RESUMO
Single-cell RNA sequencing (scRNA-seq) offers new possibilities to address biological and medical questions. However, systematic comparisons of the performance of diverse scRNA-seq protocols are lacking. We generated data from 583 mouse embryonic stem cells to evaluate six prominent scRNA-seq methods: CEL-seq2, Drop-seq, MARS-seq, SCRB-seq, Smart-seq, and Smart-seq2. While Smart-seq2 detected the most genes per cell and across cells, CEL-seq2, Drop-seq, MARS-seq, and SCRB-seq quantified mRNA levels with less amplification noise due to the use of unique molecular identifiers (UMIs). Power simulations at different sequencing depths showed that Drop-seq is more cost-efficient for transcriptome quantification of large numbers of cells, while MARS-seq, SCRB-seq, and Smart-seq2 are more efficient when analyzing fewer cells. Our quantitative comparison offers the basis for an informed choice among six prominent scRNA-seq methods, and it provides a framework for benchmarking further improvements of scRNA-seq protocols.
Assuntos
Células-Tronco Embrionárias/química , Sequenciamento de Nucleotídeos em Larga Escala , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Sequência de Bases , Linhagem Celular , Simulação por Computador , Análise Custo-Benefício , Sequenciamento de Nucleotídeos em Larga Escala/economia , Camundongos , Modelos Econômicos , RNA/isolamento & purificação , Análise de Sequência de RNA/economia , Análise de Célula Única/economiaRESUMO
Lineage plasticity and stemness have been invoked as causes of therapy resistance in cancer, because these flexible states allow cancer cells to dedifferentiate and alter their dependencies. We investigated such resistance mechanisms in relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) carrying activating NOTCH1 mutations via full-length single-cell RNA sequencing (scRNA-seq) of malignant and microenvironmental cells. We identified 2 highly distinct stem-like states that critically differed with regard to cell cycle and oncogenic signaling. Fast-cycling stem-like leukemia cells demonstrated Notch activation and were effectively eliminated in patients by Notch inhibition, whereas slow-cycling stem-like cells were Notch independent and rather relied on PI3K signaling, likely explaining the poor efficacy of Notch inhibition in this disease. Remarkably, we found that both stem-like states could differentiate into a more mature leukemia state with prominent immunomodulatory functions, including high expression of the LGALS9 checkpoint molecule. These cells promoted an immunosuppressive leukemia ecosystem with clonal accumulation of dysfunctional CD8+ T cells that expressed HAVCR2, the cognate receptor for LGALS9. Our study identified complex interactions between signaling programs, cellular plasticity, and immune programs that characterize ETP-ALL, illustrating the multidimensionality of tumor heterogeneity. In this scenario, combination therapies targeting diverse oncogenic states and the immune ecosystem seem most promising to successfully eliminate tumor cells that escape treatment through coexisting transcriptional programs.
Assuntos
Carcinogênese , Galectinas/metabolismo , Regulação Leucêmica da Expressão Gênica , Evasão da Resposta Imune , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Análise de Célula Única/métodos , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Criança , Pré-Escolar , Resistencia a Medicamentos Antineoplásicos , Feminino , Seguimentos , Galectinas/genética , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Prognóstico , RNA-Seq/métodos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Single-cell RNA sequencing (scRNA-seq) has significantly deepened our insights into complex tissues, with the latest techniques capable of processing tens of thousands of cells simultaneously. Analyzing increasing numbers of cells, however, generates extremely large data sets, extending processing time and challenging computing resources. Current scRNA-seq analysis tools are not designed to interrogate large data sets and often lack sensitivity to identify marker genes. With bigSCale, we provide a scalable analytical framework to analyze millions of cells, which addresses the challenges associated with large data sets. To handle the noise and sparsity of scRNA-seq data, bigSCale uses large sample sizes to estimate an accurate numerical model of noise. The framework further includes modules for differential expression analysis, cell clustering, and marker identification. A directed convolution strategy allows processing of extremely large data sets, while preserving transcript information from individual cells. We evaluated the performance of bigSCale using both a biological model of aberrant gene expression in patient-derived neuronal progenitor cells and simulated data sets, which underlines the speed and accuracy in differential expression analysis. To test its applicability for large data sets, we applied bigSCale to assess 1.3 million cells from the mouse developing forebrain. Its directed down-sampling strategy accumulates information from single cells into index cell transcriptomes, thereby defining cellular clusters with improved resolution. Accordingly, index cell clusters identified rare populations, such as reelin (Reln)-positive Cajal-Retzius neurons, for which we report previously unrecognized heterogeneity associated with distinct differentiation stages, spatial organization, and cellular function. Together, bigSCale presents a solution to address future challenges of large single-cell data sets.
Assuntos
RNA/genética , Análise de Célula Única/métodos , Software , Transcriptoma/genética , Animais , Moléculas de Adesão Celular Neuronais/genética , Diferenciação Celular/genética , Análise por Conglomerados , Proteínas da Matriz Extracelular/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteína Reelina , Serina Endopeptidases/genéticaRESUMO
The brain is the most complex tissue in terms of cell types that it comprises, to the extent that it is still poorly understood. Single cell genome and transcriptome profiling allow to disentangle the neuronal heterogeneity, enabling the categorization of individual neurons into groups with similar molecular signatures. Herein, we unravel the current state of knowledge in single cell neurogenomics. We describe the molecular understanding of the cellular architecture of the mammalian nervous system in health and in disease; from the discovery of unrecognized cell types to the validation of known ones, applying these state-of-the-art technologies.
Assuntos
Encéfalo/citologia , Genômica/métodos , Neurônios/metabolismo , Análise de Célula Única , Animais , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Código das Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurogênese , Neurônios/classificação , Oligodendroglia/metabolismo , Processamento de Proteína Pós-Traducional/genética , Retina/citologia , Retina/metabolismo , Análise de Sequência/métodos , Análise de Célula Única/métodos , Olfato/genética , TranscriptomaRESUMO
Genomic imprinting is the epigenetic process that results in monoallelic expression of genes depending on parental origin. These genes are known to be critical for placental development and fetal growth in mammals. Aberrant epigenetic profiles at imprinted loci, such as DNA methylation defects, are surprisingly rare in pregnancies with compromised fetal growth, while variations in transcriptional output from the expressed alleles of imprinted genes are more commonly reported in pregnancies complicated with intrauterine growth restriction (IUGR). To determine if PLAGL1 and HYMAI, two imprinted transcripts deregulated in Transient Neonatal Diabetes Mellitus, are involved in non-syndromic IUGR we compared the expression and DNA methylation levels in a large cohort of placental biopsies from IUGR and uneventful pregnancies. This revealed that despite appropriate maternal methylation at the shared PLAGL1/HYMAI promoter, there was a loss of correlation between PLAGL1 and HYMAI expression in IUGR. This incongruity was due to higher HYMAI expression in IUGR gestations, coupled with PLAGL1 down-regulation in placentas from IUGR girls, but not boys. The PLAGL1 protein is a zinc-finger transcription factor that has been shown to be a master coordinator of a genetic growth network in mice. We observe PLAGL1 binding to the H19/IGF2 shared enhancers in placentae, with significant correlations between PLAGL1 levels with H19 and IGF2 expression levels. In addition, PLAGL1 binding and expression also correlate with expression levels of metabolic regulator genes SLC2A4, TCF4 and PPARγ1. Our results strongly suggest that fetal growth can be influenced by altered expression of the PLAGL1 gene network in human placenta.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Retardo do Crescimento Fetal/metabolismo , Redes Reguladoras de Genes , Impressão Genômica , Placenta/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Ciclo Celular/genética , Estudos de Coortes , Metilação de DNA , Epigênese Genética , Feminino , Retardo do Crescimento Fetal/genética , Humanos , Masculino , Gravidez , Fatores Sexuais , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Paternal duplications of chromosome 6q24, a region that contains the imprinted PLAGL1 and HYMAI transcripts, are associated with transient neonatal diabetes mellitus. A common feature of imprinted genes is that they tend to cluster together, presumably as a result of sharing common cis-acting regulatory elements. To determine the extent of this imprinted cluster in human and mouse, we have undertaken a systematic analysis of allelic expression and DNA methylation of the genes mapping within an â¼1.4-Mb region flanking PLAGL1/Plagl1. We confirm that all nine neighbouring genes are biallelically expressed in both species. In human we identify two novel paternally expressed PLAGL1 coding transcripts that originate from unique promoter regions. Chromatin immunoprecipitation for CTCF and the cohesin subunits RAD21 and SMC3 reveals evolutionarily conserved binding sites within unmethylated regions â¼5 kb downstream of the PLAGL1 differentially methylated region and within the PLAGL1 3' untranslated region (UTR). Higher-order chromatin looping occurs between these regions in both expressing and non-expressing tissues, forming a non-allelic chromatin loop around the PLAGL1/Plagl1 gene. In placenta and brain tissues, we identify an additional interaction between the PLAGL1 P3/P4 promoters and the unmethylated element downstream of the PLAGL1 differentially methylated region that we propose facilitates imprinted expression of these alternative isoforms.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Impressão Genômica , Proteínas Repressoras/metabolismo , Alelos , Animais , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Cromossomos Humanos Par 6 , Proteínas de Ligação a DNA/metabolismo , Feminino , Genes Supressores de Tumor , Genótipo , Humanos , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Placenta/metabolismo , Gravidez , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição/genética , CoesinasRESUMO
Imprinted retrotransposed genes share a common genomic organization including a promoter-associated differentially methylated region (DMR) and a position within the intron of a multi-exonic 'host' gene. In the mouse, at least one transcript of the host gene is also subject to genomic imprinting. Human retrogene orthologues are imprinted and we reveal that human host genes are not imprinted. This coincides with genomic rearrangements that occurred during primate evolution, which increase the separation between the retrogene DMRs and the host genes. To address the mechanisms governing imprinted retrogene expression, histone modifications were assayed at the DMRs. For the mouse retrogenes, the active mark H3K4me2 was associated with the unmethylated paternal allele, while the methylated maternal allele was enriched in repressive marks including H3K9me3 and H4K20me3. Two human retrogenes showed monoallelic enrichment of active, but not of repressive marks suggesting a partial uncoupling of the relationship between DNA methylation and repressive histone methylation, possibly due to the smaller size and lower CpG density of these DMRs. Finally, we show that the genes immediately flanking the host genes in mouse and human are biallelically expressed in a range of tissues, suggesting that these loci are distinct from large imprinted clusters.
Assuntos
Impressão Genômica , Histonas/metabolismo , Retroelementos , Alelos , Animais , Cromatina/metabolismo , Metilação de DNA , Humanos , Camundongos , Regiões Promotoras GenéticasRESUMO
Chimeric antigen receptor (CAR) T-cell therapy has led to tremendous successes in the treatment of B-cell malignancies. However, a large fraction of treated patients relapse, often with disease expressing reduced levels of the target antigen. Here, we report that exposing CD19+ B-cell acute lymphoblastic leukemia (B-ALL) cells to CD19 CAR T cells reduced CD19 expression within hours. Initially, CD19 CAR T cells caused clustering of CD19 at the T cell-leukemia cell interface followed by CD19 internalization and decreased CD19 surface expression on the B-ALL cells. CD19 expression was then repressed by transcriptional rewiring. Using single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing, we demonstrated that a subset of refractory CD19low cells sustained decreased CD19 expression through transcriptional programs of physiologic B-cell activation and germinal center reaction. Inhibiting B-cell activation programs with the Bruton's tyrosine kinase inhibitor ibrutinib increased the cytotoxicity of CD19 CAR T cells without affecting CAR T-cell viability. These results demonstrate transcriptional plasticity as an underlying mechanism of escape from CAR T cells and highlight the importance of combining CAR T-cell therapy with targeted therapies that aim to overcome this plasticity. See related Spotlight by Zhao and Melenhorst, p. 1040.
Assuntos
Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Antígenos CD19/imunologia , Centro Germinativo/imunologia , Humanos , Imunoterapia Adotiva/métodos , Linfoma de Células B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologiaRESUMO
PURPOSE: Although remarkably effective in some patients, precision medicine typically induces only transient responses despite initial absence of resistance-conferring mutations. Using BRAF-mutated myeloma as a model for resistance to precision medicine we investigated if BRAF-mutated cancer cells have the ability to ensure their survival by rapidly adapting to BRAF inhibitor treatment. EXPERIMENTAL DESIGN: Full-length single-cell RNA (scRNA) sequencing (scRNA-seq) was conducted on 3 patients with BRAF-mutated myeloma and 1 healthy donor. We sequenced 1,495 cells before, after 1 week, and at clinical relapse to BRAF/MEK inhibitor treatment. We developed an in vitro model of dabrafenib resistance using genetically homogeneous single-cell clones from two cell lines with established BRAF mutations (U266, DP6). Transcriptional and epigenetic adaptation in resistant cells were defined by RNA-seq and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq). Mitochondrial metabolism was characterized by metabolic flux analysis. RESULTS: Profiling by scRNA-seq revealed rapid cellular state changes in response to BRAF/MEK inhibition in patients with myeloma and cell lines. Transcriptional adaptation preceded detectable outgrowth of genetically discernible drug-resistant clones and was associated with widespread enhancer remodeling. As a dominant vulnerability, dependency on oxidative phosphorylation (OxPhos) was induced. In treated individuals, OxPhos was activated at the time of relapse and showed inverse correlation to MAPK activation. Metabolic flux analysis confirmed OxPhos as a preferential energetic resource of drug-persistent myeloma cells. CONCLUSIONS: This study demonstrates that cancer cells have the ability to rapidly adapt to precision treatments through transcriptional state changes, epigenetic adaptation, and metabolic rewiring, thus facilitating the development of refractory disease while simultaneously exposing novel vulnerabilities.
Assuntos
Melanoma , Mieloma Múltiplo , Resistencia a Medicamentos Antineoplásicos , Humanos , Melanoma/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mutação , Recidiva Local de Neoplasia/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf , Análise de Célula ÚnicaRESUMO
Understanding the origins of animal multicellularity is a fundamental biological question. Recent genome data have unravelled the role that co-option of pre-existing genes played in the origin of animals. However, there were also some important genetic novelties at the onset of Metazoa. To have a clear understanding of the specific genetic innovations and how they appeared, we need the broadest taxon sampling possible, especially among early-branching animals and their unicellular relatives. Here, we take advantage of single-cell genomics to expand our understanding of the genomic diversity of choanoflagellates, the sister-group to animals. With these genomes, we have performed an updated and taxon-rich reconstruction of protein evolution from the Last Eukaryotic Common Ancestor (LECA) to animals. Our novel data re-defines the origin of some genes previously thought to be metazoan-specific, like the POU transcription factor, which we show appeared earlier in evolution. Moreover, our data indicate that the acquisition of new genes at the stem of Metazoa was mainly driven by duplications and protein domain rearrangement processes at the stem of Metazoa. Furthermore, our analysis allowed us to reveal protein domains that are essential to the maintenance of animal multicellularity. Our analyses also demonstrate the utility of single-cell genomics from uncultured taxa to address evolutionary questions. This article is part of a discussion meeting issue 'Single cell ecology'.
Assuntos
Coanoflagelados/genética , Evolução Molecular , Genoma de Protozoário , Domínios Proteicos/genéticaRESUMO
Forced transcription factor expression can transdifferentiate somatic cells into other specialised cell types or reprogram them into induced pluripotent stem cells (iPSCs) with variable efficiency. To better understand the heterogeneity of these processes, we used single-cell RNA sequencing to follow the transdifferentation of murine pre-B cells into macrophages as well as their reprogramming into iPSCs. Even in these highly efficient systems, there was substantial variation in the speed and path of fate conversion. We predicted and validated that these differences are inversely coupled and arise in the starting cell population, with Mychigh large pre-BII cells transdifferentiating slowly but reprogramming efficiently and Myclow small pre-BII cells transdifferentiating rapidly but failing to reprogram. Strikingly, differences in Myc activity predict the efficiency of reprogramming across a wide range of somatic cell types. These results illustrate how single cell expression and computational analyses can identify the origins of heterogeneity in cell fate conversion processes.
Assuntos
Linhagem da Célula , Transdiferenciação Celular , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células Precursoras de Linfócitos B/citologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RNA-Seq , Transdução de Sinais , Análise de Célula Única , TranscriptomaRESUMO
Aging is a multifactorial process characterized by the progressive loss of physiological functions, leading to an increased vulnerability to age-associated diseases and finally to death. Several theories have been proposed to explain the nature of aging. One of the most known identifies the free radicals produced by the mitochondrial metabolism as the cause of cellular and DNA damage. However, there are also several evidences supporting that epigenetic modifications, such as DNA methylation, noncoding RNAs, and histone modifications, play a critical role in the molecular mechanism of aging. In this review, we explore the significance of these findings and argue how the interlinked effects of oxidative stress and epigenetics can explain the cause of age-related declines.
Assuntos
Envelhecimento , Epigênese Genética , Estresse Oxidativo , Animais , Dano ao DNA , Histonas/metabolismo , Humanos , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , RNA não Traduzido/metabolismoRESUMO
Single-cell genomics (SCG) appeared as a powerful technique to get genomic information from uncultured organisms. However, SCG techniques suffer from biases at the whole genome amplification step that can lead to extremely variable numbers of genome recovery (5-100%). Thus, it is unclear how useful can SCG be to address evolutionary questions on uncultured microbial eukaryotes. To provide some insights into this, we here analysed 3 single-cell amplified genomes (SAGs) of the choanoflagellate Monosiga brevicollis, whose genome is known. Our results show that each SAG has a different, independent bias, yielding different levels of genome recovery for each cell (6-36%). Genes often appear fragmented and are split into more genes during annotation. Thus, analyses of gene gain and losses, gene architectures, synteny and other genomic features can not be addressed with a single SAG. However, the recovery of phylogenetically-informative protein domains can be up to 55%. This means SAG data can be used to perform accurate phylogenomic analyses. Finally, we also confirm that the co-assembly of several SAGs improves the general genomic recovery. Overall, our data show that, besides important current limitations, SAGs can still provide interesting and novel insights from poorly-known, uncultured organisms.
Assuntos
Coanoflagelados/genética , Coanoflagelados/isolamento & purificação , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Genômica/métodos , Análise de Célula Única/métodos , Coanoflagelados/classificação , Biologia Computacional , Sequenciamento Completo do GenomaRESUMO
A variety of single-cell RNA preparation procedures have been described. So far, protocols require fresh material, which hinders complex study designs. We describe a sample preservation method that maintains transcripts in viable single cells, allowing one to disconnect time and place of sampling from subsequent processing steps. We sequence single-cell transcriptomes from >1000 fresh and cryopreserved cells using 3'-end and full-length RNA preparation methods. Our results confirm that the conservation process did not alter transcriptional profiles. This substantially broadens the scope of applications in single-cell transcriptomics and could lead to a paradigm shift in future study designs.
Assuntos
Criopreservação , Perfilação da Expressão Gênica , Análise de Célula Única , Transcriptoma , Animais , Linhagem Celular , Análise por Conglomerados , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Xenoenxertos , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , RNA/genética , Estabilidade de RNA , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Análise de Célula Única/métodosRESUMO
Highly proliferative Lgr5+ stem cells maintain the intestinal epithelium and are thought to be largely homogeneous. Although quiescent intestinal stem cell (ISC) populations have been described, the identity and features of such a population remain controversial. Here we report unanticipated heterogeneity within the Lgr5+ ISC pool. We found that expression of the RNA-binding protein Mex3a labels a slowly cycling subpopulation of Lgr5+ ISCs that contribute to all intestinal lineages with distinct kinetics. Single-cell transcriptome profiling revealed that Lgr5+ cells adopt two discrete states, one of which is defined by a Mex3a expression program and relatively low levels of proliferation genes. During homeostasis, Mex3a+ cells continually shift into the rapidly dividing, self-renewing ISC pool. Chemotherapy and radiation preferentially target rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, helping to promote regeneration of the intestinal epithelium following toxic insults. Thus, Mex3a defines a reserve-like ISC population within the Lgr5+ compartment.
Assuntos
Proliferação de Células/fisiologia , Mucosa Intestinal/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Animais , Mucosa Intestinal/citologia , Camundongos , Camundongos Transgênicos , Proteínas de Ligação a RNA/genética , Receptores Acoplados a Proteínas G/genética , Células-Tronco/citologiaRESUMO
BACKGROUND: Wilms tumor 1 (WT1) is over-expressed in numerous cancers with respect to normal cells, and has either a tumor suppressor or oncogenic role depending on cellular context. This gene is associated with numerous alternatively spliced transcripts, which initiate from two different unique first exons within the WT1 and the alternative (A)WT1 promoter intervals. Within the hematological system, WT1 expression is restricted to CD34+/CD38- cells and is undetectable after differentiation. Detectable expression of this gene is an excellent marker for minimal residual disease in acute myeloid leukemia (AML), but the underlying epigenetic alterations are unknown. METHODS: To determine the changes in the underlying epigenetic landscape responsible for this expression, we characterized expression, DNA methylation and histone modification profiles in 28 hematological cancer cell lines and confirmed the methylation signature in 356 cytogenetically well-characterized primary hematological malignancies. RESULTS: Despite high expression of WT1 and AWT1 transcripts in AML-derived cell lines, we observe robust hypermethylation of the AWT1 promoter and an epigenetic switch from a permissive to repressive chromatin structure between normal cells and AML cell lines. Subsequent methylation analysis in our primary leukemia and lymphoma cohort revealed that the epigenetic signature identified in cell lines is specific to myeloid-lineage malignancies, irrespective of underlying mutational status or translocation. In addition to being a highly specific marker for AML diagnosis (positive predictive value 100%; sensitivity 86.1%; negative predictive value 89.4%), we show that AWT1 hypermethylation also discriminates patients that relapse from those achieving complete remission after hematopoietic stem cell transplantation, with similar efficiency to WT1 expression profiling. CONCLUSIONS: We describe a methylation signature of the AWT1 promoter CpG island that is a promising marker for classifying myeloid-derived leukemias. In addition AWT1 hypermethylation is ideally suited to monitor the recurrence of disease during remission in patients undergoing allogeneic stem cell transfer.
Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas/genética , Leucemia Mieloide/genética , Regiões Promotoras Genéticas/genética , Proteínas WT1/genética , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Criança , Pré-Escolar , Ilhas de CpG/genética , Feminino , Células HL-60 , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Humanos , Células K562 , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células U937 , Proteínas WT1/metabolismo , Adulto JovemRESUMO
BACKGROUND: Genomic imprinting is the epigenetic marking of genes that results in parent-of-origin monoallelic expression. Most imprinted domains are associated with differentially DNA methylated regions (DMRs) that originate in the gametes, and are maintained in somatic tissues after fertilization. This allelic methylation profile is associated with a plethora of histone tail modifications that orchestrates higher order chromatin interactions. The mouse chromosome 15 imprinted cluster contains multiple brain-specific maternally expressed transcripts including Ago2, Chrac1, Trappc9 and Kcnk9 and a paternally expressed gene, Peg13. The promoter of Peg13 is methylated on the maternal allele and is the sole DMR within the locus. To determine the extent of imprinting within the human orthologous region on chromosome 8q24, a region associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome, we have undertaken a systematic analysis of allelic expression and DNA methylation of genes mapping within an approximately 2 Mb region around TRAPPC9. RESULTS: Utilizing allele-specific RT-PCR, bisulphite sequencing, chromatin immunoprecipitation and chromosome conformation capture (3C) we show the reciprocal expression of the novel, paternally expressed, PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and the biallelic expression of flanking transcripts in a range of tissues. We identify a tandem-repeat region overlapping the PEG13 transcript that is methylated on the maternal allele, which binds CTCF-cohesin in chromatin immunoprecipitation experiments and possesses enhancer-blocker activity. Using 3C, we identify mutually exclusive approximately 58 and 500 kb chromatin loops in adult frontal cortex between a novel brain-specific enhancer, marked by H3K4me1 and H3K27ac, with the KCNK9 and PEG13 promoters which we propose regulates brain-specific expression. CONCLUSIONS: We have characterised the molecular mechanism responsible for reciprocal allelic expression of the PEG13 and KCNK9 transcripts. Therefore, our observations may have important implications for identifying the cause of intellectual disabilities associated with the 8q24 locus.
RESUMO
Genomic imprinting is a complex epigenetic mechanism of transcriptional control that utilizes DNA methylation and histone modifications to bring about parent-of-origin specific monoallelic expression in mammals. Genes subject to imprinting are often organised in clusters associated with large non-coding RNAs (ncRNAs), some of which have cis-regulatory functions. Here we have undertaken a detailed allelic expression analysis of an imprinted domain on mouse proximal chromosome 10 comprising the paternally expressed Plagl1 gene. We identified three novel Plagl1 transcripts, only one of which contains protein-coding exons. In addition, we characterised two unspliced ncRNAs, Hymai, the mouse orthologue of HYMAI, and Plagl1it (Plagl1 intronic transcript), a transcript located in intron 5 of Plagl1. Imprinted expression of these novel ncRNAs requires DNMT3L-mediated maternal DNA methylation, which is also indispensable for establishing the correct chromatin profile at the Plagl1 DMR. Significantly, the two ncRNAs are retained in the nucleus, consistent with a potential regulatory function at the imprinted domain. Analysis with catRAPID, a protein-ncRNA association prediction algorithm, suggests that Hymai and Plagl1it RNAs both have potentially high affinity for Trithorax chromatin regulators. The two ncRNAs could therefore help to protect the paternal allele from DNA methylation by attracting Trithorax proteins that mediate H3 lysine-4 methylation.
Assuntos
Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , RNA Mensageiro/genética , RNA não Traduzido/genética , Fatores de Transcrição/genética , Alelos , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Regulação da Expressão Gênica , Genes Supressores de Tumor , Impressão Genômica , Camundongos , Dados de Sequência Molecular , Isoformas de RNA , Estabilidade de RNA , Transporte de RNA , Transcrição GênicaRESUMO
CETP inhibition increases HDL cholesterol levels and presumably could contribute to human atheroprotection via increasing macrophage-specific reverse cholesterol transport (RCT) and antioxidant properties of HDL. However, the impact of CETP activity variation on these two antiatherogenic functions of HDL remain unknown. In this study, we assessed the effects of overexpressing CETP in transgenic (Tg) mice on macrophage-specific RCT and HDL ability to protect against LDL oxidative modification. [(3)H]cholesterol-labeled macrophages were injected intraperitoneally into mice maintained on a chow diet or an atherogenic diet, after which the appearance of [(3)H]cholesterol in plasma, liver and feces over 48 h was determined. The degree of protection of oxidative modification of LDL coincubated with HDL was evaluated by measuring relative electrophoretic mobility and dichlorofluorescein fluorescence. CETP-Tg mice presented decreased radiolabeled HDL-bound [(3)H]cholesterol 24 and 48 h after the label injection. However, the magnitude of macrophage-derived [(3)H]cholesterol in liver and feces did not differ between CETP-Tg and control mice on either diet. Similar results were found when [(3)H]cholesterol-labeled endogenous peritoneal macrophages were injected into the CETP-Tg and control mice. Further, the injection of endogenous macrophages from CETP-Tg mice did not alter macrophage RCT in control mice. HDL from CETP-Tg and control mice protected LDL from oxidative modification similarly, and paraoxonase 1, platelet activated factor acetyl-hydrolase and lecithin-cholesterol acyl transferase activities of transgenic mice did not differ from those of control mice. In conclusion, CETP overexpression in transgenic mice does not affect RCT from macrophages to feces in vivo or the protection conferred by HDL against LDL oxidative modification.