Browsing by MeSH term "Chromatin Immunoprecipitation"
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Publication A molecular role for lysyl oxidase-like 2 enzyme in snail regulation and tumor progression.(EMBO Press, 2005-10-05) Peinado, Héctor; Del Carmen Iglesias-de la Cruz, Maria; Olmeda, David; Csiszar, Katalin; Fong, Keith S K; Vega, Sonia; Nieto, Maria Angela; Cano, Amparo; Portillo, FranciscoThe transcription factor Snail controls epithelial-mesenchymal transitions (EMT) by repressing E-cadherin expression and other epithelial genes. However, the mechanisms involved in the regulation of Snail function are not fully understood. Here we show that lysyl-oxidase-like 2 and 3 (LOXL2 and LOXL3), two members of the lysyl-oxidase gene family, interact and cooperate with Snail to downregulate E-cadherin expression. Snail's lysine residues 98 and 137 are essential for Snail stability, functional cooperation with LOXL2/3 and induction of EMT. Overexpression of LOXL2 or LOXL3 in epithelial cells induces an EMT process, supporting their implication in tumor progression. The biological importance of LOXL2 is further supported by RNA interference of LOXL2 in Snail-expressing metastatic carcinoma cells, which led to a strong decrease of tumor growth associated to increased apoptosis and reduced expression of mesenchymal and invasive/angiogenic markers. Taken together, these results establish a direct link between LOXL2 and Snail in carcinoma progression.Publication Analysis of URI nuclear interaction with RPB5 and components of the R2TP/prefoldin-like complex.(Public Library of Science (PLOS), 2013-05-08) Mita, Paolo; Savas, Jeffrey N; Ha, Susan; Djouder, Nabil; Yates, John R; Logan, Susan K; NIH - National Cancer Institute (NCI) (Estados Unidos); Urology department at New York University School of MedicineUnconventional prefoldin RPB5 Interactor (URI) was identified as a transcriptional repressor that binds RNA polymerase II (pol II) through interaction with the RPB5/POLR2E subunit. Despite the fact that many other proteins involved in transcription regulation have been shown to interact with URI, its nuclear function still remains elusive. Previous mass spectrometry analyses reported that URI is part of a novel protein complex called R2TP/prefoldin-like complex responsible for the cytoplasmic assembly of RNA polymerase II. We performed a mass spectrometry (MS)-based proteomic analysis to identify nuclear proteins interacting with URI in prostate cells. We identified all the components of the R2TP/prefoldin-like complex as nuclear URI interactors and we showed that URI binds and regulates RPB5 protein stability and transcription. Moreover, we validated the interaction of URI to the P53 and DNA damage-Regulated Gene 1 (PDRG1) and show that PDRG1 protein is also stabilized by URI binding. We present data demonstrating that URI nuclear/cytoplasmic shuttling is affected by compounds that stall pol II on the DNA (α-amanitin and actinomycin-D) and by leptomycin B, an inhibitor of the CRM1 exportin that mediates the nuclear export of pol II subunits. These data suggest that URI, and probably the entire R2TP/prefoldin-like complex is exported from the nucleus through CRM1. Finally we identified putative URI sites of phosphorylation and acetylation and confirmed URI sites of post-transcriptional modification identified in previous large-scale analyses the importance of which is largely unknown. However URI post-transcriptional modification was shown to be essential for URI function and therefore characterization of novel sites of URI modification will be important to the understanding of URI function.Publication Chromatin immunoprecipitation improvements for the processing of small frozen pieces of adipose tissue.(2018-02-14) Castellano-Castillo, Daniel; Denechaud, Pierre-Damien; Moreno-Indias, Isabel; Tinahones, Francisco; Fajas, Lluis; Queipo-Ortuño, María Isabel; Cardona, FernandoChromatin immunoprecipitation (ChIP) has gained importance to identify links between the genome and the proteome. Adipose tissue has emerged as an active tissue, which secretes a wide range of molecules that have been related to metabolic and obesity-related disorders, such as diabetes, cardiovascular failure, metabolic syndrome, or cancer. In turn, epigenetics has raised the importance in discerning the possible relationship between metabolic disorders, lifestyle and environment. However, ChIP application in human adipose tissue is limited by several factors, such as sample size, frozen sample availability, high lipid content and cellular composition of the tissue. Here, we optimize the standard protocol of ChIP for small pieces of frozen human adipose tissue. In addition, we test ChIP for the histone mark H3K4m3, which is related to active promoters, and validate the performance of the ChIP by analyzing gene promoters for factors usually studied in adipose tissue using qPCR. Our improvements result in a higher performance in chromatin shearing and DNA recovery of adipocytes from the tissue, which may be useful for ChIP-qPCR or ChIP-seq analysis.Publication CSF1R Protein Expression in Reactive Lymphoid Tissues and Lymphoma: Its Relevance in Classical Hodgkin Lymphoma.(Public Library of Science (PLOS), 2015-06-12) Martín-Moreno, Ana M; Mata, Elena; Jiménez, Scherezade; Reyes-García, Ana I; Rubio, Carmen; Tomás, José F; Estévez, Mónica; Pulford, Karen; Piris, Miguel A; García, Juan F; Roncador, Giovanna; Maestre, Maestre L; Martinez Torrecuadrada, Jorge Luis; Asociación Española Contra el Cáncer; Unión Europea; Ministerio de Ciencia e Innovación (España)Tumour-associated macrophages (TAMs) have been associated with survival in classic Hodgkin lymphoma (cHL) and other lymphoma types. The maturation and differentiation of tissue macrophages depends upon interactions between colony-stimulating factor 1 receptor (CSF1R) and its ligands. There remains, however, a lack of consistent information on CSF1R expression in TAMs. A new monoclonal antibody, FER216, was generated to investigate CSF1R protein distribution in formalin fixed tissue samples from 24 reactive lymphoid tissues and 187 different lymphoma types. We also analysed the distribution of CSF1R+, CD68+ and CD163+ macrophages by double immunostaining, and studied the relationship between CSF1R expression and survival in an independent series of 249 cHL patients. CSF1R+ TAMs were less frequent in B-cell lymphocytic leukaemia and lymphoblastic B-cell lymphoma than in diffuse large B-cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma and cHL. HRS cells in cHL and, with the exception of three cases of anaplastic large cell lymphoma, the neoplastic cells in NHLs, lacked detectable CSF1R protein. A CSF1R+ enriched microenvironment in cHL was associated with shorter survival in an independent series of 249 cHL patients. CSF1R pathway activation was evident in the cHL and inactivation of this pathway could be a potential therapeutic target in cHL cases.Publication Epigenetic silencing of Oct4 by a complex containing SUV39H1 and Oct4 pseudogene lncRNA(Nature Publishing Group, 2015-07-09) Scarola, Michele; Comisso, Elisa; Pascolo, Rhena; Chiaradia, Riccardo; Marion, RM; Schneider, Claudio; Blasco, MA; Schoeftner, Stefan; Benetti, Roberta; Italian Association for Cancer Research; Fondazione Umberto Veronesi; Ministero dell Istruzione, dell Universita e della Ricerca (Italia)Pseudogene-derived, long non-coding RNAs (lncRNAs) act as epigenetic regulators of gene expression. Here we present a panel of new mouse Oct4 pseudogenes and demonstrate that the X-linked Oct4 pseudogene Oct4P4 critically impacts mouse embryonic stem cells (mESCs) self-renewal. Sense Oct4P4 transcription produces a spliced, nuclear-restricted lncRNA that is efficiently upregulated during mESC differentiation. Oct4P4 lncRNA forms a complex with the SUV39H1 HMTase to direct the imposition of H3K9me3 and HP1α to the promoter of the ancestral Oct4 gene, located on chromosome 17, leading to gene silencing and reduced mESC self-renewal. Targeting Oct4P4 expression in primary mouse embryonic fibroblasts causes the re-acquisition of self-renewing features of mESC. We demonstrate that Oct4P4 lncRNA plays an important role in inducing and maintaining silencing of the ancestral Oct4 gene in differentiating mESCs. Our data introduces a sense pseudogene-lncRNA-based mechanism of epigenetic gene regulation that controls the cross-talk between pseudogenes and their ancestral genes.Publication Genome Profiling of H3k4me3 Histone Modification in Human Adipose Tissue during Obesity and Insulin Resistance(Multidisciplinary Digital Publishing Institute (MDPI), 2021-09-30) Castellano-Castillo, Daniel; Ramos-Molina, Bruno; Oliva-Olivera, Wilfredo; Ocaña-Wilhelmi, Luis; Queipo-Ortuño, María Isabel; Cardona, Fernando; [Castellano-Castillo,D; Oliva-Olivera,W] Instituto de Investigación Biomédica de Málaga, Universidad de Málaga, Málaga, Spain. [Ramos-Molina,B] Grupo de Obesidad y Metabolismo, Instituto Murciano de Investigación Biosanitaria (IMIB-Arrixaca), Murcia, Spain. [Ocaña-Wilhelmi,L] Unidad de Cirugía Metabólica, Hospital Clínico Virgen de la Victoria, Málaga, Spain. [Queipo-Ortuño,MI] Unidad de Gestión Clínica Intercentros de Oncología Médica, Hospitales Universitarios Regional y Virgen de la Victoria, Instituto de Investigación Biomédica de Málaga (IBIMA)-CIMES-UMA, Málaga, Spain. [Cardona,F] UGC Pediatría, Instituto de Investigación Biomédica de Málaga, Hospital Regional de Málaga, Málaga, Spain. [Cardona,F] Department of Surgical Specialties, Biochemistry and Immunology School of Medicine, University of Malaga, Málaga, Spain.Background: Adipose tissue (AT) dysfunction is involved in obesity-related comorbidities. Epigenetic alterations have been recently associated with AT deterioration in obesity conditions.In this work, we profiled the H3K4me3 histone mark in human AT, with special emphasis on the changes in the pattern of histone modification in obesity and insulin resistance (IR). Visceral AT (VAT) was collected and subjected to chromatin immunoprecipitation (ChIP) using anti-H3K4me3 antibody and then sequenced to obtain the H3K4me3 genome profile. Results: We found that most of the H3K4me3 enriched regions were located in gene promoters of pathways related to AT biology and function. H3K4me3 enrichment at gene promoters was strongly related to higher mRNA levels. Differentially expressed genes in AT of patients classified as non-obese, obese with low IR, and obese with high IR could be regulated by differentially enriched H3K4me3; these genes encoded for pathways that could in part explain AT functioning during obesity and insulin resistance (e.g., extracellular matrix organization, PPARG signaling or inflammation). Conclusions: In conclusion, we emphasize the importance of the epigenetic mark H3K4me3 in VAT dysfunction in obesity and IR. The understanding of such mechanisms could give rise to the development of new epigenetic-based pharmacological strategies to ameliorate obesity-related comorbiditiesPublication MCRS1 binds and couples Rheb to amino acid-dependent mTORC1 activation.(Elsevier, 2015-04-06) Fawal, Mohamad-Ali; Brandt, Marta; Djouder, Nabil; Fundación Caja Navarra; Fundación La Caixa; Ministerio de Ciencia e Innovación. Centro de Excelencia Severo Ochoa (España)Ras homolog enriched in brain (Rheb) is critical for mechanistic target of rapamycin complex 1 (mTORC1) activation in response to growth factors and amino acids (AAs). Whereas growth factors inhibit the tuberous sclerosis complex (TSC1-TSC2), a negative Rheb regulator, the role of AAs in Rheb activation remains unknown. Here, we identify microspherule protein 1 (MCRS1) as the essential link between Rheb and mTORC1 activation. MCRS1, in an AA-dependent manner, maintains Rheb at lysosome surfaces, connecting Rheb to mTORC1. MCRS1 suppression in human cancer cells using small interference RNA or mouse embryonic fibroblasts using an inducible-Cre/Lox system reduces mTORC1 activity. MCRS1 depletion promotes Rheb/TSC2 interaction, rendering Rheb inactive and delocalizing it from lysosomes to recycling endocytic vesicles, leading to mTORC1 inactivation. These findings have important implications for signaling mechanisms in various pathologies, including diabetes mellitus and cancer.Publication Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells(Nature Publishing Group, 2015-01-06) The, Inge; Ruijtenberg, Suzan; Bouchet, Benjamin P; Cristobal, Alba; Prinsen, Martine B W; van Mourik, Tim; Koreth, John; Xu, Huihong; Heck, Albert J R; Akhmanova, Anna; Cuppen, Edwin; Boxem, Mike; Muñoz, Javier; van den Heuvel, Sander; NIH - Office of Research Infrastructure Programs; Unión Europea; Dutch Research Council (Holanda)Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/C(FZR1) activity as an important determinant in response to CDK4/6-inhibitors.Publication Regulation of androgen receptor-mediated transcription by RPB5 binding protein URI/RMP.(Taylor & Francis, 2011-09) Mita, Paolo; Savas, Jeffrey N; Djouder, Nabil; Yates, John R; Ha, Susan; Ruoff, Rachel; Schafler, Eric D; Nwachukwu, Jerome C; Tanese, Naoko; Cowan, Nicholas J; Zavadil, Jiri; Garabedian, Michael J; Logan, Susan K; NIH - National Cancer Institute (NCI) (Estados Unidos); Urologic Disease Center of Excellence at the New York University School of MedicineAndrogen receptor (AR)-mediated transcription is modulated by interaction with coregulatory proteins. We demonstrate that the unconventional prefoldin RPB5 interactor (URI) is a new regulator of AR transcription and is critical for antagonist (bicalutamide) action. URI is phosphorylated upon androgen treatment, suggesting communication between the URI and AR signaling pathways. Whereas depletion of URI enhances AR-mediated gene transcription, overexpression of URI suppresses AR transcriptional activation and anchorage-independent prostate cancer cell growth. Repression of AR-mediated transcription is achieved, in part, by URI binding and regulation of androgen receptor trapped clone 27 (Art-27), a previously characterized AR corepressor. Consistent with this idea, genome-wide expression profiling in prostate cancer cells upon depletion of URI or Art-27 reveals substantially overlapping patterns of gene expression. Further, depletion of URI increases the expression of the AR target gene NKX-3.1, decreases the recruitment of Art-27, and increases AR occupancy at the NKX-3.1 promoter. While Art-27 can bind AR directly, URI is bound to chromatin prior to hormone-dependent recruitment of AR, suggesting a role for URI in modulating AR recruitment to target genes.Publication Regulation of HIV-1 env mRNA translation by Rev protein(Elsevier, 2005-03-22) Perales, Celia; Carrasco, Luis; Gonzalez Portal, Maria Eugenia; Fundación para la Investigación y la Prevención del Sida en España; Instituto de Salud Carlos III; Dirección General de Investigación Científica y Técnica (España)We have examined the effect of Rev on the regulation of the expression of RRE containing mRNAs when they were synthesised in the nucleus or directly in the cytoplasm. In the nuclear expression system, Rev enhanced env mRNA transport by about 1.6-fold, while translation of this mRNA was increased more than a 100-fold. These findings indicate that the target of Rev activity is located mainly at the translational level. Synthesis of Env using a recombinant vaccinia virus system, which synthesised env mRNA directly in the cytoplasm, is also enhanced by Rev. Finally, RRE functioning was examined using a luciferase mRNA bearing this element. Rev stimulated the synthesis of Luciferase both when the luc mRNA was made in the nucleus or in cytoplasm. Our results indicate that the effect of Rev on env mRNA transport is low compared with the enhancement of translation of this mRNA.Publication Systems analysis identifies melanoma-enriched pro-oncogenic networks controlled by the RNA binding protein CELF1(Nature Publishing Group, 2017) Cifdaloz, Metehan; Osterloh, Lisa; Graña Castro, Osvaldo; Riveiro-Falkenbach, Erica; Ximenez-Embun, Pilar; Muoz Peralta, Javier; Tejedo, Cristina; Calvo, Tonantzin G; Karras, Panagiotis; Olmeda, David; Miñana, Belén; Gómez-López, Gonzalo; Cañón, Estela; Eyras, Eduardo; Guo, Haihong; Kappes, Ferdinand; Ortiz-Romero, Pablo L; Rodríguez-Peralto, Jose L; Megias Vazquez, Diego; Valcárcel, Juan; Soengas, MS; Ministerio de Ciencia e Innovación (España); Botín Foundation; Unión Europea. Comisión Europea. European Research Council (ERC); Ministerio de Sanidad y Consumo (España); Fundación La Caixa; Fundación La Marató TV3; Asociación Española Contra el Cáncer; Fundación Mutua MadrileñaMelanomas are well-known for their altered mRNA expression profiles. Yet, the specific contribution of mRNA binding proteins (mRBPs) to melanoma development remains unclear. Here we identify a cluster of melanoma-enriched genes under the control of CUGBP Elav-like family member 1 (CELF1). CELF1 was discovered with a distinct prognostic value in melanoma after mining the genomic landscape of the 692 known mRBPs across different cancer types. Genome-wide transcriptomic, proteomic, and RNA-immunoprecipitation studies, together with loss-of-function analyses in cell lines, and histopathological evaluation in clinical biopsies, revealed an intricate repertoire of CELF1-RNA interactors with minimal overlap with other malignancies. This systems approach uncovered the oncogene DEK as an unexpected target and downstream effector of CELF1. Importantly, CELF1 and DEK were found to represent early-induced melanoma genes and adverse indicators of overall patient survival. These results underscore novel roles of CELF1 in melanoma, illustrating tumor type-restricted functions of RBPs in cancer.Publication The proto-oncogene c-myc regulates antibody secretion and Ig class switch recombination.(American Association of Immunologists (AAI), 2013-06-15) Fernández, David; Ortiz, Maitane; Rodríguez, Lorena; García, Arancha; Martinez Garcia, Maria Dolores; Moreno de Alborán, Ignacio; Ministerio de Ciencia e Innovación (España); Unión Europea. Fondo Social Europeo (ESF/FSE); Comunidad de Madrid (España)The immune response involves the generation of Ab-secreting cells and memory B cells through a process called terminal B lymphocyte differentiation. This program requires the transcriptional repressor Blimp-1, which inhibits c-myc expression and terminates proliferation. Although the role of c-Myc in cell proliferation is well characterized, it is not known whether it has other functions in terminal differentiation. In this study, we show that c-Myc not only regulates cell proliferation, but it is also essential for Ab-secreting cell function and differentiation in vivo. c-Myc-deficient B lymphocytes hypersecrete IgM and do not undergo Ig class switch recombination (CSR). CSR has been previously linked to proliferation, and in this study we mechanistically link class switching and proliferation via c-Myc. We observed that c-Myc regulates CSR by transcriptionally activating the B cell-specific factor activation-induced cytidine deaminase. By linking cell proliferation and CSR, c-Myc is thus a critical component for a potent immune response.