RESUMO
Current therapies for medulloblastoma, a highly malignant childhood brain tumour, impose debilitating effects on the developing child, and highlight the need for molecularly targeted treatments with reduced toxicity. Previous studies have been unable to identify the full spectrum of driver genes and molecular processes that operate in medulloblastoma subgroups. Here we analyse the somatic landscape across 491 sequenced medulloblastoma samples and the molecular heterogeneity among 1,256 epigenetically analysed cases, and identify subgroup-specific driver alterations that include previously undiscovered actionable targets. Driver mutations were confidently assigned to most patients belonging to Group 3 and Group 4 medulloblastoma subgroups, greatly enhancing previous knowledge. New molecular subtypes were differentially enriched for specific driver events, including hotspot in-frame insertions that target KBTBD4 and 'enhancer hijacking' events that activate PRDM6. Thus, the application of integrative genomics to an extensive cohort of clinical samples derived from a single childhood cancer entity revealed a series of cancer genes and biologically relevant subtype diversity that represent attractive therapeutic targets for the treatment of patients with medulloblastoma.
Assuntos
Análise Mutacional de DNA , Genoma Humano/genética , Meduloblastoma/classificação , Meduloblastoma/genética , Sequenciamento Completo do Genoma , Carcinogênese/genética , Proteínas de Transporte/genética , Estudos de Coortes , Metilação de DNA , Conjuntos de Dados como Assunto , Epistasia Genética , Genômica , Humanos , Terapia de Alvo Molecular , Proteínas Musculares/genética , Mutação , Oncogenes/genética , Fatores de Transcrição/genética , Proteínas Wnt/genéticaRESUMO
Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.
Assuntos
Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica/genética , Meduloblastoma/classificação , Meduloblastoma/patologia , Fatores de Transcrição/metabolismo , Animais , Neoplasias Cerebelares/classificação , Feminino , Redes Reguladoras de Genes/genética , Genes Neoplásicos/genética , Genes Reporter/genética , Humanos , Masculino , Meduloblastoma/genética , Camundongos , Reprodutibilidade dos Testes , Peixe-Zebra/genéticaRESUMO
Epigenetic alterations, that is, disruption of DNA methylation and chromatin architecture, are now acknowledged as a universal feature of tumorigenesis. Medulloblastoma, a clinically challenging, malignant childhood brain tumour, is no exception. Despite much progress from recent genomics studies, with recurrent changes identified in each of the four distinct tumour subgroups (WNT-pathway-activated, SHH-pathway-activated, and the less-well-characterized Group 3 and Group 4), many cases still lack an obvious genetic driver. Here we present whole-genome bisulphite-sequencing data from thirty-four human and five murine tumours plus eight human and three murine normal controls, augmented with matched whole-genome, RNA and chromatin immunoprecipitation sequencing data. This comprehensive data set allowed us to decipher several features underlying the interplay between the genome, epigenome and transcriptome, and its effects on medulloblastoma pathophysiology. Most notable were highly prevalent regions of hypomethylation correlating with increased gene expression, extending tens of kilobases downstream of transcription start sites. Focal regions of low methylation linked to transcription-factor-binding sites shed light on differential transcriptional networks between subgroups, whereas increased methylation due to re-normalization of repressed chromatin in DNA methylation valleys was positively correlated with gene expression. Large, partially methylated domains affecting up to one-third of the genome showed increased mutation rates and gene silencing in a subgroup-specific fashion. Epigenetic alterations also affected novel medulloblastoma candidate genes (for example, LIN28B), resulting in alternative promoter usage and/or differential messenger RNA/microRNA expression. Analysis of mouse medulloblastoma and precursor-cell methylation demonstrated a somatic origin for many alterations. Our data provide insights into the epigenetic regulation of transcription and genome organization in medulloblastoma pathogenesis, which are probably also of importance in a wider developmental and disease context.
Assuntos
Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Meduloblastoma/genética , Análise de Sequência de DNA/métodos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Feminino , Genoma/genética , Histonas/metabolismo , Humanos , Meduloblastoma/patologia , Camundongos , Regiões Promotoras Genéticas/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer.
Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genética , Variação Estrutural do Genoma/genética , Meduloblastoma/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Criança , Cromossomos Humanos Par 9/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Meduloblastoma/classificação , Meduloblastoma/patologia , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.
Assuntos
Neoplasias Cerebelares/genética , Genoma Humano/genética , Meduloblastoma/genética , Envelhecimento/genética , Sequência de Aminoácidos , Transformação Celular Neoplásica , Neoplasias Cerebelares/classificação , Neoplasias Cerebelares/diagnóstico , Neoplasias Cerebelares/patologia , Criança , Cromatina/metabolismo , Cromossomos Humanos/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genômica , Proteínas Hedgehog/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Histona Desmetilases/genética , Humanos , Meduloblastoma/classificação , Meduloblastoma/diagnóstico , Meduloblastoma/patologia , Metilação , Mutação/genética , Taxa de Mutação , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Receptores Patched , Receptor Patched-1 , Fosfoproteínas Fosfatases/genética , Poliploidia , Receptores de Superfície Celular/genética , Análise de Sequência de RNA , Transdução de Sinais , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMO
A substantial fraction of phenotypic differences between closely related species are likely caused by differences in gene regulation. While this has already been postulated over 30 years ago, only few examples of evolutionary changes in gene regulation have been verified. Here, we identified and investigated binding sites of the transcription factor GA-binding protein alpha (GABPa) aiming to discover cis-regulatory adaptations on the human lineage. By performing chromatin immunoprecipitation-sequencing experiments in a human cell line, we found 11,619 putative GABPa binding sites. Through sequence comparisons of the human GABPa binding regions with orthologous sequences from 34 mammals, we identified substitutions that have resulted in 224 putative human-specific GABPa binding sites. To experimentally assess the transcriptional impact of those substitutions, we selected four promoters for promoter-reporter gene assays using human and African green monkey cells. We compared the activities of wild-type promoters to mutated forms, where we have introduced one or more substitutions to mimic the ancestral state devoid of the GABPa consensus binding sequence. Similarly, we introduced the human-specific substitutions into chimpanzee and macaque promoter backgrounds. Our results demonstrate that the identified substitutions are functional, both in human and nonhuman promoters. In addition, we performed GABPa knock-down experiments and found 1,215 genes as strong candidates for primary targets. Further analyses of our data sets link GABPa to cognitive disorders, diabetes, KRAB zinc finger (KRAB-ZNF), and human-specific genes. Thus, we propose that differences in GABPa binding sites played important roles in the evolution of human-specific phenotypes.
Assuntos
Fator de Transcrição de Proteínas de Ligação GA/genética , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Regulação da Expressão Gênica , Animais , Sítios de Ligação , Evolução Biológica , Células COS , Chlorocebus aethiops , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Evolução Molecular , Especiação Genética , Células HEK293 , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Alinhamento de Sequência , Dedos de Zinco/genéticaRESUMO
The regulation of gene expression in response to environmental signals and metabolic imbalances is a key step in maintaining cellular homeostasis. BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAF recognition elements, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we combined chromatin immunoprecipitation sequencing analysis of BACH1 target genes in HEK 293 cells with knockdown of BACH1 using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. The 59 BACH1 target genes identified by chromatin immunoprecipitation sequencing were found highly enriched in genes showing expression changes after BACH1 knockdown, demonstrating the impact of BACH1 repression on transcription. In addition to known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1, and SLC48A1) and redox regulation (GCLC, GCLM, and SLC7A11), we also discovered BACH1 target genes affecting cell cycle and apoptosis pathways (ITPR2, CALM1, SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, and MAFG) as well as subcellular transport processes (CLSTN1, PSAP, MAPT, and vault RNA). The newly identified impact of BACH1 on genes involved in neurodegenerative processes and proliferation provides an interesting basis for future dissection of BACH1-mediated gene repression in neurodegeneration and virus-induced cancerogenesis.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Ciclo Celular/fisiologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Regulação da Expressão Gênica/fisiologia , Estresse Oxidativo/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Elementos de Resposta/fisiologiaRESUMO
Given the inherent limitations of in silico studies relying solely on DNA sequence analysis, the functional characterization of mammalian promoters and associated cis-regulatory elements requires experimental support, which demands cloning and analysis of putative promoter regions. Focusing on human chromosome 21, we cloned 182 gene promoters of 2500 bp in length and conducted reporter gene assays on transfected-cell arrays. We found 56 promoters that were active in HEK293 cells, while another 49 promoters could be activated by treatment of cells with Trichostatin A or depletion of serum. We observed high correlations between promoter activities and endogenous transcript levels, RNA polymerase II occupancy, CpG islands and core promoter elements. Truncation of a subset of 62 promoters to â¼500 bp revealed that truncation rarely resulted in loss of activity, but rather in loss of responses to external stimuli, suggesting the presence of cis-regulatory response elements within distal promoter regions. In these regions, we found a strong enrichment of transcription factor binding sites that could potentially activate gene expression in the presence of stimuli. This study illustrates the modular functional architecture of chromosome 21 promoters and helps to reveal the complex mechanisms governing transcriptional regulation.
Assuntos
Cromossomos Humanos Par 21 , Regiões Promotoras Genéticas , Elementos Reguladores de Transcrição , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Humanos , RNA Polimerase II/metabolismoRESUMO
Most relapses of acute lymphoblastic leukemia (ALL) occur in patients with a medium risk (MR) for relapse on the Associazione Italiana di Ematologia e Oncologia Pediatrica and Berlin-Frankfurt-Münster (AIEOP-BFM) ALL protocol, based on persistence of minimal residual disease (MRD). New insights into biological features that are associated with MRD are needed. Here, we identify the glycosylphosphatidylinositol-anchored cell surface protein vanin-2 (VNN2; GPI-80) by charting the cell surface proteome of MRD very high-risk (HR) B-cell precursor (BCP) ALL using a chemoproteomics strategy. The correlation between VNN2 transcript and surface protein expression enabled a retrospective analysis (ALL-BFM 2000; N = 770 cases) using quantitative polymerase chain reaction to confirm the association of VNN2 with MRD and independent prediction of worse outcome. Using flow cytometry, we detected VNN2 expression in 2 waves, in human adult bone marrow stem and progenitor cells and in the mature myeloid compartment, in line with proposed roles for fetal hematopoietic stem cells and inflammation. Prospective validation by flow cytometry in the ongoing clinical trial (AIEOP-BFM 2009) identified 10% (103/1069) of VNN2+ BCP ALL patients at first diagnosis, primarily in the MRD MR (48/103, 47%) and HR (37/103, 36%) groups, across various cytogenetic subtypes. We also detected frequent mutations in epigenetic regulators in VNN2+ ALLs, including histone H3 methyltransferases MLL2, SETD2, and EZH2 and demethylase KDM6A. Inactivation of the VNN2 gene did not impair leukemia repopulation capacity in xenografts. Taken together, VNN2 marks a cellular state of increased resistance to chemotherapy that warrants further investigations. Therefore, this marker should be included in diagnostic flow cytometry panels.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Amidoidrolases/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Linfócitos B , Moléculas de Adesão Celular , Criança , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Ligadas por GPI , Células-Tronco Hematopoéticas , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Estudos Prospectivos , Estudos RetrospectivosRESUMO
The chimeric transcription factor TCF3-HLF defines an incurable acute lymphoblastic leukemia subtype. Here we decipher the regulome of endogenous TCF3-HLF and dissect its essential transcriptional components and targets by functional genomics. We demonstrate that TCF3-HLF recruits HLF binding sites at hematopoietic stem cell/myeloid lineage associated (super-) enhancers to drive lineage identity and self-renewal. Among direct targets, hijacking an HLF binding site in a MYC enhancer cluster by TCF3-HLF activates a conserved MYC-driven transformation program crucial for leukemia propagation in vivo. TCF3-HLF pioneers the cooperation with ERG and recruits histone acetyltransferase p300 (EP300), conferring susceptibility to EP300 inhibition. Our study provides a framework for targeting driving transcriptional dependencies in this fatal leukemia.
Assuntos
Proteína p300 Associada a E1A/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Ligação a DNA/genética , Humanos , Translocação GenéticaRESUMO
Existing methods for paired antibody heavy- and light-chain repertoire sequencing rely on specialized equipment and are limited by their commercial availability and high costs. Here, we report a novel simple and cost-effective emulsion-based single-cell paired antibody repertoire sequencing method that employs only basic laboratory equipment. We performed a proof-of-concept using mixed mouse hybridoma cells and we also showed that our method can be used for discovery of novel antigen-specific monoclonal antibodies by sequencing human CD19+ B cell IgM and IgG repertoires isolated from peripheral whole blood before and seven days after Td (Tetanus toxoid/Diphtheria toxoid) booster immunization. We anticipate broad applicability of our method for providing insights into adaptive immune responses associated with various diseases, vaccinations, and cancer immunotherapies.
Assuntos
Anticorpos/metabolismo , Retículo Endoplasmático/metabolismo , Microssomos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos Monoclonais/metabolismo , Linfócitos B/metabolismo , Células HEK293 , Humanos , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/genética , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de ProteínaRESUMO
Despite the increasing use of humanized mouse models to study new approaches of graft-versus-host disease (GVHD) prevention, the pathogenesis of xenogeneic GVHD (xGVHD) in these models remains misunderstood. The aim of this study is to describe this pathogenesis in NOD/LtSz-PrkdcscidIL2rγtm1Wjl (NSG) mice infused with human PBMCs and to assess the impact of the expression of HLA-A0201 by NSG mice cells (NSG-HLA-A2/HHD mice) on xGVHD and graft-versus-leukemia (GvL) effects, by taking advantage of next-generation technologies. We found that T cells recovered from NSG mice after transplantation had upregulated expression of genes involved in cell proliferation, as well as in TCR, co-stimulatory, IL-2/STAT5, mTOR and Aurora kinase A pathways. T cells had mainly an effector memory or an effector phenotype and exhibited a Th1/Tc1-skewed differentiation. TCRß repertoire diversity was markedly lower both in the spleen and lungs (a xGVHD target organ) than at infusion. There was no correlation between the frequencies of specific clonotypes at baseline and in transplanted mice. Finally, expression of HLA-A0201 by NSG mice led to more severe xGVHD and enhanced GvL effects toward HLA-A2+ leukemic cells. Altogether our data demonstrate that the pathogenesis of xGVHD shares important features with human GVHD and that NSG-HLA-A2/HHD mice could serve as better model to study GVHD and GvL effects.
Assuntos
Regulação da Expressão Gênica/imunologia , Doença Enxerto-Hospedeiro/imunologia , Antígeno HLA-A2/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/transplante , Animais , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Efeito Enxerto vs Leucemia/genética , Efeito Enxerto vs Leucemia/imunologia , Antígeno HLA-A2/genética , Xenoenxertos , Humanos , Leucócitos Mononucleares/patologia , CamundongosRESUMO
Congenital Cytomegalovirus infection (cCMV) is the leading infection in determining permanent long-term impairments (LTI), and its pathogenesis is largely unknown due to the complex interplay between viral, maternal, placental, and child factors. The cellular activity, considered to be the result of the response to exogenous and endogenous factors, is captured by the determination of gene expression profiles. In this study, we determined whole blood transcriptomes in relation to cCMV, CMV viral load and LTI development at 6 years of age by using RNA isolated from neonatal dried blood spots (DBS) stored at room temperature for 8 years. As DBS were assumed to mainly reflect the neonatal immune system, particular attention was given to the immune pathways using the global test. Additionally, differential expression of individual genes was performed using the voom/limma function packages. We demonstrated feasibility of RNA sequencing from archived neonatal DBS of children with cCMV, and non-infected controls, in relation to LTI and CMV viral load. Despite the lack of statistical power to detect individual genes differences, pathway analysis suggested the involvement of innate immune response with higher CMV viral loads, and of anti-inflammatory markers in infected children that did not develop LTI. Finally, the T cell exhaustion observed in infected neonates, in particular with higher viral load, did not correlate with LTI, therefore other mechanisms are likely to be involved in the long-term immune dysfunction. Despite these data demonstrate limitation in determining prognostic markers for LTI by means of transcriptome analysis, this exploratory study represents a first step in unraveling the pathogenesis of cCMV, and the aforementioned pathways certainly merit further evaluation.
Assuntos
Preservação de Sangue/métodos , Infecções por Citomegalovirus/genética , Teste em Amostras de Sangue Seco/métodos , Transcriptoma , Criança , Pré-Escolar , Disfunção Cognitiva/diagnóstico , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/virologia , Feminino , Humanos , Recém-Nascido , Masculino , Doença dos Neurônios Motores/diagnóstico , Fatores de Tempo , Carga ViralRESUMO
Identifying the earliest somatic changes in prostate cancer can give important insights into tumor evolution and aids in stratifying high- from low-risk disease. We integrated whole genome, transcriptome and methylome analysis of early-onset prostate cancers (diagnosis ≤55 years). Characterization across 292 prostate cancer genomes revealed age-related genomic alterations and a clock-like enzymatic-driven mutational process contributing to the earliest mutations in prostate cancer patients. Our integrative analysis identified four molecular subgroups, including a particularly aggressive subgroup with recurrent duplications associated with increased expression of ESRP1, which we validate in 12,000 tissue microarray tumors. Finally, we combined the patterns of molecular co-occurrence and risk-based subgroup information to deconvolve the molecular and clinical trajectories of prostate cancer from single patient samples.
Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Transcriptoma , Adulto , Biomarcadores Tumorais/metabolismo , Evolução Molecular , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Risco , Sequenciamento Completo do Genoma/métodosRESUMO
Molecular mechanisms of endocytosis in the genetically and biochemically tractable professional phagocyte Dictyostelium discoideum reveal a striking degree of similarity to higher eukaryotic cells. Pulse-chase feeding with latex beads allowed purification of phagosomes at different stages of maturation. Gentle ATP stripping of an actin meshwork entrapping contaminating organelles resulted in a 10-fold increase in yield and purity, as confirmed by electron microscopy. Temporal profiling of signaling, cytoskeletal, and trafficking proteins resulted in a complex molecular fingerprint of phagosome biogenesis and maturation. First, nascent phagosomes were associated with coronin and rapidly received a lysosomal glycoprotein, LmpB. Second, at least two phases of delivery of lysosomal hydrolases (cathepsin D [CatD] and cysteine protease [CPp34]) were accompanied by removal of plasma membrane components (PM4C4 and biotinylated surface proteins). Third, a phase of late maturation, preparing for final exocytosis of undigested material, included quantitative recycling of hydrolases and association with vacuolin. Also, lysosomal glycoproteins of the Lmp family showed distinct trafficking kinetics. The delivery and recycling of CatD was directly visualized by confocal microscopy. This heavy membrane traffic of cargos was precisely accompanied by regulatory proteins such as the Rab7 GTPases and the endosomal SNAREs Vti1 and VAMP7. This initial molecular description of phagocytosis demonstrates the feasibility of a comprehensive analysis of phagosomal lipids and proteins in genetically modified strains.
Assuntos
Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Dictyostelium/metabolismo , Fagossomos/metabolismo , Proteínas de Transporte Vesicular , Trifosfato de Adenosina/metabolismo , Animais , Biomarcadores , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Proteínas de Transporte/metabolismo , Catepsina D/metabolismo , Membrana Celular/química , Dictyostelium/citologia , Endocitose/fisiologia , Corantes Fluorescentes/metabolismo , Carioferinas/metabolismo , Metabolismo dos Lipídeos , Lipídeos/química , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fagossomos/química , Fagossomos/ultraestrutura , Proteínas/química , Proteínas/metabolismo , Proteínas Qb-SNARE , Proteínas R-SNARE , Proteínas SNARE , Tiazóis/metabolismo , Tiazolidinas , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.
Assuntos
Biomarcadores Tumorais/genética , Cetuximab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Trisomy of human chromosome 21 (Chr21) results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins. RESULTS: We chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb) to MCM3AP (46.6 Mb), with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His6 tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized. CONCLUSION: The cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome.
Assuntos
Cromossomos Humanos Par 21 , Análise Serial de Proteínas/métodos , Análise Serial de Tecidos/métodos , Distribuição Tecidual/genética , Ciclo Celular , Núcleo Celular/metabolismo , Células Cultivadas , Análise Custo-Benefício , Citosol/metabolismo , Síndrome de Down/genética , Síndrome de Down/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Transporte Proteico , Vesículas Secretórias/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3's interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5'UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex. Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3's role in this process. Arsenite-induced stress shifts DDX3 binding from the 5'UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, medulloblastoma-associated DDX3R534H variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in chromatin organization in DDX3R534H-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation.
Assuntos
Neoplasias Cerebelares/genética , RNA Helicases DEAD-box/genética , Meduloblastoma/genética , Biossíntese de Proteínas/genética , Estresse Fisiológico/genética , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , TranscriptomaRESUMO
The Prep1 (Pknox1) homeodomain transcription factor is essential at multiple stages of embryo development. In the E11.5 embryo trunk, we previously estimated that Prep1 binds about 3,300 genomic sites at a highly specific decameric consensus sequence, mainly governing basal cellular functions. We now show that in embryonic stem (ES) cells Prep1 binding pattern only partly overlaps that of the embryo trunk, with about 2,000 novel sites. Moreover, in ES cells Prep1 still binds mostly to promoters, as in total embryo trunk but, among the peaks bound exclusively in ES cells, the percentage of enhancers was three-fold higher. RNA-seq identifies about 1800 genes down-regulated in Prep1-/- ES cells which belong to gene ontology categories not enriched in the E11.5 Prep1i/i differentiated embryo, including in particular essential components of the Wnt and Fgf pathways. These data agree with aberrant Wnt and Fgf expression levels in the Prep1-/- ES cells with a deficient embryoid bodies (EBs) formation and differentiation. Re-establishment of the Prep1 level rescues the phenotype.
Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Homeodomínio/biossíntese , Células-Tronco Embrionárias Murinas , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Fatores de Crescimento de Fibroblastos/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Proteínas de Homeodomínio/genética , Camundongos , Via de Sinalização Wnt/genéticaRESUMO
Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients' outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.