RESUMO
How cells respond to different external cues to develop along defined cell lineages to form complex tissues is a major question in systems biology. Here, we investigated the potential of retinoic acid receptor (RAR)-selective synthetic agonists to activate the gene regulatory programs driving cell specialization during nervous tissue formation from embryonic carcinoma (P19) and mouse embryonic (E14) stem cells. Specifically, we found that the synergistic activation of the RARß and RARγ by selective ligands (BMS641 or BMS961) induces cell maturation to specialized neuronal subtypes, and to astrocytes and oligodendrocyte precursors. Using RAR isotype knockout lines exposed to RAR-specific agonists, interrogated by global transcriptome landscaping and in silico modeling of transcription regulatory signal propagation, revealed major RARα-driven gene programs essential for optimal neuronal cell specialization and hijacked by the synergistic activation of the RARß and RARγ receptors. Overall, this study provides a systems biology view of the gene programs accounting for the previously observed redundancy between RARs, paving the way toward their potential use for directing cell specialization during nervous tissue formation.
Assuntos
Diferenciação Celular , Receptores do Ácido Retinoico , Células-Tronco , Animais , Camundongos , Diferenciação Celular/genética , Linhagem da Célula/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Ácido Retinoico/genética , Células-Tronco/fisiologia , Receptor gama de Ácido RetinoicoRESUMO
Cancer genetics has led to major discoveries, including protooncogene and tumor-suppressor concepts, and cancer genomics generated concepts like driver and passenger genes, revealed tumor heterogeneity and clonal evolution. Reconstructing trajectories of tumorigenesis using spatial and single-cell genomics is possible. Patient stratification and prognostic parameters have been improved. Yet, despite these advances, successful translation into targeted therapies has been scarce and mostly limited to kinase inhibitors. Here, we argue that current cancer research may be on the wrong track, by considering cancer more as a "monogenic" disease, trying to extract common information from thousands of patients, while not properly considering complexity and individual diversity. We suggest to empower a systems cancer approach which reconstructs the information network that has been altered by the tumorigenic events, to analyze hierarchies and predict (druggable) key nodes that could interfere with/block the aberrant information transfer. We also argue that the interindividual variability between patients of similar cohorts is too high to extract common polygenic network information from large numbers of patients and argue in favor of an individualized approach. The analysis we propose would require a structured multinational and multidisciplinary effort, in which clinicians, and cancer, developmental, cell and computational biologists together with mathematicians and informaticians develop dynamic regulatory networks which integrate the entire information transfer in and between cells and organs in (patho)physiological conditions, revealing hierarchies and available drugs to interfere with key regulators. Based on this blueprint, the altered information transfer in individual cancers could be modeled and possible targeted (combo)therapies proposed.
Assuntos
Genômica , Neoplasias , Carcinogênese , Humanos , Neoplasias/genética , PrognósticoRESUMO
Prostate cancer (PrCa) is the second most common malignancy in men. More than 50% of advanced prostate cancers display the TMPRSS2-ERG fusion. Despite extensive cancer genome/transcriptome data, little is known about the impact of mutations and altered transcription on regulatory networks in the PrCa of individual patients. Using patient-matched normal and tumor samples, we established somatic variations and differential transcriptome profiles of primary ERG-positive prostate cancers. Integration of protein-protein interaction and gene-regulatory network databases defined highly diverse patient-specific network alterations. Different components of a given regulatory pathway were altered by novel and known mutations and/or aberrant gene expression, including deregulated ERG targets, and were validated by using a novel in silico methodology. Consequently, different sets of pathways were altered in each individual PrCa. In a given PrCa, several deregulated pathways share common factors, predicting synergistic effects on cancer progression. Our integrated analysis provides a paradigm to identify druggable key deregulated factors within regulatory networks to guide personalized therapies.
Assuntos
Antineoplásicos/farmacologia , Epigênese Genética/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Metilação de DNA/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Neoplasias/genéticaRESUMO
Epigenetic deregulation of gene transcription is central to cancer cell plasticity and malignant progression but remains poorly understood. We found that the uncharacterized epigenetic factor chromodomain on Y-like 2 (CDYL2) is commonly over-expressed in breast cancer, and that high CDYL2 levels correlate with poor prognosis. Supporting a functional role for CDYL2 in malignancy, it positively regulated breast cancer cell migration, invasion, stem-like phenotypes, and epithelial-to-mesenchymal transition. CDYL2 regulation of these plasticity-associated processes depended on signaling via p65/NF-κB and STAT3. This, in turn, was downstream of CDYL2 regulation of MIR124 gene transcription. CDYL2 co-immunoprecipitated with G9a/EHMT2 and GLP/EHMT1 and regulated the chromatin enrichment of G9a and EZH2 at MIR124 genes. We propose that CDYL2 contributes to poor prognosis in breast cancer by recruiting G9a and EZH2 to epigenetically repress MIR124 genes, thereby promoting NF-κB and STAT3 signaling, as well as downstream cancer cell plasticity and malignant progression.
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The enormous amount of freely accessible functional genomics data is an invaluable resource for interrogating the biological function of multiple DNA-interacting players and chromatin modifications by large-scale comparative analyses. However, in practice, interrogating large collections of public data requires major efforts for (i) reprocessing available raw reads, (ii) incorporating quality assessments to exclude artefactual and low-quality data, and (iii) processing data by using high-performance computation. Here, we present qcGenomics, a user-friendly online resource for ultrafast retrieval, visualization, and comparative analysis of tens of thousands of genomics datasets to gain new functional insight from global or focused multidimensional data integration.
Assuntos
Visualização de Dados , Processamento Eletrônico de Dados/métodos , Genômica/métodos , Armazenamento e Recuperação da Informação/métodos , Montagem e Desmontagem da Cromatina/genética , Bases de Dados Genéticas , Código das Histonas/genética , Histonas/genética , Humanos , Células MCF-7 , Software , Fluxo de TrabalhoRESUMO
The acetyltransferases CBP and P300 have been implicated in myogenesis in mouse immortalized cell lines but these studies focused only on the expression of a handful of myogenic factors. Hence, the respective role of these two related cofactors and their impact at global scale on gene expression rewiring during primary myoblast differentiation remain unknown. Here, we characterised the gene networks regulated by these two epigenetic enzymes during human primary myoblast differentiation (HPM). We found that CBP and p300 play a critical role in the activation of the myogenic program and mostly regulate distinct gene sets to control several aspects of HPM biology, even though they also exhibit some degree of redundancy. Moreover, CBP or P300 knockdown strongly impaired muscle cell adhesion and resulted in the activation of inflammation markers, two hallmarks of dystrophic disease. This was further validated in zebrafish where inhibition of CBP and P300 enzymatic activities led to cell adhesion defects and muscle fiber detachment. Our data highlight an unforeseen link between CBP/P300 activity and the emergence of dystrophic phenotypes. They thereby identify CBP and P300 as mediators of adult muscle integrity and suggest a new lead for intervention in muscular dystrophy.
Assuntos
Proteína p300 Associada a E1A/genética , Redes Reguladoras de Genes , Mioblastos/fisiologia , Fragmentos de Peptídeos/genética , Sialoglicoproteínas/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Proteína p300 Associada a E1A/metabolismo , Humanos , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Mioblastos/citologia , Mioblastos/metabolismo , Fragmentos de Peptídeos/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Sialoglicoproteínas/metabolismo , Transativadores/metabolismo , Transcrição Gênica , Ativação Transcricional , Peixe-ZebraRESUMO
Complex organisms originate from and are maintained by the information encoded in the genome. A major challenge of systems biology is to develop algorithms that describe the dynamic regulation of genome functions from large omics datasets. Here, we describe TETRAMER, which reconstructs gene-regulatory networks from temporal transcriptome data during cell fate transitions to predict "master" regulators by simulating cascades of temporal transcription-regulatory events.
RESUMO
Herein, we report the rational design, synthesis and biological evaluation of conjugates consisting of the synthetic retinoid Am580 and biotin connected via a linker moiety. We found that the linking substructure between the retinoid part and the biotin part is critical for retaining the biological activity. Conjugate 4 with a shorter linker showed similar potency to endogenous retinoid ATRA (1) and the parent compound Am580 (2) for neural differentiation of mouse embryotic carcinoma P19 cells, and showed the same pattern of induction of gene expression. It is expected to be useful as a probe for investigations of retinoid function. The design rationale and structure-activity relationship of the linker moiety are expected to be helpful for developing biotin conjugates of other nuclear receptor ligands.
Assuntos
Biotina/química , Sondas Moleculares/química , Retinoides/análise , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ligantes , Camundongos , Modelos Moleculares , Sondas Moleculares/síntese química , Estrutura Molecular , Neurônios/efeitos dos fármacos , Neurônios/patologia , RNA Mensageiro/genética , Retinoides/metabolismo , Relação Estrutura-AtividadeRESUMO
Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16INK4a, p21CIP1 and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed 'stemness'). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eµ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of cancer cells.
Assuntos
Reprogramação Celular , Senescência Celular , Linfoma de Células B/patologia , Células-Tronco Neoplásicas/patologia , Animais , Biomarcadores/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Células Clonais/efeitos dos fármacos , Células Clonais/patologia , Feminino , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/genética , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Fenótipo , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Besides its tumor-selective apoptotic activity, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) promotes pro-survival, proliferative or migratory signaling (NF-κB, PI3K/Akt, MAPK and JNK; referred to as 'non-apoptotic' cascades). Indeed, apoptosis and non-apoptotic signaling can be activated in clonal populations of cancer cells in response to treatment and, as a result, only a part of the initial cellular population dies while a fraction survives and develops resistance to TRAIL-induced apoptosis (referred to as 'fractional survival'). Notably, the molecular characterization of the protein platforms streaming into tumoricidal versus tumor-promoting cascades that control fractional survival remained elusive. Here we demonstrate that, in the context of DR4-DR5-DcR2 hetero-oligomeric complexes, a single death receptor (DR5) suffices to assemble composite plasma membrane-proximal pro-apoptotic/pro-survival platforms that propagate TRAIL signaling to both death and survival pathways in clonal populations of cancer cells. Moreover, we show that while all members of TRAIL-induced complexes support survival, none of them acted exclusively pro-apoptotic. Indeed, key apoptotic proteins as FADD and procaspase-8 were also involved in transducing non-apoptotic signaling in response to this cytokine. Collectively, this study reveals the Janus faces of DR5, and the contributions of other death complex components in fractional survival that foster the generation of resistance. Our data highlight a new level of complexity in TRAIL signaling and point to an improved therapeutic rationale in view of hitherto disappointing results.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Fibroblastos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/genética , Receptores Chamariz do Fator de Necrose Tumoral/metabolismoRESUMO
BACKGROUND: Exponentially increasing numbers of NGS-based epigenomic datasets in public repositories like GEO constitute an enormous source of information that is invaluable for integrative and comparative studies of gene regulatory mechanisms. One of today's challenges for such studies is to identify functionally informative local and global patterns of chromatin states in order to describe the regulatory impact of the epigenome in normal cell physiology and in case of pathological aberrations. Critically, the most preferred Chromatin ImmunoPrecipitation-Sequencing (ChIP-Seq) is inherently prone to significant variability between assays, which poses significant challenge on comparative studies. One challenge concerns data normalization to adjust sequencing depth variation. RESULTS: Currently existing tools either apply linear scaling corrections and/or are restricted to specific genomic regions, which can be prone to biases. To overcome these restrictions without any external biases, we developed Epimetheus, a genome-wide quantile-based multi-profile normalization tool for histone modification data and related datasets. CONCLUSIONS: Epimetheus has been successfully used to normalize epigenomics data in previous studies on X inactivation in breast cancer and in integrative studies of neuronal cell fate acquisition and tumorigenic transformation; Epimetheus is freely available to the scientific community.
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Epigenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Células Hep G2 , Histonas/genética , Histonas/metabolismo , Humanos , Tretinoína/farmacologiaRESUMO
The KDM5 family of histone demethylases removes the H3K4 tri-methylation (H3K4me3) mark frequently found at promoter regions of actively transcribed genes and is therefore generally considered to contribute to corepression. In this study, we show that knockdown (KD) of all expressed members of the KDM5 family in white and brown preadipocytes leads to deregulated gene expression and blocks differentiation to mature adipocytes. KDM5 KD leads to a considerable increase in H3K4me3 at promoter regions; however, these changes in H3K4me3 have a limited effect on gene expression per se. By contrast, genome-wide analyses demonstrate that KDM5A is strongly enriched at KDM5-activated promoters, which generally have high levels of H3K4me3 and are associated with highly expressed genes. We show that KDM5-activated genes include a large set of cell cycle regulators and that the KDM5s are necessary for mitotic clonal expansion in 3T3-L1 cells, indicating that KDM5 KD may interfere with differentiation in part by impairing proliferation. Notably, the demethylase activity of KDM5A is required for activation of at least a subset of pro-proliferative cell cycle genes. In conclusion, the KDM5 family acts as dual modulators of gene expression in preadipocytes and is required for early stage differentiation and activation of pro-proliferative cell cycle genes.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Ciclo Celular/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica , Histona Desmetilases/genética , Família Multigênica , Adipogenia/genética , Animais , Linhagem Celular , Proliferação de Células , Ativação Enzimática , Histona Desmetilases/metabolismo , Histonas/metabolismo , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Cell lineages, which shape the body architecture and specify cell functions, derive from the integration of a plethora of cell intrinsic and extrinsic signals. These signals trigger a multiplicity of decisions at several levels to modulate the activity of dynamic gene regulatory networks (GRNs), which ensure both general and cell-specific functions within a given lineage, thereby establishing cell fates. Significant knowledge about these events and the involved key drivers comes from homogeneous cell differentiation models. Even a single chemical trigger, such as the morphogen all-trans retinoic acid (RA), can induce the complex network of gene-regulatory decisions that matures a stem/precursor cell to a particular step within a given lineage. Here we have dissected the GRNs involved in the RA-induced neuronal or endodermal cell fate specification by integrating dynamic RXRA binding, chromatin accessibility, epigenetic promoter epigenetic status, and the transcriptional activity inferred from RNA polymerase II mapping and transcription profiling. Our data reveal how RA induces a network of transcription factors (TFs), which direct the temporal organization of cognate GRNs, thereby driving neuronal/endodermal cell fate specification. Modeling signal transduction propagation using the reconstructed GRNs indicated critical TFs for neuronal cell fate specification, which were confirmed by CRISPR/Cas9-mediated genome editing. Overall, this study demonstrates that a systems view of cell fate specification combined with computational signal transduction models provides the necessary insight in cellular plasticity for cell fate engineering. The present integrated approach can be used to monitor the in vitro capacity of (engineered) cells/tissues to establish cell lineages for regenerative medicine.
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Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Neurogênese , Animais , Linhagem Celular Tumoral , Linhagem da Célula , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Endoderma/citologia , Epigênese Genética , Camundongos , Ativação Transcricional , Tretinoína/farmacologiaRESUMO
We have established a certification system for antibodies to be used in chromatin immunoprecipitation assays coupled to massive parallel sequencing (ChIP-seq). This certification comprises a standardized ChIP procedure and the attribution of a numerical quality control indicator (QCi) to biological replicate experiments. The QCi computation is based on a universally applicable quality assessment that quantitates the global deviation of randomly sampled subsets of ChIP-seq dataset with the original genome-aligned sequence reads. Comparison with a QCi database for >28,000 ChIP-seq assays were used to attribute quality grades (ranging from 'AAA' to 'DDD') to a given dataset. In the present report we used the numerical QC system to assess the factors influencing the quality of ChIP-seq assays, including the nature of the target, the sequencing depth and the commercial source of the antibody. We have used this approach specifically to certify mono and polyclonal antibodies obtained from Active Motif directed against the histone modification marks H3K4me3, H3K27ac and H3K9ac for ChIP-seq. The antibodies received the grades AAA to BBC ( www.ngs-qc.org). We propose to attribute such quantitative grading of all antibodies attributed with the label "ChIP-seq grade".
RESUMO
BACKGROUND: Alterations in genetic and epigenetic landscapes are known to contribute to the development of different types of cancer. However, the mechanistic links between transcription factors and the epigenome which coordinate the deregulation of gene networks during cell transformation are largely unknown. METHODS: We used an isogenic model of stepwise tumorigenic transformation of human primary cells to monitor the progressive deregulation of gene networks upon immortalization and oncogene-induced transformation. We applied a systems biology approach by combining transcriptome and epigenome data for each step during transformation and integrated transcription factor-target gene associations in order to reconstruct the gene regulatory networks that are at the basis of the transformation process. RESULTS: We identified 142 transcription factors and 24 chromatin remodelers/modifiers (CRMs) which are preferentially associated with specific co-expression pathways that originate from deregulated gene programming during tumorigenesis. These transcription factors are involved in the regulation of divers processes, including cell differentiation, the immune response, and the establishment/modification of the epigenome. Unexpectedly, the analysis of chromatin state dynamics revealed patterns that distinguish groups of genes which are not only co-regulated but also functionally related. Decortication of transcription factor targets enabled us to define potential key regulators of cell transformation which are engaged in RNA metabolism and chromatin remodeling. CONCLUSIONS: We reconstructed gene regulatory networks that reveal the alterations occurring during human cellular tumorigenesis. Using these networks we predicted and validated several transcription factors as key players for the establishment of tumorigenic traits of transformed cells. Our study suggests a direct implication of CRMs in oncogene-induced tumorigenesis and identifies new CRMs involved in this process. This is the first comprehensive view of the gene regulatory network that is altered during the process of stepwise human cellular tumorigenesis in a virtually isogenic system.