RESUMEN
In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.
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Cromatina , Epigénesis Genética , Genotipo , Mutación , Análisis de la Célula Individual , Animales , Femenino , Humanos , Masculino , Ratones , Antígenos CD34/metabolismo , Diferenciación Celular/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Epigénesis Genética/genética , Epigenoma/genética , Genoma Mitocondrial/genética , Técnicas de Genotipaje , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Inflamación/genética , Inflamación/patología , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Megacariocitos/metabolismo , Megacariocitos/patología , Proteínas de la Membrana/genética , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , ARN/genética , Células Clonales/metabolismoRESUMEN
Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.
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Inestabilidad Cromosómica , Segregación Cromosómica , Cromosomas , Epigénesis Genética , Micronúcleos con Defecto Cromosómico , Neoplasias , Animales , Humanos , Ratones , Cromatina/genética , Inestabilidad Cromosómica/genética , Cromosomas/genética , Cromosomas/metabolismo , Histonas/química , Histonas/metabolismo , Neoplasias/genética , Neoplasias/patología , Mitosis , Variaciones en el Número de Copia de ADN , Procesamiento Proteico-PostraduccionalRESUMEN
To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.
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Biocatálisis , Histonas/metabolismo , Oncogenes , Transcripción Genética , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Línea Celular , Cromatina/metabolismo , Proteínas Co-Represoras/metabolismo , Secuencia Conservada , Evolución Molecular , Redes Reguladoras de Genes , Genoma , Histona Desacetilasas/metabolismo , Humanos , Cinética , Metilación , Modelos Biológicos , ARN Polimerasa II/metabolismoRESUMEN
Polycomb repressive complex 2 (PRC2) maintains gene silencing by catalyzing methylation of histone H3 at lysine 27 (H3K27me2/3) within chromatin. By designing a system whereby PRC2-mediated repressive domains were collapsed and then reconstructed in an inducible fashion in vivo, a two-step mechanism of H3K27me2/3 domain formation became evident. First, PRC2 is stably recruited by the actions of JARID2 and MTF2 to a limited number of spatially interacting "nucleation sites," creating H3K27me3-forming Polycomb foci within the nucleus. Second, PRC2 is allosterically activated via its binding to H3K27me3 and rapidly spreads H3K27me2/3 both in cis and in far-cis via long-range contacts. As PRC2 proceeds further from the nucleation sites, its stability on chromatin decreases such that domains of H3K27me3 remain proximal, and those of H3K27me2 distal, to the nucleation sites. This study demonstrates the principles of de novo establishment of PRC2-mediated repressive domains across the genome.
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Complejo Represivo Polycomb 2/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Animales , Cromatina/metabolismo , Silenciador del Gen , Código de Histonas , Histonas/metabolismo , Lisina/metabolismo , Metilación , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
In 2021, the World Health Organization reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH)-wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intratumoral heterogeneity is a key contributor to therapeutic failure. To better define this heterogeneity, genome-wide chromatin accessibility and transcription profiles of clinical samples of glioblastomas and G4 IDHm astrocytomas were analyzed at single-cell resolution. These profiles afforded resolution of intratumoral genetic heterogeneity, including delineation of cell-to-cell variations in distinct cell states, focal gene amplifications, as well as extrachromosomal circular DNAs. Despite differences in IDH mutation status and significant intratumoral heterogeneity, the profiled tumor cells shared a common chromatin structure defined by open regions enriched for nuclear factor 1 transcription factors (NFIA and NFIB). Silencing of NFIA or NFIB suppressed in vitro and in vivo growths of patient-derived glioblastomas and G4 IDHm astrocytoma models. These findings suggest that despite distinct genotypes and cell states, glioblastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intratumoral heterogeneity.
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Astrocitoma , Neoplasias Encefálicas , Glioblastoma , Adulto , Humanos , Glioblastoma/genética , Glioblastoma/patología , Cromatina/genética , Transcriptoma , Astrocitoma/genética , Astrocitoma/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Mutación , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismoRESUMEN
Precision oncology hinges on linking tumour genotype with molecularly targeted drugs1; however, targeting the frequently dysregulated metabolic landscape of cancer has proven to be a major challenge2. Here we show that tissue context is the major determinant of dependence on the nicotinamide adenine dinucleotide (NAD) metabolic pathway in cancer. By analysing more than 7,000 tumours and 2,600 matched normal samples of 19 tissue types, coupled with mathematical modelling and extensive in vitro and in vivo analyses, we identify a simple and actionable set of 'rules'. If the rate-limiting enzyme of de novo NAD synthesis, NAPRT, is highly expressed in a normal tissue type, cancers that arise from that tissue will have a high frequency of NAPRT amplification and be completely and irreversibly dependent on NAPRT for survival. By contrast, tumours that arise from normal tissues that do not express NAPRT highly are entirely dependent on the NAD salvage pathway for survival. We identify the previously unknown enhancer that underlies this dependence. Amplification of NAPRT is shown to generate a pharmacologically actionable tumour cell dependence for survival. Dependence on another rate-limiting enzyme of the NAD synthesis pathway, NAMPT, as a result of enhancer remodelling is subject to resistance by NMRK1-dependent synthesis of NAD. These results identify a central role for tissue context in determining the choice of NAD biosynthetic pathway, explain the failure of NAMPT inhibitors, and pave the way for more effective treatments.
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Elementos de Facilitación Genéticos/genética , Amplificación de Genes , NAD/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Animales , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/metabolismo , Muerte Celular , Línea Celular Tumoral , Citocinas/antagonistas & inhibidores , Citocinas/genética , Citocinas/metabolismo , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Neoplasias/enzimología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/genética , Nicotinamida Fosforribosiltransferasa/metabolismo , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
Oncogenes are commonly amplified on particles of extrachromosomal DNA (ecDNA) in cancer1,2, but our understanding of the structure of ecDNA and its effect on gene regulation is limited. Here, by integrating ultrastructural imaging, long-range optical mapping and computational analysis of whole-genome sequencing, we demonstrate the structure of circular ecDNA. Pan-cancer analyses reveal that oncogenes encoded on ecDNA are among the most highly expressed genes in the transcriptome of the tumours, linking increased copy number with high transcription levels. Quantitative assessment of the chromatin state reveals that although ecDNA is packaged into chromatin with intact domain structure, it lacks higher-order compaction that is typical of chromosomes and displays significantly enhanced chromatin accessibility. Furthermore, ecDNA is shown to have a significantly greater number of ultra-long-range interactions with active chromatin, which provides insight into how the structure of circular ecDNA affects oncogene function, and connects ecDNA biology with modern cancer genomics and epigenetics.
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Cromatina/genética , ADN Circular/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/genética , Oncogenes/genética , Línea Celular Tumoral , Cromatina/química , ADN Circular/genética , Humanos , Microscopía Electrónica de Rastreo , Neoplasias/fisiopatologíaRESUMEN
Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated 'regulatory hotspots' around genes closely associated with progenitor programs. To examine their functional significance, we deleted 'hotspot' enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.
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Diferenciación Celular/genética , Redes Reguladoras de Genes , Nefronas/embriología , Organogénesis/genética , Células Madre/fisiología , Factores de Transcripción/fisiología , Animales , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Proteína Wnt4/genética , Proteína Wnt4/fisiologíaRESUMEN
Hi-C and chromatin immunoprecipitation (ChIP) have been combined to identify long-range chromatin interactions genome-wide at reduced cost and enhanced resolution, but extracting information from the resulting datasets has been challenging. Here we describe a computational method, MAPS, Model-based Analysis of PLAC-seq and HiChIP, to process the data from such experiments and identify long-range chromatin interactions. MAPS adopts a zero-truncated Poisson regression framework to explicitly remove systematic biases in the PLAC-seq and HiChIP datasets, and then uses the normalized chromatin contact frequencies to identify significant chromatin interactions anchored at genomic regions bound by the protein of interest. MAPS shows superior performance over existing software tools in the analysis of chromatin interactions from multiple PLAC-seq and HiChIP datasets centered on different transcriptional factors and histone marks. MAPS is freely available at https://github.com/ijuric/MAPS.
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Ensamble y Desensamble de Cromatina/fisiología , Mapeo Cromosómico/métodos , Biología Computacional/métodos , Cromatina/metabolismo , Cromatina/fisiología , Inmunoprecipitación de Cromatina/métodos , Simulación por Computador , Genoma , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Código de Histonas , Humanos , Análisis de Secuencia de ADN/métodos , Programas InformáticosRESUMEN
Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these. Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEκ, 3'Eκ and Edκ, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings.
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Cromosomas/metabolismo , Sitios Genéticos , Conformación de Ácido Nucleico , Análisis de Secuencia de ADN/métodos , Cromatina/metabolismo , Elementos de Facilitación Genéticos , Receptor beta de Estrógeno/metabolismo , Genoma , Receptores de Antígenos de Linfocitos T alfa-betaRESUMEN
4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or "bait") that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes.
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ADN Catalítico/química , ADN Catalítico/genética , Genoma/fisiología , Mapeo Restrictivo/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Algoritmos , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Somatic mosaicism is a hallmark of malignancy that is also pervasively observed in human physiological aging, with clonal expansions of cells harboring mutations in recurrently mutated driver genes. Bulk sequencing of tissue microdissection captures mutation frequencies, but cannot distinguish which mutations co-occur in the same clones to reconstruct clonal architectures, nor phenotypically profile clonal populations to delineate how driver mutations impact cellular behavior. To address these challenges, we developed single-cell Genotype-to-Phenotype sequencing (scG2P) for high-throughput, highly-multiplexed, single-cell joint capture of recurrently mutated genomic regions and mRNA phenotypic markers in cells or nuclei isolated from solid tissues. We applied scG2P to aged esophagus samples from five individuals with high alcohol and tobacco exposure and observed a clonal landscape dominated by a large number of clones with a single driver event, but only rare clones with two driver mutations. NOTCH1 mutants dominate the clonal landscape and are linked to stunted epithelial differentiation, while TP53 mutants and double-driver mutants promote clonal expansion through both differentiation biases and increased cell cycling. Thus, joint single-cell highly multiplexed capture of somatic mutations and mRNA transcripts enables high resolution reconstruction of clonal architecture and associated phenotypes in solid tissue somatic mosaicism.
RESUMEN
A subset of patients with IDH-mutant glioma respond to inhibitors of mutant IDH (IDHi), yet the molecular underpinnings of such responses are not understood. Here, we profiled by single-cell or single-nucleus RNA-sequencing three IDH-mutant oligodendrogliomas from patients who derived clinical benefit from IDHi. Importantly, the tissues were sampled on-drug, four weeks from treatment initiation. We further integrate our findings with analysis of single-cell and bulk transcriptomes from independent cohorts and experimental models. We find that IDHi treatment induces a robust differentiation toward the astrocytic lineage, accompanied by a depletion of stem-like cells and a reduction of cell proliferation. Furthermore, mutations in NOTCH1 are associated with decreased astrocytic differentiation and may limit the response to IDHi. Our study highlights the differentiating potential of IDHi on the cellular hierarchies that drive oligodendrogliomas and suggests a genetic modifier that may improve patient stratification.
Asunto(s)
Neoplasias Encefálicas , Diferenciación Celular , Isocitrato Deshidrogenasa , Mutación , Oligodendroglioma , Oligodendroglioma/genética , Oligodendroglioma/patología , Oligodendroglioma/tratamiento farmacológico , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Humanos , Diferenciación Celular/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/tratamiento farmacológico , Linaje de la Célula/efectos de los fármacos , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proliferación Celular/efectos de los fármacos , Animales , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/patología , Ratones , Análisis de la Célula Individual/métodosRESUMEN
In meiotic prophase, chromosomes are organized into compacted loop arrays to promote homolog pairing and recombination. Here, we probe the architecture of the mouse spermatocyte genome in early and late meiotic prophase using chromosome conformation capture (Hi-C). Our data support the established loop array model of meiotic chromosomes, and infer loops averaging 0.8-1.0 megabase pairs (Mb) in early prophase and extending to 1.5-2.0 Mb in late prophase as chromosomes compact and homologs undergo synapsis. Topologically associating domains (TADs) are lost in meiotic prophase, suggesting that assembly of the meiotic chromosome axis alters the activity of chromosome-associated cohesin complexes. While TADs are lost, physically separated A and B compartments are maintained in meiotic prophase. Moreover, meiotic DNA breaks and interhomolog crossovers preferentially form in the gene-dense A compartment, revealing a role for chromatin organization in meiotic recombination. Finally, direct detection of interhomolog contacts genome-wide reveals the structural basis for homolog alignment and juxtaposition by the synaptonemal complex.
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Emparejamiento Cromosómico/genética , Recombinación Homóloga/genética , Profase Meiótica I/genética , Espermatogénesis/genética , Animales , Cromatina/metabolismo , Cromosomas/metabolismo , Roturas del ADN , Genoma/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Espermatocitos/citología , Complejo Sinaptonémico/metabolismoRESUMEN
Long-range enhancers govern the temporal and spatial control of gene expression; however, the mechanisms that regulate enhancer activity during normal and malignant development remain poorly understood. Here, we demonstrate a role for aberrant chromatin accessibility in the regulation of MYC expression in T-cell lymphoblastic leukemia (T-ALL). Central to this process, the NOTCH1-MYC enhancer (N-Me), a long-range T cell-specific MYC enhancer, shows dynamic changes in chromatin accessibility during T-cell specification and maturation and an aberrant high degree of chromatin accessibility in mouse and human T-ALL cells. Mechanistically, we demonstrate that GATA3-driven nucleosome eviction dynamically modulates N-Me enhancer activity and is strictly required for NOTCH1-induced T-ALL initiation and maintenance. These results directly implicate aberrant regulation of chromatin accessibility at oncogenic enhancers as a mechanism of leukemic transformation. SIGNIFICANCE: MYC is a major effector of NOTCH1 oncogenic programs in T-ALL. Here, we show a major role for GATA3-mediated enhancer nucleosome eviction as a driver of MYC expression and leukemic transformation. These results support the role of aberrant chromatin accessibility and consequent oncogenic MYC enhancer activation in NOTCH1-induced T-ALL.This article is highlighted in the In This Issue feature, p. 1631.
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Elementos de Facilitación Genéticos , Factor de Transcripción GATA3/metabolismo , Leucemia de Células T/patología , Nucleosomas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Células Jurkat , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Ratones , Trasplante de Neoplasias , Receptor Notch1/metabolismoRESUMEN
BACKGROUND: The organization of chromatin in the nucleus plays an essential role in gene regulation. About half of the mammalian genome comprises transposable elements. Given their repetitive nature, reads associated with these elements are generally discarded or randomly distributed among elements of the same type in genome-wide analyses. Thus, it is challenging to identify the activities and properties of individual transposons. As a result, we only have a partial understanding of how transposons contribute to chromatin folding and how they impact gene regulation. RESULTS: Using PCR and Capture-based chromosome conformation capture (3C) approaches, collectively called 4Tran, we take advantage of the repetitive nature of transposons to capture interactions from multiple copies of endogenous retrovirus (ERVs) in the human and mouse genomes. With 4Tran-PCR, reads are selectively mapped to unique regions in the genome. This enables the identification of transposable element interaction profiles for individual ERV families and integration events specific to particular genomes. With this approach, we demonstrate that transposons engage in long-range intra-chromosomal interactions guided by the separation of chromosomes into A and B compartments as well as topologically associated domains (TADs). In contrast to 4Tran-PCR, Capture-4Tran can uniquely identify both ends of an interaction that involve retroviral repeat sequences, providing a powerful tool for uncovering the individual transposable element insertions that interact with and potentially regulate target genes. CONCLUSIONS: 4Tran provides new insight into the manner in which transposons contribute to chromosome architecture and identifies target genes that transposable elements can potentially control.
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Elementos Transponibles de ADN , Regulación de la Expresión Génica , Genómica/métodos , Animales , Retrovirus Endógenos , Humanos , Ratones , Polimorfismo GenéticoRESUMEN
The inheritance of gene expression patterns is dependent on epigenetic regulation, but the establishment and maintenance of epigenetic landscapes during T cell differentiation are incompletely understood. Here we show that two stage-specific Cd4 cis-elements, the previously characterized enhancer E4p and a novel enhancer E4m, coordinately promote Cd4 transcription in mature thymic MHC-II-specific T cells, in part through the canonical Wnt pathway. Specifically, E4p licenses E4m to orchestrate DNA demethylation by TET1 and TET3, which in turn poises the Cd4 locus for transcription in peripheral T cells. Cd4 locus demethylation is important for subsequent Cd4 transcription in activated peripheral T cells wherein these cis-elements become dispensable. By contrast, in developing thymocytes the loss of TET1/3 does not affect Cd4 transcription, highlighting an uncoupled event between transcription and epigenetic modifications. Together our findings reveal an important function for thymic cis-elements in governing gene expression in the periphery via a heritable epigenetic mechanism.
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Antígenos CD4/genética , Linfocitos T CD4-Positivos/fisiología , Elementos de Facilitación Genéticos/fisiología , Epigénesis Genética/fisiología , Regulación de la Expresión Génica/fisiología , Animales , Antígenos CD4/metabolismo , Diferenciación Celular/genética , Quimera , Desmetilación del ADN , Metilación de ADN/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dioxigenasas , Femenino , Técnicas de Inactivación de Genes , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Timo/citología , Timo/fisiologíaRESUMEN
In the version of this article initially published online, the accession code was given as GSE1000333. The correct code is GSE100033. The error has been corrected in the print, HTML and PDF versions of the article.
RESUMEN
Analysis of chromatin accessibility can reveal transcriptional regulatory sequences, but heterogeneity of primary tissues poses a significant challenge in mapping the precise chromatin landscape in specific cell types. Here we report single-nucleus ATAC-seq, a combinatorial barcoding-assisted single-cell assay for transposase-accessible chromatin that is optimized for use on flash-frozen primary tissue samples. We apply this technique to the mouse forebrain through eight developmental stages. Through analysis of more than 15,000 nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell types. We further define cell-type-specific transcriptional regulatory sequences, infer potential master transcriptional regulators and delineate developmental changes in forebrain cellular composition. Our results provide insight into the molecular and cellular dynamics that underlie forebrain development in the mouse and establish technical and analytical frameworks that are broadly applicable to other heterogeneous tissues.
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Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Prosencéfalo/crecimiento & desarrollo , Animales , Línea Celular , Proteínas de Unión al ADN , Femenino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Proteínas Nucleares/metabolismo , Embarazo , Prosencéfalo/citología , Prosencéfalo/metabolismo , Análisis de la Célula IndividualRESUMEN
Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.