonly interact with several Polycomb complex subunits, and promote H3K27me3 deposition, we now know that their regulatory functions diverged substantially during plant evolution. Moreover, our work experimentally illustrates specific targets of CTF4/EOL1/DRW1, their protein–proteininteraction partners, and their chromatin/epigenetic effects in plants.
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Lee, Youngsook, Eunjin Cho, and Matthew Mysliwiec. "Abstract 118: Epigenetic Regulation of Ventricular Development." Circulation Research 115, suppl_1 (July 18, 2014). http://dx.doi.org/10.1161/res.115.suppl_1.118.
Full textAbstract:
Perturbations of the epigenetic machinery can lead to the deregulation of cardiac gene expression, resulting in defective cardiac development and cardiac hypertrophy. Due to a groundbreaking discovery of histone demethylases such as Jumonji (Jmj) family factors, histone methylation is now considered as a reversible epigenetic mark. Jarid2/Jumonji is the founding member of the JMJ family. Jarid2 is enzymatically inactive, but functions as a transcriptional regulator. Jarid2 critically regulates cardiovascular development as well as ES cell differentiation and generation of iPS cells. Jarid2 knockout (KO) mice exhibit cardiac defects including hyper-trabeculation with noncompaction of the ventricular wall. Although Jarid2 interacts with Polycomb Repressor Complex in ES cells, the precise function of Jarid2 in cardiac development remains to be determined. Therefore, we set out to determine molecular mechanisms of Jarid2 critical for cardiac development. To identify cardiac-specific roles of Jarid2, we generated deletion of Jarid2 in early cardiac progenitors using Nkx2.5-Cre Knock-in mice (Jarid2Nkx-KI). Jarid2Nkx-KI mice recapitulate partial phenotypic defects observed in Jarid2 KO including hyper-trabeculation, thin myocardium and ventricular septal defects. By overlapping ChIP-chip and microarray analyses, we have identified potential transcriptional targets of Jarid2, which are occupied by Jarid2, SETDB1, H3K9me3 or H3K27me3, and upregulated in Jarid2 mutant hearts. 174 genes including Isl1 were identified as dysregulated genes that showed accumulation of Jarid2 and H3K27me3. 172 genes including Bmp10 were identified as dysregulated genes and were occupied by Jarid2, SETDB1 and H3K9me3. Isl1, Bmp10/p-Smad1/5/8, and Ifgbp2 were upregulated in Jarid2Nkx-KI hearts by qRT-PCR and Western blotting. Islet1 plays crucial roles in early cardiac development and a marker for cardiac progenitors. Jarid2 occupancy was observed at the Isl1 promoter region by ChIP assays in WT hearts, which was reduced in Jarid2Nkx-KI. All together, our data indicate that Jarid2 regulates target gene expression by interacting with different histone modifiers depending on the cell/promoter context, which is critical for ventricular wall maturation.
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Mohan, Dipika R., Isabella Finco, Christopher Ryan LaPensee, Juilee Rege, Tobias Else, Madson Q. Almeida, Michelle Vinco, et al. "SAT-LB34 Repressive Epigenetic Programs Reinforce Steroidogenic Differentiation and Wnt/β-Catenin Signaling in Aggressive Adrenocortical Carcinoma." Journal of the Endocrine Society 4, Supplement_1 (April 2020). http://dx.doi.org/10.1210/jendso/bvaa046.2265.
Full textAbstract:
Abstract Adrenocortical carcinoma (ACC) is a rare, aggressive cancer. Up to 75% of patients develop incurable metastatic disease, highlighting an urgent need for novel medical therapies. We recently identified a rapidly progressive ACC subtype characterized by CpG island hypermethylation (CIMP-high), sustained Wnt/β-catenin signaling, steroidogenic differentiation, and cell cycle activation. CIMP-high status alone accounts for 40% of ACC, but predicts 70% of recurrences and >50% of deaths. Intriguingly, hypermethylated CpG islands in CIMP-high ACC are unmethylated in fetal and adult adrenal cortex, suggesting DNA methylation is supported by cancer-specific mechanisms. We therefore sought to investigate how aberrant epigenetic programming contributes to ACC biology. In embryonic stem cells, the Polycomb repressive complex 2 (PRC2) represses differentiation programs through EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) deposition in promoter CpG islands free of DNA methylation. Gain or loss of EZH2/PRC2 function prevails in a variety of human cancers, enabling proliferation in a tissue-specific manner. Here, we identify that CIMP-high ACC exhibit high expression of EZH2/H3K27me3, but paradoxically bear DNA hypermethylation in annotated PRC2 target regions. To determine if DNA methylation of PRC2 targets disrupts or is controlled by EZH2, we characterized EZH2’s role in CIMP-high ACC cell line NCI-H295R at baseline and in response to EZH2 inhibition (EZH2i). EZH2-directed IP-MS revealed EZH2 interacts with PRC2 members and DNA methylation-sensitive accessory proteins, but no DNA methyltransferase machinery. ChIP-seq revealed EZH2 and H3K27me3 colocalize in repressive domains genome-wide, but DNA methylation and H3K27me3 are mutually exclusive. EZH2i induced H3K27 demethylation and loss of viability, but with no effect on CIMP-high DNA methylation. These data suggest PRC2 target DNA methylation in CIMP-high ACC is maintained independently of EZH2, enabling EZH2/PRC2 to coordinate alternative programs required for cell survival. We then measured the consequences of EZH2i on the NCI-H295R transcriptome (RNA-seq), EZH2/H3K27me3 deposition genome-wide (ChIP-seq), and chromatin accessibility landscape (ATAC-seq). EZH2i led to global downregulation of cell cycle, Wnt/β-catenin transcriptional programming, and steroidogenic differentiation, partially explained by EZH2i-induced offloading of EZH2 from H3K27me3 domains to accessible promoters genome-wide. Taken together, our studies illustrate how aberrant CpG island hypermethylation in CIMP-high ACC participates in a targetable repressive epigenetic cascade that reinforces oncogenic adrenocortical transcriptional programs. Ultimately, we hope to illuminate novel strategies for tissue-specific disruption of the aberrant epigenetic wiring that defines CIMP-high ACC.