29 results on '"Laura Ciudad"'
Search Results
2. Vitamin C triggers NF-κB-driven epigenomic reprogramming and enhanced immunogenic responses of dendritic cells
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Octavio Morante-Palacios, Gerard Godoy-Tena, Josep Calafell-Segura, Laura Ciudad, Eva M. Martínez-Cáceres, José Luis Sardina, and Esteban Ballestar
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Dendritic cells (DCs) are central in the immune system, bridging the adaptive and innate immune responses. Research on in vitro differentiation of DCs from monocytes provides both in-depth understanding of the analogous in vivo process and potential sources for cancer cell therapy. Active DNA demethylation is crucial in DC differentiation. Vitamin C is a known cofactor of ten-eleven translocation (TET) enzymes, which drive active demethylation. Currently, the effects of vitamin C treatment on human immune cells are poorly understood. In this study, we have studied the epigenomic and transcriptomic reprogramming orchestrated by vitamin C in monocyte-derived DC differentiation and maturation. Vitamin C triggers extensive demethylation at NF-kB/p65 binding sites, together with concordant upregulation of antigen-presentation immune response-related genes during DC maturation. p65 interacts with TET2 and mediates the aforementioned vitamin C-mediated changes, as demonstrated by pharmacological inhibition. Moreover, vitamin C increases TNFβ production in DCs through NF-kB, in concordance with the upregulation of its coding gene and the demethylation of adjacent CpGs. Finally, vitamin C enhances DC’s ability to stimulate the proliferation of autologous antigen-specific T cells. We propose that vitamin C can improve monocyte-derived DC-based cell therapies. Finally, our results provide a feasible mechanism of action for intravenous high-dose vitamin C treatment in patients.
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- 2022
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3. Methylome and transcriptome profiling of giant cell arteritis monocytes reveals novel pathways involved in disease pathogenesis and molecular response to glucocorticoids
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Elkyn Estupiñán-Moreno, Lourdes Ortiz-Fernández, Tianlu Li, Jose Hernández-Rodríguez, Laura Ciudad, Eduardo Andrés-León, Laura Carmen Terron-Camero, Sergio Prieto-González, Georgina Espígol-Frigolé, Maria Cinta Cid, Ana Márquez, Esteban Ballestar, and Javier Martín
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Inflammation ,Rheumatology ,Immunology ,Giant Cell Arteritis ,Immunology and Allergy ,Immune Complex Diseases ,Glucocorticoids ,General Biochemistry, Genetics and Molecular Biology - Abstract
ObjectivesGiant cell arteritis (GCA) is a complex systemic vasculitis mediated by the interplay between both genetic and epigenetic factors. Monocytes are crucial players of the inflammation occurring in GCA. Therefore, characterisation of the monocyte methylome and transcriptome in GCA would be helpful to better understand disease pathogenesis.MethodsWe performed an integrated epigenome-and transcriptome-wide association study in CD14+ monocytes from 82 patients with GCA, cross-sectionally classified into three different clinical statuses (active, in remission with or without glucocorticoid (GC) treatment), and 31 healthy controls.ResultsWe identified a global methylation and gene expression dysregulation in GCA monocytes. Specifically, monocytes from active patients showed a more proinflammatory phenotype compared with healthy controls and patients in remission. In addition to inflammatory pathways known to be involved in active GCA, such as response to IL-6 and IL-1, we identified response to IL-11 as a new pathway potentially implicated in GCA. Furthermore, monocytes from patients in remission with treatment showed downregulation of genes involved in inflammatory processes as well as overexpression of GC receptor-target genes. Finally, we identified changes in DNA methylation correlating with alterations in expression levels of genes with a potential role in GCA pathogenesis, such asITGA7andCD63, as well as genes mediating the molecular response to GC, includingFKBP5, ETS2,ZBTB16andADAMTS2.ConclusionOur results revealed profound alterations in the methylation and transcriptomic profiles of monocytes from GCA patients, uncovering novel genes and pathways involved in GCA pathogenesis and in the molecular response to GC treatment.
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- 2022
4. Coordinated glucocorticoid receptor and MAFB action induces tolerogenesis and epigenome remodeling in dendritic cells
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Octavio Morante-Palacios, Laura Ciudad, Raphael Micheroli, Carlos de la Calle-Fabregat, Tianlu Li, Gisela Barbisan, Miranda Houtman, Sam G Edalat, Mojca Frank-Bertoncelj, Caroline Ospelt, Esteban Ballestar, and University of Zurich
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Adult ,Male ,AcademicSubjects/SCI00010 ,MafB Transcription Factor ,610 Medicine & health ,Dioxygenases ,Epigenesis, Genetic ,03 medical and health sciences ,Receptors, Glucocorticoid ,0302 clinical medicine ,Immune Tolerance ,Genetics ,Humans ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,Gene regulation, Chromatin and Epigenetics ,10051 Rheumatology Clinic and Institute of Physical Medicine ,Dendritic Cells ,DNA Methylation ,Middle Aged ,3. Good health ,DNA-Binding Proteins ,030220 oncology & carcinogenesis ,Female - Abstract
Glucocorticoids (GCs) exert potent anti-inflammatory effects in immune cells through the glucocorticoid receptor (GR). Dendritic cells (DCs), central actors for coordinating immune responses, acquire tolerogenic properties in response to GCs. Tolerogenic DCs (tolDCs) have emerged as a potential treatment for various inflammatory diseases. To date, the underlying cell type-specific regulatory mechanisms orchestrating GC-mediated acquisition of immunosuppressive properties remain poorly understood. In this study, we investigated the transcriptomic and epigenomic remodeling associated with differentiation to DCs in the presence of GCs. Our analysis demonstrates a major role of MAFB in this process, in synergy with GR. GR and MAFB both interact with methylcytosine dioxygenase TET2 and bind to genomic loci that undergo specific demethylation in tolDCs. We also show that the role of MAFB is more extensive, binding to thousands of genomic loci in tolDCs. Finally, MAFB knockdown erases the tolerogenic properties of tolDCs and reverts the specific DNA demethylation and gene upregulation. The preeminent role of MAFB is also demonstrated in vivo for myeloid cells from synovium in rheumatoid arthritis following GC treatment. Our results imply that, once directly activated by GR, MAFB plays a critical role in orchestrating the epigenomic and transcriptomic remodeling that define the tolerogenic phenotype.
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- 2022
5. Circulating Cell-Free DNA Methylation Mirrors Alterations In Cerebral Patterns in Epilepsy
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Ricardo Martins-Ferreira, Bárbara Leal, João Chaves, Laura Ciudad, Raquel Samões, António Martins da Silva, Paulo Pinho Costa, and Esteban Ballestar
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History ,Polymers and Plastics ,Business and International Management ,Industrial and Manufacturing Engineering - Published
- 2022
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6. The DNA Methylomes of Synovial and Peripheral Blood Monocytes Associate and Evolve With Prognosis and Treatment in Undifferentiated Arthritis
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Carlos de la Calle-Fabregat, Javier Rodríguez-Ubreva, Laura Ciudad, Julio Ramírez, Raquel Celis, Ana Belén Azuaga, Andrea M. Cuervo, Eduard Graell, Carolina Pérez-García, César Díaz-Torné, Georgina Salvador, José A. Gómez-Puerta, Isabel Haro, Raimon Sanmartí, Juan D. Cañete, and Esteban Ballestar
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- 2022
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7. MAFB surrogates the glucocorticoid receptor ability to induce tolerogenesis in dendritic cells
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Miranda Houtman, Caroline Ospelt, Carlos de la Calle-Fabregat, Gisela Barbisan, Octavio Morante-Palacios, Tianlu Li, Mojca Frank-Bertoncelj, Raphael Micheroli, Esteban Ballestar, Sam Edalat, and Laura Ciudad
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Transcriptome ,Gene knockdown ,DNA demethylation ,Glucocorticoid receptor ,Immune system ,Downregulation and upregulation ,MAFB ,Biology ,Epigenomics ,Cell biology - Abstract
Glucocorticoids (GCs) exert potent anti-inflammatory effects in immune cells through the glucocorticoid receptor (GR). Dendritic cells (DCs), central actors for coordinating immune responses, acquire tolerogenic properties in response to GCs. Tolerogenic DCs (tolDCs) have emerged as a potential treatment for various inflammatory diseases. To date, the underlying cell type-specific regulatory mechanisms orchestrating GC-mediated acquisition of immunosuppressive properties remain poorly understood. In this study, we investigated the transcriptomic and epigenomic remodeling associated with differentiation to DCs in the presence of GCs. Our analysis demonstrates a major role of MAFB in this process, in synergy with GR. GR and MAFB both interact with methylcytosine dioxygenase TET2 and bind to genomic loci that undergo specific demethylation in tolDCs. We also show that the role of MAFB is more extensive, binding to thousands of genomic loci in tolDCs. Finally, MAFB knockdown erases the tolerogenic properties of tolDCs and reverts the specific DNA demethylation and gene upregulation. The preeminent role of MAFB is also demonstrated in vivo for myeloid cells from synovium in rheumatoid arthritis following GC treatment. Our results imply that, once directly activated by GR, MAFB takes over the main roles to orchestrate the epigenomic and transcriptomic remodeling that define the tolerogenic phenotype.
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- 2021
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8. Epilepsy progression is associated with cumulative DNA methylation changes in inflammatory genes
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Ricardo Martins-Ferreira, Bárbara Leal, João Chaves, Tianlu Li, Laura Ciudad, Rui Rangel, Agostinho Santos, António Martins da Silva, Paulo Pinho Costa, and Esteban Ballestar
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DNA Methylation ,Inflammation ,Adult ,Epilepsy ,Sclerosis ,Epilepsy, Temporal Lobe ,General Neuroscience ,Humans ,Neocortex ,Hippocampus ,Doenças Genéticas - Abstract
Mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) is the most common focal epilepsy in adults. It is characterized by alarming rates of pharmacoresistance. Epileptogenesis is associated with the occurrence of epigenetic alterations, and the few epigenetic studies carried out in MTLE-HS have mainly focused on the hippocampus. In this study, we obtained the DNA methylation profiles from both the hippocampus and anterior temporal neocortex of MTLE-HS patients subjected to resective epilepsy surgery and autopsied non-epileptic controls. We assessed the progressive nature of DNA methylation changes in relation to epilepsy duration. We identified significantly altered hippocampal DNA methylation patterns encompassing multiple pathways known to be involved in epileptogenesis. DNA methylation changes were even more striking in the neocortex, wherein pathogenic pathways and genes were common to both tissues. Most importantly, DNA methylation changes at many genomic sites varied significantly with epilepsy duration. Such progressive changes were associated with inflammation-related genes in the hippocampus. Our results suggest that the neocortex, relatively spared of extensive histopathological damage, may also be involved in epilepsy development. These results also open the possibility that the observed neocortical impairment could represent a preliminary stage of epileptogenesis before the establishment of chronic lesions or a consequence of prolonged seizure exposure. Our two-tissue multi-level characterization of the MTLE-HS DNA methylome suggests the occurrence of a self-propagating inflammatory wave of epigenetic dysregulation. Highlights: DNA methylation of epileptogenic pathways is altered in MTLE-HS brain; Neocortical DNA methylation changes are more striking; DNA methylation correlates with epileptic transcriptomic traits; DNA methylation may modulate a self-propagating inflammatory cycle in epilepsy. E.B. is funded by the Spanish Ministry of Science and Innovation (MICINN) [SAF2017-88086-R, PID2020117212RB-I00; AEI/10.13039/501100011033]. This work was supported by a BICE Tecnifar Grant. R.M.-F was funded by an FCT (Fundação para a Ciência e Tecnologia) fellowship (grant number SFRH/BD/137900/2018). Unit for Multidisciplinary Research in Biomedicine (UMIB) is funded by FCT Portugal (grant numbers UIDB/00215/2020, and UIDP/00215/2020), and Laboratory for Integrative and Translational Research in Population Health (ITR) (LA/P/0064/2020). info:eu-repo/semantics/publishedVersion
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- 2021
9. Inflammatory cytokines shape a changing DNA methylome in monocytes mirroring disease activity in rheumatoid arthritis
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Juan D. Cañete, Frances Humby, Javier Rodríguez-Ubreva, Costantino Pitzalis, Javier Martín, Antonio Garcia-Gomez, Octavio Morante-Palacios, Laura Ciudad, Tianlu Li, Carlos de la Calle-Fabregat, Raquel Celis, Maria Luisa Ballestar, Alessandra Nerviani, Esteban Ballestar, Francesc Català-Moll, Generalitat de Catalunya, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, and European Commission
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rheumatoid arthritis ,0301 basic medicine ,DAS28 ,Immunology ,disease activity ,General Biochemistry, Genetics and Molecular Biology ,Proinflammatory cytokine ,Arthritis, Rheumatoid ,Pathogenesis ,Epigenome ,03 medical and health sciences ,0302 clinical medicine ,Rheumatology ,medicine ,Humans ,Immunology and Allergy ,Epigenomics ,030203 arthritis & rheumatology ,DNA methylation ,Tumor Necrosis Factor-alpha ,business.industry ,Macrophages ,Monocyte ,TNFa ,Methylation ,DNA Methylation ,medicine.disease ,030104 developmental biology ,medicine.anatomical_structure ,Rheumatoid arthritis ,Leukocytes, Mononuclear ,Cytokines ,Tumor necrosis factor alpha ,Inflammation Mediators ,business ,Biomarkers - Abstract
Objective: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that mainly targets joints. Monocytes and macrophages are critical in RA pathogenesis and contribute to inflammatory lesions. These extremely plastic cells respond to extracellular signals which cause epigenomic changes that define their pathogenic phenotype. Here, we interrogated how DNA methylation alterations in RA monocytes are determined by extracellular signals. Methods: High-throughput DNA methylation analyses of patients with RA and controls and in vitro cytokine stimulation were used to investigate the underlying mechanisms behind DNA methylation alterations in RA as well as their relationship with clinical parameters, including RA disease activity. Results: The DNA methylomes of peripheral blood monocytes displayed significant changes and increased variability in patients with RA with respect to healthy controls. Changes in the monocyte methylome correlate with DAS28, in which high-activity patients are divergent from healthy controls in contrast to remission patients whose methylome is virtually identical to healthy controls. Indeed, the notion of a changing monocyte methylome is supported after comparing the profiles of same individuals at different stages of activity. We show how these changes are mediated by an increase in disease activity-associated cytokines, such as tumour necrosis factor alpha and interferons, as they recapitulate the DNA methylation changes observed in patients in vitro. Conclusion: We demonstrate a direct link between RA disease activity and the monocyte methylome through the action of inflammation-associated cytokines. Finally, we have obtained a DNA methylation-based mathematical formula that predicts inflammation-mediated disease activity for RA and other chronic immune-mediated inflammatory diseases., We thank CERCA Programme/Generalitat de Catalunya for institutional support. EB was funded by the Spanish Ministry of Economy and Competitiveness (MINECO; grant numbers SAF2014-55942-R and SAF2017-88086-R). JDC was funded by FIS grant (PI17/00993) from Institute of Health Carlos III (ISCIII). JDC, JM and EB are supported by RETICS network grant from ISCIII (RIER, RD16/0012/0013), FEDER 'Una manera de hacer Europa'
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- 2019
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10. Targeting aberrant DNA methylation in mesenchymal stromal cells as a treatment for myeloma bone disease
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Antonio Garcia-Gomez, Laura San-Segundo, Laura Ciudad, Manel Esteller, Xabier Morales, Esteban Ballestar, Javier Rodríguez-Ubreva, Carlos Ortiz-de-Solorzano, Julen Oyarzabal, Felipe Prosper, Mercedes Garayoa, Tianlu Li, Sandra Muntión, Montserrat Martín-Sánchez, Xabier Agirre, Edurne San José-Enériz, Carlos de la Calle-Fabregat, Gerard Godoy-Tena, Francesc Català-Moll, Generalitat de Catalunya, Josep Carreras Leukemia Foundation, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Multiple Myeloma Research Foundation, European Commission, Instituto de Salud Carlos III, Asociación Española Contra el Cáncer, Fundació La Marató de TV3, Associazione Italiana per la Ricerca sul Cancro, and Fundación Ramón Areces
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0301 basic medicine ,Male ,Bone disease ,ADN ,General Physics and Astronomy ,Myeloma ,Epigenesis, Genetic ,Mice ,0302 clinical medicine ,Bone Marrow ,Osteogenesis ,Multiple myeloma ,Histocompatibility Antigens ,DNA (Cytosine-5-)-Methyltransferases ,Femur ,Enzyme Inhibitors ,Regulation of gene expression ,Aged, 80 and over ,Multidisciplinary ,DNA methylation ,Osteoblast ,Middle Aged ,Gene Expression Regulation, Neoplastic ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Female ,Bone Diseases ,Multiple Myeloma ,Adult ,Science ,Antineoplastic Agents ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Target identification ,medicine ,Animals ,Humans ,Epigenetics ,Aged ,Mesenchymal stem cell ,Mieloma múltiple ,Mesenchymal Stem Cells ,General Chemistry ,Histone-Lysine N-Methyltransferase ,DNA ,DNA Methylation ,medicine.disease ,Xenograft Model Antitumor Assays ,030104 developmental biology ,Cancer research ,Bone marrow - Abstract
© The Author(s) 2021., Multiple myeloma (MM) progression and myeloma-associated bone disease (MBD) are highly dependent on bone marrow mesenchymal stromal cells (MSCs). MM-MSCs exhibit abnormal transcriptomes, suggesting the involvement of epigenetic mechanisms governing their tumor-promoting functions and prolonged osteoblast suppression. Here, we identify widespread DNA methylation alterations of bone marrow-isolated MSCs from distinct MM stages, particularly in Homeobox genes involved in osteogenic differentiation that associate with their aberrant expression. Moreover, these DNA methylation changes are recapitulated in vitro by exposing MSCs from healthy individuals to MM cells. Pharmacological targeting of DNMTs and G9a with dual inhibitor CM-272 reverts the expression of hypermethylated osteogenic regulators and promotes osteoblast differentiation of myeloma MSCs. Most importantly, CM-272 treatment prevents tumor-associated bone loss and reduces tumor burden in a murine myeloma model. Our results demonstrate that epigenetic aberrancies mediate the impairment of bone formation in MM, and its targeting by CM-272 is able to reverse MBD., We thank CERCA Program/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. E.B. was funded by the Spanish Ministry of Science and Innovation (grant numbers SAF2014-55942-R and SAF2017-88086-R), co-funded by FEDER funds/European Regional Development Fund (ERDF)—a way to build Europe, and a Senior Research Award from the Multiple Myeloma Research Foundation (MMRF). C.O.-d.-S. was funded by the Spanish Ministry of Science, Innovation and Universities, under grant RTI2018-094494-B-C22 (MCIU/AEI/FEDER, UE). M.G. received financial support from the Spanish FIS-ISCIII (PI15/02156 and PI19/01384) and FEDER. A.G.G is funded by a postdoctoral contract of the Asociación Española Contra el Cáncer (AECC). F.P. was funded by grants from Instituto de Salud Carlos III (ISCIII), PI17/00701 and PI19/01352, TRASCAN (EPICA and Immunocell), Fundació La Marató de TV3, the Accelerator award CRUK/AIRC/AECC joint funder-partnership, CIBERONC (CB16/12/00489) and co-financed with FEDER funds and Fundación Ramón Areces (PREMAMM).
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- 2021
11. Vitamin D Receptor, STAT3 and TET2 Cooperate to Establish Tolerogenesis
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Javier Rodríguez-Ubreva, Laura Barberà, Octavio Morante-Palacios, Anna G. Ferreté-Bonastre, Francesc Català-Moll, Federico Fondelli, Gerard Godoy-Tena, Esteban Ballestar, Eva Martínez-Cáceres, Tianlu Li, and Laura Ciudad
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STAT3 Transcription Factor ,Tolerogenesis ,Dendritic cells ,Calcitriol receptor ,General Biochemistry, Genetics and Molecular Biology ,Dioxygenases ,STAT3 ,Immune Tolerance ,Vitamin D and neurology ,Humans ,Epigenetics ,Vitamin D ,Cells, Cultured ,VDR ,Epigenomics ,TET2 ,DNA methylation ,biology ,Chemistry ,Dendritic Cells ,Cell biology ,DNA-Binding Proteins ,Methylcytosine Dioxygenase TET2 ,DNA demethylation ,JAK2 ,biology.protein ,Receptors, Calcitriol ,Phosphorylation ,IL-6-JAK-STAT - Abstract
We are very grateful to Dr. José Luis Sardina for useful feedback. We thank CERCA Programme/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. E.B. was funded by the Spanish Ministry of Science and Innovation (MICINN ; grant number PID2020-117212RB-I00/AEI/10.13038/501100011033). E.M.-C. is funded with RESTORE project (EU H2020 Research and Innovation Programme, number 779316) and Spanish projects PI17/01521 and PI20/01313, integrated in the Plan Nacional de I+D+I and co-supported by the ISCIII-Subdirección General de Evaluación and FEDER. O.M.-P. holds an i-PFIS PhD fellowship (grant number IFI17/00034) from Acción Estratégica en Salud 2013-2016 ISCIII, co-financed by Fondo Social Europeo. F.F. holds a PhD fellowship from the INsTRuCT Consortium, which receives. Innovative Training Network subsidy from the EU H2020 program. We are very grateful to Dr. Jos? Luis Sardina for useful feedback. We thank CERCA Programme/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. E.B. was funded by the Spanish Ministry of Science and Innovation (MICINN; grant number PID2020-117212RB-I00/AEI/10.13038/501100011033). E.M.-C. is funded with RESTORE project (EU H2020 Research and Innovation Programme, number 779316) and Spanish projects PI17/01521 and PI20/01313, integrated in the Plan Nacional de I+D+I and co-supported by the ISCIII-Subdirecci?n General de Evaluaci?n and FEDER. O.M.-P. holds an i-PFIS PhD fellowship (grant number IFI17/00034) from Acci?n Estrat?gica en Salud 2013?2016 ISCIII, co-financed by Fondo Social Europeo. F.F. holds a PhD fellowship from the INsTRuCT Consortium, which receives. Innovative Training Network subsidy from the EU H2020 program. F.C.-M. and E.B. conceived and designed the study; F.C.-M. A.G.F.-B. G.G.-T. O.M.-P. L.C. L.B. F.F. and T.L. performed the differentiation, chromatin immunoprecipitation, co-immunoprecipitation experiments, and immunological assays; F.C.-M. performed the bioinformatic analyses; F.C.-M. A.G.F.-B. G.G.-T. E.M.-C. and E.B. analyzed results; J.R.-U. and E.B. supervised the study; F.C.-M. T.L. and E.B. wrote the manuscript; all authors participated in discussions and interpreting the results. The authors declare no competing interests. The active form of vitamin D, 1,25-dihydroxyvitamin D3, induces a stable tolerogenic phenotype in dendritic cells (DCs). This process involves the vitamin D receptor (VDR), which translocates to the nucleus, binds its cognate genomic sites, and promotes epigenetic and transcriptional remodeling. In this study, we report the occurrence of vitamin D-specific DNA demethylation and transcriptional activation at VDR binding sites associated with the acquisition of tolerogenesis in vitro. Differentiation to tolerogenic DCs associates with activation of the IL-6-JAK-STAT3 pathway. We show that JAK2-mediated STAT3 phosphorylation is specific to vitamin D stimulation. VDR and the phosphorylated form of STAT3 interact with each other to form a complex with methylcytosine dioxygenase TET2. Most importantly, pharmacological inhibition of JAK2 reverts vitamin D-induced tolerogenic properties of DCs. This interplay among VDR, STAT3, and TET2 opens up possibilities for modulating DC immunogenic properties in clinics.
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- 2021
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12. The JAK2-STAT pathway epigenetically regulates tolerized genes during the first encounter with bacterial antigens
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Damiana Álvarez-Errico, Esteban Ballestar, Antonio Garcia-Gomez, Octavio Morante-Palacios, Francesc Català-Moll, Mónica Martínez-Gallo, Juan Carlos Ruiz-Rodríguez, Adolfo Ruiz-Sanmartín, Ricard Ferrer-Roca, Laura Ciudad, and Clara Lorente-Sorolla
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chemistry.chemical_compound ,biology ,Lipopolysaccharide ,chemistry ,biology.protein ,TLR4 ,STAT protein ,JAK-STAT signaling pathway ,STAT1 ,Bacterial antigen ,STAT3 ,STAT5 ,Cell biology - Abstract
Microbial challenges, such as widespread bacterial infection, induce endotoxin tolerance. This state of hyporesponsiveness to subsequent infections is mainly displayed by monocytes and macrophages. Endotoxin tolerance is generally acquired following a septic episode. In this study, we investigated DNA methylation changes during the acquisition of in vitro tolerance. We identified a set of TET2-mediated demethylation events that are specific to toll-like receptor (TLR) 2 and TLR4 stimulation. Lipopolysaccharide (LPS)-specific demethylation occurs at genomic sites that have low accessibility in quiescent monocytes, concomitantly with the transcriptional activation of many inflammation-related genes, and they are enriched in binding motifs for several signal transducer and activator of transcription (STAT) family members. Indeed, STAT1, STAT3 and STAT5, elements of the JAK2 pathway, are phosphorylated in association with the acquisition of endotoxin tolerance. Inhibition of the JAK2 pathway impairs the activation of tolerized genes on the first encounter with LPS. This is evidence of a crucial role for this pathway in determining the initial response of these genes to bacterial antigens and provides a pharmacological target to prevent exacerbated responses, allowing regulated responses upon subsequent challenges. Finally, we assess the pathological relevance of the JAK2 pathway in monocytes from patients with sepsis.
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- 2020
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13. Epigenomics and Transcriptomics of Systemic Sclerosis CD4+ T cells reveal Long Range Dysregulation of Key Inflammatory Pathways mediated by disease-associated Susceptibility Loci
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Biola M. Javierre, Elena López-Isac, Laura Ciudad, Javier Martín, Tianlu Li, Alfredo Guillén-Del-Castillo, Eduardo Andrés-León, Lourdes Ortiz, Carmen Pilar Simeón-Aznar, and Esteban Ballestar
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Genetics ,integumentary system ,T cell ,Inflammation ,Biology ,Transcriptome ,medicine.anatomical_structure ,Immune system ,CTCF ,DNA methylation ,medicine ,Epigenetics ,medicine.symptom ,Epigenomics - Abstract
System sclerosis (SSc) is a genetically complex autoimmune disease mediated by the interplay between genetic and epigenetic factors in a multitude of immune cells, with CD4+ T lymphocytes as one of the principle drivers of pathogenesis. In this study, we obtained DNA methylation and expression profiles of CD4+ T cells from 48 SSc patients and 16 healthy controls. Consequently, we identified 9112 and 3929 differentially methylated CpGs positions (DMPs) and differentially expressed genes (DEGs) respectively. These DMPs and DEGs are enriched in functional categories related to inflammation and T cell biology. Furthermore, correlation analysis identified 17,500 possible DMP-DEG interaction pairs within a window of 5 Mb, and utilizing promoter capture Hi-C data, we confirmed that 212 CD4+ T cel specific pairs of DMP-DEG physically interact involving CTCF. Finally, utilizing SSc GWAS data, we identified four important SSc-associated susceptibility loci, TNIP1 (rs3792783), GSDMB (rs9303277), IL12RB1 (rs2305743) and CSK (rs1378942), that physically interact with DMP-DEG pairs cg17239269-ANXA6, cg19458020-CCR7, cg10808810-JUND and cg11062629-ULK3 respectively. Overall, our study reveals a solid link between genetic, epigenetic and transcriptional deregulation in CD4+ T cells of SSc patients, providing a novel integrated view of SSc pathogenic determinants.
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- 2020
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14. Vitamin D receptor and STAT3 cooperate to establish TET2-mediated tolerogenesis
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Laura Ciudad, Tianlu Li, Francesc Català-Moll, Javier Rodríguez-Ubreva, and Esteban Ballestar
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DNA demethylation ,biology ,Chemistry ,DNA methylation ,Vitamin D and neurology ,biology.protein ,Phosphorylation ,Epigenetics ,STAT3 ,Calcitriol receptor ,Transcription factor ,Cell biology - Abstract
SUMMARYThe active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), induces stable tolerogenesis in dendritic cells (DCs). This process involves the vitamin D receptor (VDR), which translocates to the nucleus, binds its cognate genomic sites, and promotes epigenetic and transcriptional remodeling. In this study, we investigated the interplay between the VDR and other transcription factors to induce DNA methylation changes that might provide phenotypic stability to the tolerogenic phenotype of DCs. Our study reveals the occurrence of vitamin D-specific DNA demethylation and transcriptional activation at VDR binding sites associated with the acquisition of tolerogenesis. Tolerogenic properties in DCs are acquired together with activation of the IL6-JAK-STAT3 pathway. In fact, VDR directly binds the IL6 gene, and JAK2-mediated STAT3 phosphorylation is specific to vitamin D stimulation. VDR and the phosphorylated form of STAT3 interact with each other and with methylcytosine dioxygenase TET2 following vitamin D treatment. Most importantly, pharmacological inhibition of STAT3 phosphorylation reverts the vitamin-induced tolerogenic properties of DCs. Our results reveal an interplay between VDR and STAT3 leading to the DNA demethylation-dependent induction of tolerogenesis by vitamin D.
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- 2020
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15. Targeting aberrant DNA methylation in mesenchymal stromal cells as a treatment for myeloma bone disease
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Antonio Garcia-Gomez, Esteban Ballestar, Xabier Morales, Francesc Català-Moll, Felipe Prosper, Carlos Ortiz de Solórzano, Laura San-Segundo, Xabier Agirre, Julen Oyarzabal, Laura Ciudad, Edurne San José-Enériz, Montserrat Martín-Sánchez, Javier Rodríguez-Ubreva, Tianlu Li, and Mercedes Garayoa
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Bone disease ,business.industry ,Mesenchymal stem cell ,Osteoblast ,medicine.disease ,medicine.anatomical_structure ,DNA methylation ,Cancer research ,medicine ,Epigenetics ,Bone marrow ,business ,Monoclonal gammopathy of undetermined significance ,Multiple myeloma - Abstract
Multiple myeloma (MM) progression and myeloma-associated bone disease (MBD) are highly dependent on the bone marrow (BM) microenvironment, in particular on mesenchymal stromal cells (MSCs). MSCs from MM patients exhibit an abnormal transcriptional profile, suggesting that epigenetic alterations could be governing the tumor-promoting functions of MSCs and their prolonged osteoblast (OB) suppression in MM. In this study, we analyzed the DNA methylome of BM-derived MSCs from patients with monoclonal gammopathy of undetermined significance, smoldering myeloma and symptomatic MM at diagnosis in comparison with their normal counterparts. DNA methylation alterations were found at each of the myeloma stage in association with deregulated expression levels of Homeobox genes involved in osteogenic differentiation. Moreover, these DNA methylation changes were recapitulatedin vitroby exposing MSCs from healthy individuals to MM plasma cells. Pharmacological targeting of DNMTs and G9a with the dual inhibitor CM-272, reverted the expression of aberrantly methylated osteogenic regulators and promoted OB differentiation of MSCs from myeloma patients. Most importantly, in a mouse model of bone marrow-disseminated MM, administration of CM-272 prevented tumor-associated bone loss and reduced tumor burden. Our results demonstrated that not only was aberrant DNA methylation a main contributor to bone formation impairment found in MM patients, but also its targeting by CM-272 was able to reverse MM-associated bone loss.KEY POINTS- Bone marrow-derived mesenchymal stromal cells (MSCs) from monoclonal gammopathy of undetermined significance, smoldering myeloma and myeloma patients exhibit an aberrant DNA methylome compared to their healthy counterparts.- These DNA methylation changes are associated with an altered expression of genes of the Homeobox loci that orchestrate osteogenic differentiation of mesenchymal precursors.- MM plasma cell-exposed healthy MSCs recapitulate the DNA methylation alterations observed in MSCs isolated from myeloma patients.- Dual targeting of DNMTs and the histone methyltransferase G9a with CM-272 not only controls MM tumor burden but also prevents myeloma-associated bone loss.
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- 2019
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16. Inflammatory cytokines and organ dysfunction associate with the aberrant DNA methylome of monocytes in sepsis
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Damiana Álvarez-Errico, Antonio Garcia-Gomez, Álvaro García del Campo, Ricard Ferrer-Roca, Laura Ciudad, Victor Toledano, Clara Lorente-Sorolla, Mónica Martínez-Gallo, Eduardo López-Collazo, Francesc Català-Moll, Juan Carlos Ruiz-Rodríguez, Adolfo Ruiz-Sanmartín, Esteban Ballestar, Alejandro Martín-Quirós, Charbel Maroun-Eid, and José Avendaño-Ortiz
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Male ,0301 basic medicine ,lcsh:QH426-470 ,Multiple Organ Failure ,lcsh:Medicine ,Endotoxin tolerance ,Biology ,Monocytes ,Proinflammatory cytokine ,Sepsis ,03 medical and health sciences ,0302 clinical medicine ,Citoquines ,Genetics ,medicine ,Humans ,Epigenetics ,Molecular Biology ,Genetics (clinical) ,Aged ,Inflammation ,DNA methylation ,Research ,lcsh:R ,Organ dysfunction ,DNA ,Methylation ,Middle Aged ,medicine.disease ,Systemic inflammatory response syndrome ,lcsh:Genetics ,Phenotype ,030104 developmental biology ,Insuficiència respiratòria ,Case-Control Studies ,030220 oncology & carcinogenesis ,Immunology ,Molecular Medicine ,Cytokines ,Female ,Inflammation Mediators ,medicine.symptom ,Respiratory insufficiency ,Reprogramming ,Signal Transduction - Abstract
Background Sepsis, a life-threatening organ dysfunction caused by a dysregulated systemic immune response to infection, associates with reduced responsiveness to subsequent infections. How such tolerance is acquired is not well understood but is known to involve epigenetic and transcriptional dysregulation. Methods Bead arrays were used to compare global DNA methylation changes in patients with sepsis, non-infectious systemic inflammatory response syndrome, and healthy controls. Bioinformatic analyses were performed to dissect functional reprogramming and signaling pathways related to the acquisition of these specific DNA methylation alterations. Finally, in vitro experiments using human monocytes were performed to test the induction of similar DNA methylation reprogramming. Results Here, we focused on DNA methylation changes associated with sepsis, given their potential role in stabilizing altered phenotypes. Tolerized monocytes from patients with sepsis display changes in their DNA methylomes with respect to those from healthy controls, affecting critical monocyte-related genes. DNA methylation profiles correlate with IL-10 and IL-6 levels, significantly increased in monocytes in sepsis, as well as with the Sequential Organ Failure Assessment score; the observed changes associate with TFs and pathways downstream to toll-like receptors and inflammatory cytokines. In fact, in vitro stimulation of toll-like receptors in monocytes results in similar gains and losses of methylation together with the acquisition of tolerance. Conclusion We have identified a DNA methylation signature associated with sepsis that is downstream to the response of monocytes to inflammatory signals associated with the acquisition of a tolerized phenotype and organic dysfunction.
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- 2019
17. 05 / Ventriculoperitoneal shunt due to Paracoccidiomicosis: Anaesthetic management in the obstetric patient
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Laura Ciudad Morales
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- 2018
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18. 03 / Facial Palsy and cranial nerve injuries during pregnancy: a predictive sign of preeclampsia: two cases
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Laura Ciudad Morales
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- 2018
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19. C/EBPa-Mediated Activation of MicroRNAs 34a and 223 Inhibits Lef1 Expression To Achieve Efficient Reprogramming into Macrophages
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Esteban Ballestar, Thomas Graf, Laura Ciudad, Javier Rodríguez-Ubreva, Chris van Oevelen, and Maribel Parra
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Lymphoid Enhancer-Binding Factor 1 ,PAX5 Transcription Factor ,Antigens, CD19 ,Down-Regulation ,Biology ,Ikaros Transcription Factor ,Mice ,Downregulation and upregulation ,Animals ,Molecular Biology ,Transcription factor ,B-Lymphocytes ,Ccaat-enhancer-binding proteins ,Macrophages ,Precursor Cells, B-Lymphoid ,Transdifferentiation ,Articles ,Cell Biology ,Molecular biology ,Up-Regulation ,Cell biology ,MicroRNAs ,Cell Transdifferentiation ,CCAAT-Enhancer-Binding Proteins ,Trans-Activators ,Ectopic expression ,Transcription Factors ,Lymphoid enhancer-binding factor 1 - Abstract
MicroRNAs (miRNAs) exert negative effects on gene expression and influence cell lineage choice during hematopoiesis. C/EBPa-induced pre-B cell-to-macrophage transdifferentiation provides an excellent model to investigate the contribution of miRNAs to hematopoietic cell identity, especially because the two cell types involved fall into separate lymphoid and myeloid branches. In this process, efficient repression of the B cell-specific program is essential to ensure transdifferentation and macrophage function. miRNA profiling revealed that upregulation of miRNAs is highly predominant compared with downregulation and that C/EBPa directly regulates several upregulated miRNAs. We also determined that miRNA 34a (miR-34a) and miR-223 sharply accelerate C/EBPa-mediated transdifferentiation, whereas their depletion delays this process. These two miRNAs affect the transdifferentiation efficiency and activity of macrophages, including their lipopolysaccharide (LPS)-dependent inflammatory response. miR-34a and miR-223 directly target and downregulate the lymphoid transcription factor Lef1, whose ectopic expression delays transdifferentiation to an extent similar to that seen with miR-34a and miR-223 depletion. In addition, ectopic introduction of Lef1 in macrophages causes upregulation of B cell markers, including CD19, Pax5, and Ikzf3. Our report demonstrates the importance of these miRNAs in ensuring the erasure of key B cell transcription factors, such as Lef1, and reinforces the notion of their essential role in fine-tuning the control required for establishing cell identity.
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- 2014
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20. Activation-induced cytidine deaminase targets SUV4-20-mediated histone H4K20 trimethylation to class-switch recombination sites
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Esteban Ballestar, Javier Rodríguez-Ubreva, Laura Ciudad, Almudena R. Ramiro, Francesc Català-Moll, Alejandro Vaquero, Ganesh Poorani-Subramani, Henar Hernando, Antonio Garcia-Gomez, Arantxa Pérez-García, Virginia C. Rodríguez-Cortez, Javier M. Di Noia, Jose Urquiza, Paloma Martinez-Redondo, Instituto de Salud Carlos III, Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF), Canada Research Chairs, and Canadian Institutes of Health Research
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0301 basic medicine ,Lipopolysaccharides ,Methyltransferase ,DOUBLE-STRAND BREAKS ,Hyper-IgM Immunodeficiency Syndrome ,Lymphocyte Activation ,RECRUIT AID ,Epigenesis, Genetic ,Histones ,Mice ,Activation-induced (cytidine) deaminase ,Regulation of gene expression ,Genetics ,B-Lymphocytes ,Multidisciplinary ,biology ,METHYLATION ,Cytidine deaminase ,REGIONS ,Histone ,B-CELLS ,Medicine ,Epigenetics ,RNA Polymerase II ,Protein Binding ,Signal Transduction ,Science ,Models, Biological ,DEMETHYLATION ,Article ,03 medical and health sciences ,Cell Line, Tumor ,Cytidine Deaminase ,SOMATIC HYPERMUTATION ,Animals ,Humans ,Binding site ,REPAIR ,Binding Sites ,DNA ,Histone-Lysine N-Methyltransferase ,Epigenètica ,SUPER-ENHANCERS ,Immunoglobulin Class Switching ,Mice, Inbred C57BL ,030104 developmental biology ,HEK293 Cells ,Immunoglobulin class switching ,Gene Expression Regulation ,Immunoglobulin G ,Mutation ,biology.protein ,RNA ,HeLa Cells - Abstract
Activation-induced cytidine deaminase (AID) triggers antibody diversification in B cells by catalysing deamination and subsequently mutating immunoglobulin (Ig) genes. Association of AID with RNA Pol II and occurrence of epigenetic changes during Ig gene diversification suggest participation of AID in epigenetic regulation. AID is mutated in hyper-IgM type 2 (HIGM2) syndrome. Here, we investigated the potential role of AID in the acquisition of epigenetic changes. We discovered that AID binding to the IgH locus promotes an increase in H4K20me3. In 293F cells, we demonstrate interaction between co-transfected AID and the three SUV4-20 histone H4K20 methyltransferases, and that SUV4-20H1.2, bound to the IgH switch (S) mu site, is replaced by SUV4-20H2 upon AID binding. Analysis of HIGM2 mutants shows that the AID truncated form W68X is impaired to interact with SUV4-20H1.2 and SUV4-20H2 and is unable to bind and target H4K20me3 to the Smu site. We finally show in mouse primary B cells undergoing class-switch recombination (CSR) that AID deficiency associates with decreased H4K20me3 levels at the Smu site. Our results provide a novel link between SUV4-20 enzymes and CSR and offer a new aspect of the interplay between AID and histone modifications in setting the epigenetic status of CSR sites. This work was supported by grant SAF2014-55942-R from the Instituto de Salud Carlos III, organisms ascribed to the Ministerio de Economia y Competitividad and cofunded by FEDER funds/European Regional Development Fund (ERDF) - a way to build Europe, and the EU FP7 306000 STATegra project. JMDN is supported by a Canada Research Chair tier II. Work by JMDN and SPG was supported by the Canadian Institutes of Health Research, MOP 125991. Sí
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- 2016
21. Epigenetic regulation of PRAME in acute myeloid leukemia is different compared to CD34+ cells from healthy donors: Effect of 5-AZA treatment
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Carmen Herrero-Sánchez, Silvia Gutierrez-Cosio, Carlos Santamaría, J F San Miguel, Luis Ignacio Sánchez-Abarca, Belén Blanco, C Cañizo, Teresa Caballero-Velázquez, L. de la Rica, Laura Ciudad, Cristina Calderón, José A. Pérez-Simón, Soraya Carrancio, and Esteban Ballestar
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Antimetabolites, Antineoplastic ,Cancer Research ,Myeloid ,Azacitidine ,CD34 ,Antigens, CD34 ,Blood Donors ,Bone Marrow Cells ,Epigenesis, Genetic ,Antigens, Neoplasm ,hemic and lymphatic diseases ,Biomarkers, Tumor ,Humans ,Medicine ,Epigenetics ,Cells, Cultured ,PRAME ,Gene Expression Regulation, Leukemic ,business.industry ,Stem Cells ,Myeloid leukemia ,Hematology ,Leukemia, Myeloid, Acute ,medicine.anatomical_structure ,Oncology ,Health ,Cytogenetic Analysis ,DNA methylation ,Immunology ,CpG Islands ,Stem cell ,business ,medicine.drug - Abstract
PRAME is a tumor associated antigen (TAA) of particular interest since it is widely expressed by lymphoid and myeloid malignancies. Several studies have associated high PRAME RNA levels with good prognosis in acute myeloid leukemia (AML). PRAME expression is regulated at the epigenetic level. For this reason inhibitors of DNA methylation, such as 5-azacytidine, can modulate the expression of this TAAs. In the current study we analyzed the effect of 5-azaC on the expression of PRAME in blasts versus CD34+ cells from healthy donors in an attempt to increase its expression, thus inducing a potential target for therapeutic strategies.
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- 2012
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22. Pre-B cell to macrophage transdifferentiation without significant promoter DNA methylation changes
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David Gomez-Cabrero, Javier Rodríguez-Ubreva, Maribel Parra, Eric M. Kallin, Lars H. Bussmann, Alessandro di Tullio, Esteban Ballestar, Thomas Graf, Jesper Tegnér, and Laura Ciudad
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Cèl·lules B ,Cellular differentiation ,ADN ,Gene Regulation, Chromatin and Epigenetics ,Biology ,Epigenesis, Genetic ,Histones ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Epigenetics of physical exercise ,CCAAT-Enhancer-Binding Protein-alpha ,Genetics ,Animals ,p300-CBP Transcription Factors ,Epigenetics ,Promoter Regions, Genetic ,Cells, Cultured ,030304 developmental biology ,B cells ,0303 health sciences ,Macrophages ,Precursor Cells, B-Lymphoid ,Transdifferentiation ,Promoter ,DNA ,DNA Methylation ,Molecular biology ,3. Good health ,030220 oncology & carcinogenesis ,Cell Transdifferentiation ,DNA methylation ,Reprogramming - Abstract
Transcription factor-induced lineage reprogramming or transdifferentiation experiments are essential for understanding the plasticity of differentiated cells. These experiments helped to define the specific role of transcription factors in conferring cell identity and played a key role in the development of the regenerative medicine field. We here investigated the acquisition of DNA methylation changes during C/EBP alpha-induced pre-B cell to macrophage transdifferentiation. Unexpectedly, cell lineage conversion occurred without significant changes in DNA methylation not only in key B cell- and macrophage-specific genes but also throughout the entire set of genes differentially methylated between the two parental cell types. In contrast, active and repressive histone modification marks changed according to the expression levels of these genes. We also demonstrated that C/EBP alpha and RNA Pol II are associated with the methylated promoters of macrophage-specific genes in reprogrammed macrophages without inducing methylation changes. Our findings not only provide insights about the extent and hierarchy of epigenetic events in pre-B cell to macrophage transdifferentiation but also show an important difference to reprogramming towards pluripotency where promoter DNA demethylation plays a pivotal role.
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- 2011
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23. NF-κB-direct activation of microRNAs with repressive effects on monocyte-specific genes is critical for osteoclast differentiation
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Lorenzo de la Rica, Antonio García-Gómez, Natalia R Comet, Javier Rodríguez-Ubreva, Laura Ciudad, Roser Vento-Tormo, Carlos Company, Damiana Álvarez-Errico, Mireia García, Carmen Gómez-Vaquero, Esteban Ballestar, and Universitat de Barcelona
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Transcriptional Activation ,Micro RNAs ,Cellular differentiation ,Bones cancer ,Osteoclasts ,Biology ,Artritis reumatoide ,Monocytes ,Metastasis ,Immunomodulation ,Genètica mèdica ,Downregulation and upregulation ,Metàstasi ,RNA interference ,Osteoclast ,Cell diferentiation ,medicine ,Cluster Analysis ,Humans ,Position-Specific Scoring Matrices ,Gene silencing ,Gene Silencing ,RNA, Messenger ,Epigenetics ,Rheumatoid arthritis ,Transcription factor ,Regulation of gene expression ,Binding Sites ,Research ,Gene Expression Profiling ,Medical genetics ,NF-kappa B ,Càncer d'ossos ,Cell Differentiation ,Molecular biology ,Cell biology ,MicroRNAs ,medicine.anatomical_structure ,Gene Expression Regulation ,Organ Specificity ,Multigene Family ,RNA Interference ,Diferenciació cel·lular ,Protein Binding - Abstract
Background Monocyte-to-osteoclast conversion is a unique terminal differentiation process that is exacerbated in rheumatoid arthritis and bone metastasis. The mechanisms implicated in upregulating osteoclast-specific genes involve transcription factors, epigenetic regulators and microRNAs (miRNAs). It is less well known how downregulation of osteoclast-inappropriate genes is achieved. Results In this study, analysis of miRNA expression changes in osteoclast differentiation from human primary monocytes revealed the rapid upregulation of two miRNA clusters, miR-212/132 and miR-99b/let-7e/125a. We demonstrate that they negatively target monocyte-specific and immunomodulatory genes like TNFAIP3, IGF1R and IL15. Depletion of these miRNAs inhibits osteoclast differentiation and upregulates their targets. These miRNAs are also upregulated in other inflammatory monocytic differentiation processes. Most importantly, we demonstrate for the first time the direct involvement of Nuclear Factor kappa B (NF-κB) in the regulation of these miRNAs, as well as with their targets, whereby NF-κB p65 binds the promoters of these two miRNA clusters and NF-κB inhibition or depletion results in impaired upregulation of their expression. Conclusions Our results reveal the direct involvement of NF-κB in shutting down certain monocyte-specific genes, including some anti-inflammatory activities, through a miRNA-dependent mechanism for proper osteoclast differentiation. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0561-5) contains supplementary material, which is available to authorized users.
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- 2015
24. Conservation of fruitless' role as master regulator of male courtship behaviour from cockroaches to flies
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Elke Clynen, Xavier Bellés, Maria-Dolors Piulachs, Laura Ciudad, Ministerio de Educación y Ciencia (España), Generalitat de Catalunya, European Commission, Consejo Superior de Investigaciones Científicas (España), and Research Foundation - Flanders
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Male ,media_common.quotation_subject ,RNA Splicing ,Cockroaches ,Nerve Tissue Proteins ,Fruitless ,Courtship ,Sexual Behavior, Animal ,biology.animal ,Genetics ,Melanogaster ,Animals ,Drosophila Proteins ,Drosophila ,media_common ,Cockroach ,German cockroach ,Courtship behaviour ,biology ,Courtship display ,Base Sequence ,fungi ,Zinc Fingers ,biology.organism_classification ,Biological Evolution ,Blattella germanica ,Drosophila melanogaster ,RNAi ,fruitless ,RNA Interference ,Insect ,Developmental Biology ,Transcription Factors - Abstract
In Drosophila melanogaster, male courtship behaviour is regulated by the fruitless gene. In D. melanogaster, fruitless encodes a set of putative transcription factors that are sex-specifically spliced. Male-specific variants are necessary and sufficient to elicit male courtship behaviour. Fruitless sequences have been reported in other insect species, but there are no data available on their functional role. In the present work, we cloned and sequenced fruitless in males of the German cockroach, Blattella germanica, and we studied its expression in male brain and testes. B. germanica fruitless encodes a 350-amino acid protein with BTB and Zinc finger domains typical of fruitless sequences. Upon RNAi-mediated knockdown of fruitless in B. germanica, males no longer exhibit courtship behaviour, thus implying that fruitless is necessary for male sexual behaviour in our cockroach model. This suggests that the role of fruitless as master regulator of male sexual behaviour has been conserved along insect evolution, at least from cockroaches to flies., Financial support from the Ministry of Education and Science, Spain (projects BFU2008-00484 to M-D. Piulachs and CGL2008-03517/BOS to X. Bellés), Generalitat de Catalunya (2005 SGR 00053) is gratefully acknowledged. L. Ciudad received a pre-doctoral research grant (I3P) from CSIC, and E. Clynen received a travel grant from the Fund for Scientific Research (FWO)-Flanders (Belgium) to work in the Institute of Evolutionary Biology in Barcelona. Thanks are also due to Elena Torres, who received a JAE-intro (CSIC) grant, for helping in the experimental work.
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- 2010
25. Immunomodulatory effect of 5-azacytidine (5-azaC): potential role in the transplantation setting
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Teresa Caballero-Velázquez, Jesús F. San Miguel, Laura Ciudad, José A. Pérez-Simón, Carlos Santamaría, Esteban Ballestar, Luis Ignacio Sánchez-Abarca, Consuelo del Cañizo, Soraya Carrancio, Juan Luis García, Belén Blanco, Carmen Herrero-Sánchez, Teresa Flores, Pilar Hernandez-Campo, Francisco Gonzalez, and Silvia Gutierrez-Cosio
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Male ,Cell Survival ,Immunology ,Azacitidine ,DNA Methyltransferase Inhibitor ,Graft vs Host Disease ,Biology ,Lymphocyte Activation ,Biochemistry ,T-Lymphocytes, Regulatory ,Proinflammatory cytokine ,Mice ,medicine ,Animals ,Humans ,Immunologic Factors ,Transplantation, Homologous ,Lymphocyte Count ,Promoter Regions, Genetic ,Bone Marrow Transplantation ,Cell Proliferation ,Gene Expression Profiling ,Cell Cycle ,Immunity ,FOXP3 ,Forkhead Transcription Factors ,Cell Biology ,Hematology ,Cell cycle ,DNA Methylation ,Survival Analysis ,Transplantation ,Gene Expression Regulation ,Cancer research ,Tumor necrosis factor alpha ,Female ,GADD45B ,Spleen ,medicine.drug - Abstract
Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleoside-based DNA methyltransferase inhibitor that induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in the G0 to G1 phase and decreasing the production of proinflammatory cytokines such as tumor necrosis factor-α and interferon-γ. This effect was not attributable to a proapoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes that induce cell-growth arrest, such as DCUN1D2, U2AF2, GADD45B, or p53. A longer exposure to the drug leads to demethylation of FOXP3 promoter, overexpression of FOXP3, and expansion of regulatory T cells. Finally, the administration of 5-azaC after transplantation prevented the development of graft-versus-host disease, leading to a significant increase in survival in a fully mismatched bone marrow transplantation mouse model. In conclusion, the current study shows the effect of 5-azaC in T lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways that must be explored to prevent graft-versus-host disease.
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- 2009
26. Effect of 5-Azacytidine (5-AzaC) In the Expression of PRAME In Acute Myeloid Leukemia (AML)
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Carlos Santamaría, Laura Ciudad, Jesús F. San Miguel, Silvia Gutiérrez Cosío, Luis Ignacio Sánchez Abarca, Teresa Caballero Velázquez, Belén Blanco, Esteban Ballestar, Jose Antonio Pérez Simón, Soraya Carrancio, Carmen Sánchez, and Consuelo del Cañizo
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PRAME ,Myeloid ,business.industry ,Immunology ,Azacitidine ,CD34 ,Myeloid leukemia ,Cell Biology ,Hematology ,Biochemistry ,PRAME Gene ,Demethylating agent ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,DNA methylation ,Medicine ,business ,medicine.drug - Abstract
Abstract 3615 Introduction: Preferentially expressed antigen of melanoma (PRAME) was first isolated as a human melanoma antigen by cDNA expression cloning using melanoma-reactive cytotoxic Tcells (CTL). PRAME is a tumor associated antigen (TAA) of particular interest since it is widely expressed by lymphoid and myeloid malignancies and solid tumors. Several studies have associated high PRAME RNA levels with good prognosis in acute myeloid leukemia (AML). In addition, several authors have suggested that PRAME could be used as a target for anticancer T-cell therapy. PRAME expression is regulated at the epigenetic level. For this reason inhibitors of DNA methylation, such as 5-azacytidine, can modulate the expression of this TAAs. In the current study we analyzed the effect of 5-azaC on the expression of PRAME in blasts versus CD34+ cells from healthy donors in an attempt to increase its expression, thus inducing a potential target for therapeutic strategies. Methods: We analyzed PRAME mRNA expression of blast cells from AML patients at diagnosis versus CD34+ stem cells from healthy donors by RT-PCR without treatment or after exposure to 1mM 5-azaC during the four days of culture and correlated the expression of PRAME with the methylation status of the promoter. Results: PRAME is significantly over-expressed in blasts from AML patients (n=11) compared with normal CD34+ cells (n=8) ((700±1102 vs. 1.8±2.5 p=0.002). Interestingly, we found an inverse correlation between PRAME expression and the degree of methylation in the promoter among both AML samples and healthy donors (r=-0.77 p=0.010). In order to evaluate the effect of 5-azaC on PRAME gene expression, we treated blast cells and CD34+ cells from healthy donors with the drug and we observed that the exposure to the drug induced a decrease in the percentage of methylation in the promoter and subsequently increased the expression of PRAME but, interestingly, the higher the basal methylation of the promoter the more intense the effect of the drug among AML cells. By contrast, CD34+ cells from healthy donors were resistant to the effect of the drug so that no significant changes were observed neither in terms of methylation status of the promoter nor in the expression of PRAME prior to or after exposure to the drug among healthy donors. Conclusions: The promoter region is highly methylated in normal CD34+ cells compared to AML cells and this pattern correlates with a higher expression of PRAME in blasts. Furthermore, the level of PRAME methylation was reduced in AML patients after exposure to 5-azaC which correlated with an increase in the expression of PRAME. By contrast, the effect of 5-azaC on the methylation pattern of the promoter was significantly lower in CD34+ cells from healthy donors. Disclosures: Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Jangssen-cilag: Membership on an entity's Board of Directors or advisory committees; millennium: Membership on an entity's Board of Directors or advisory committees. Off Label Use: The drug used in this study is the demethylating agent 5-azacytidine (5-azaC) and the purpose is to increase PRAME expression in blasts from AML patients and generate CTL CD8+ specific response against tumor cells.
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- 2010
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27. Effect of Demethylating Agents (5-Azacytidine/5-AzaC) On the Immune Response
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Consuelo del Cañizo, Laura Ciudad, Belén Blanco, Silvia Gutiérrez Cosío, Luis Ignacio Sánchez Abarca, Teresa Caballero Velázquez, Soraya Carrancio, Carlos Santamaría, Carmen Sánchez, Jesús F. San Miguel, Esteban Ballestar, and Jose Antonio Pérez Simón
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Immunology ,Azacitidine ,GATA3 ,FOXP3 ,Promoter ,Cell Biology ,Hematology ,Methylation ,Biology ,Biochemistry ,Demethylating agent ,chemistry.chemical_compound ,Immune system ,chemistry ,medicine ,Epigenetics ,medicine.drug - Abstract
Abstract 2771 Introduction: We have previously shown that 5-azaC inhibits T-cells proliferation and favours the development of Tregs which decreases the risk of GVHD after allogeneic transplantation. This is at least in part due to the effect of the drug on the expression of genes such as FOXP3. Nevertheless, considering the unspecific effect of 5-azaC, it could also favour the overexpression of other genes related to the regulation of the immune response such as TBET, GATA3, IFNg or IL-2, which in turn would favour the development of a Th1, Th2 or Th17 polarization instead of a Treg expansion. In the current study we have evaluated the effect of 5-azaC on these genes in order to know the mechanisms involved in the effect of the drug on the immune response. Methods: We analyzed TBET, GATA3, FOXP3, IFNg and IL-2 mRNA expression of T lymphocytes by RT-PCR after exposure to 1mM 5-azaC during eleven days of culture. These T cells were cultured in medium alone or stimulated with plate-bound anti-CD3 (5 mg/ml) plus soluble anti-CD28 (2.5 mg/ml). Furthermore, we analyzed the methylation status of the promoters of these genes before and after 5-azaC treatment. Results: The expression of TBET, GATA3 and RORγ is not significantly affected by the exposure to the drug whereas the expression of FOXP3 significantly increases along the culture. Regarding IFNg and IL-2 expression no increased expression was observed after exposure to the drug at different time-points along the 11 days of culture. Upon analyzing the mathylation status of the promoter of these genes, we observed that in steady state the promoter of TBET and GATA is demethylated, which is in contrast to FOXP3 promoter. For this reason, the exposure to the drug decreases the methylation status of the promoter of FOXP3 while there is no effect on the promoters of TBET or RORg, thus justifying the absence of effect on the expression of these genes. By contrat, the promoter of both IFNg and IL-2 is methylated prior to the exposure to 5-azaC and it is demethylated after exposure to the drug, which is in contrast to the absence of increased expression of these genes. Accordingly, other mechanisms in addition to the epigenetic regulation of the promoter of IFNg and IL-2 are responsible for their expression in this model. Conclusions: In the current study we show by the first time the effect of 5-azaC on the promoters of genes which regulate the immune response. While no effect was observed for TBET, and GATA3 5-azaC induces a strong demethylation in the promoter of IFN or IL-2. In spite of this effect there is no increase in their expression which could be due to the overexpression of FOXP3 or to additional mechanisms involved in their regulation which are currently being evaluated. Disclosures: Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Jangssen-cilag: Membership on an entity's Board of Directors or advisory committees; millennium: Membership on an entity's Board of Directors or advisory committees. Off Label Use: The drug used in this study is the demethylating agent 5-azacytidine (5-azaC) and the purpose is the inhibition of graft versus host disease.
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- 2010
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28. Structural and RNAi characterization of the German cockroach lipophorin receptor, and the evolutionary relationships of lipoprotein receptors
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Xavier Bellés, Maria-Dolors Piulachs, and Laura Ciudad
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Gene isoform ,Very low-density lipoprotein ,lcsh:QH426-470 ,Gene Expression ,Receptors, Cytoplasmic and Nuclear ,Cockroaches ,Ovary ,Biology ,Evolution, Molecular ,chemistry.chemical_compound ,medicine ,Animals ,lcsh:QH573-671 ,Receptor ,Molecular Biology ,Phylogeny ,Sequence Homology, Amino Acid ,lcsh:Cytology ,lcsh:Genetics ,medicine.anatomical_structure ,chemistry ,Biochemistry ,Low-density lipoprotein ,Juvenile hormone ,LDL receptor ,Insect Proteins ,Female ,RNA Interference ,Vitellogenesis ,Research Article - Abstract
This article is available from: http://www.biomedcentral.com/1471-2199/8/53, [Background] Lipophorin receptors (LpRs) have been described in a number of insects, but functional studies have been reported only in locusts and mosquitoes. The aim of the present work was to characterize the LpR of the cockroach Blattella germanica, not only molecularly but also functionally using RNAi techniques, and to place LpRs in a phylogenetical context among lipoprotein receptors., [Results] We cloned a putative LpR from B. germanica (BgLpR) using RT-PCR methods. Two isoforms of BgLpR that differ from each other by an insertion/deletion of 24 amino acids were obtained from the fat body and the ovary. A phylogenetical analysis of lipoprotein receptors showed that BgLpR grouped with other sequences annotated as LpR in a cluster placed as a sister group of vertebrate low density lipoprotein receptors (LDLR) + lipoprotein receptor-related proteins 8 (LPR8) + vitellogenin receptors (VgR) + very low density lipoprotein receptors (VLDLR). The two BgLpR isoforms are expressed in different adult female tissues (fat body, ovary, brain, midgut, muscle) and in embryos. In ovaries and fat body, the two isoforms are similarly expressed during the first gonadotrophic cycle. mRNA levels in the fat body increase in parallel to vitellogenesis, whereas they decrease in the ovaries. BgLpR protein levels increase in parallel to vitellogenesis in both organs. Treatment with juvenile hormone increases BgLpR protein. RNAi experiments show that females with lower BgLpR expression have less lipophorin in the growing oocytes with respect to controls., [Conclusion] The two isoforms of BgLpR are structurally similar to other LpRs. Phylogenetical analyses show that LpRs and the group formed by vertebrate LDLR + LPR8 + VgR + VLDLR, diverged from a common ancestor and diversified in parallel. The different expression patterns in the fat body and the ovary, comparing mRNA and protein, indicate that the corresponding mechanisms regulating BgLpR expression are different. Experiments with JH III suggest that such a hormone regulates the expression of BgLpR posttranscriptionally. RNAi experiments indicate that BgLpR is a functional lipophorin receptor., Financial support from the Ministry of Education and Science, Spain (projects BOS2002-03359, BFU2005-00264, AGL2002-01169, AGL2005- 00773) and the Generalitat de Catalunya (2001 SGR 003245) is gratefully acknowledged. L.C. is recipient of pre-doctoral research grants (I3P) from CSIC. We thank Prof. Coby Schal (North Carolina State University) for generous gifts of anti-lipophorin antibody.
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- 2007
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29. Epstein–Barr virus-mediated transformation of B cells induces global chromatin changes independent to the acquisition of proliferation
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Abul B. M. M. K. Islam, Axel Imhof, Esteban Ballestar, Javier Rodríguez-Ubreva, Laura Ciudad, Claire Shannon-Lowe, Ignasi Forné, and Henar Hernando
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Herpesvirus 4, Human ,Herpesviruses ,Heterochromatin ,Cèl·lules B ,Gene Regulation, Chromatin and Epigenetics ,medicine.disease_cause ,Histones ,Transformation, Genetic ,Genetics ,medicine ,Humans ,Constitutive heterochromatin ,Cell Proliferation ,B-Lymphocytes ,B cells ,CD40 ,biology ,Cell growth ,Herpesvirus ,Epstein–Barr virus ,Molecular biology ,Chromatin ,Histone ,biology.protein ,Deoxyribonuclease I - Abstract
Epstein-Barr virus (EBV) infects and transforms human primary B cells inducing indefinite proliferation. To investigate the potential participation of chromatin mechanisms during the EBV-mediated transformation of resting B cells we performed an analysis of global changes in histone modifications. We observed a remarkable decrease and redistribution of heterochromatin marks including H4K20me3, H3K27me3 and H3K9me3. Loss of H4K20me3 and H3K9me3 occurred at constitutive heterochromatin repeats. For H3K27me3 and H3K9me3, comparison of ChIP-seq data revealed a decrease in these marks in thousands of genes, including clusters of HOX and ZNF genes, respectively. Moreover, DNase-seq data comparison between resting and EBV-transformed B cells revealed increased endonuclease accessibility in thousands of genomic sites. We observed that both loss of H3K27me3 and increased accessibility are associated with transcriptional activation. These changes only occurred in B cells transformed with EBV and not in those stimulated to proliferate with CD40L/IL-4, despite their similarities in the cell pathways involved and proliferation rates. In fact, B cells infected with EBNA-2 deficient EBV, which have much lower proliferation rates, displayed similar decreases for heterochromatic histone marks. Our study describes a novel phenomenon related to transformation of B cells, and highlights its independence of the pure acquisition of proliferation.
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