RESUMEN
Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROß, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.
Asunto(s)
Movilización de Célula Madre Hematopoyética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/inmunología , Adulto , Animales , Bencilaminas , Quimiocina CXCL2/farmacología , Ciclamas , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos ICR , Polimorfismo GenéticoRESUMEN
Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.
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Epigenómica , Células Madre Hematopoyéticas/citología , Animales , Linaje de la Célula , Células Clonales/citología , Fluorescencia , Hematopoyesis , Inflamación/patología , Ratones , Transcripción GenéticaRESUMEN
Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development.
Asunto(s)
Empalme Alternativo , Corteza Cerebral/embriología , Células-Madre Neurales/citología , Neurogénesis/genética , Neuronas/citología , Animales , Centrosoma/metabolismo , Corteza Cerebral/anomalías , Corteza Cerebral/citología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Exones , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Ratones , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Dominios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Factores de Empalme de ARNRESUMEN
Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.
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Perfilación de la Expresión Génica , Enfermedades Renales , Riñón , Análisis de la Célula Individual , Transcriptoma , Humanos , Núcleo Celular/genética , Riñón/citología , Riñón/lesiones , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Transcriptoma/genética , Estudios de Casos y Controles , Imagenología TridimensionalRESUMEN
Tissue function depends on cellular organization. While the properties of individual cells are increasingly being deciphered using powerful single-cell sequencing technologies, understanding their spatial organization and temporal evolution remains a major challenge. Here, we present Image-seq, a technology that provides single-cell transcriptional data on cells that are isolated from specific spatial locations under image guidance, thus preserving the spatial information of the target cells. It is compatible with in situ and in vivo imaging and can document the temporal and dynamic history of the cells being analyzed. Cell samples are isolated from intact tissue and processed with state-of-the-art library preparation protocols. The technique therefore combines spatial information with highly sensitive RNA sequencing readouts from individual, intact cells. We have used both high-throughput, droplet-based sequencing as well as SMARTseq-v4 library preparation to demonstrate its application to bone marrow and leukemia biology. We discovered that DPP4 is a highly upregulated gene during early progression of acute myeloid leukemia and that it marks a more proliferative subpopulation that is confined to specific bone marrow microenvironments. Furthermore, the ability of Image-seq to isolate viable, intact cells should make it compatible with a range of downstream single-cell analysis tools including multi-omics protocols.
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Diagnóstico por Imagen , Leucemia , Humanos , Análisis de Secuencia de ARN , Recuento de Células , Biblioteca de Genes , Microambiente TumoralRESUMEN
Polycomb group (PcG) proteins are essential for accurate axial body patterning during embryonic development. PcG-mediated repression is conserved in metazoans and is targeted in Drosophila by Polycomb response elements (PREs). However, targeting sequences in humans have not been described. While analyzing chromatin architecture in the context of human embryonic stem cell (hESC) differentiation, we discovered a 1.8kb region between HOXD11 and HOXD12 (D11.12) that is associated with PcG proteins, becomes nuclease hypersensitive, and then shows alteration in nuclease sensitivity as hESCs differentiate. The D11.12 element repressed luciferase expression from a reporter construct and full repression required a highly conserved region and YY1 binding sites. Furthermore, repression was dependent on the PcG proteins BMI1 and EED and a YY1-interacting partner, RYBP. We conclude that D11.12 is a Polycomb-dependent regulatory region with similarities to Drosophila PREs, indicating conservation in the mechanisms that target PcG function in mammals and flies.
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Células Madre Embrionarias/metabolismo , Genes Homeobox/genética , Proteínas de Homeodominio/genética , Elementos Reguladores de la Transcripción , Proteínas Represoras/metabolismo , Diferenciación Celular , Cromatina/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
The rapid progress of protocols for sequencing single-cell transcriptomes over the past decade has been accompanied by equally impressive advances in the computational methods for analysis of such data. As capacity and accuracy of the experimental techniques grew, the emerging algorithm developments revealed increasingly complex facets of the underlying biology, from cell type composition to gene regulation to developmental dynamics. At the same time, rapid growth has forced continuous reevaluation of the underlying statistical models, experimental aims, and sheer volumes of data processing that are handled by these computational tools. Here, I review key computational steps of single-cell RNA sequencing (scRNA-seq) analysis, examine assumptions made by different approaches, and highlight successes, remaining ambiguities, and limitations that are important to keep in mind as scRNA-seq becomes a mainstream technique for studying biology.
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Biología Computacional/métodos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/fisiología , Gráficos por Computador , Bases de Datos Genéticas , Humanos , Ratones , Análisis de Componente Principal , Análisis de Secuencia de ARN/estadística & datos numéricos , Análisis de la Célula Individual/estadística & datos numéricos , Transcripción GenéticaRESUMEN
SUMMARY: scFates provides an extensive toolset for the analysis of dynamic trajectories comprising tree learning, feature association testing, branch differential expression and with a focus on cell biasing and fate splits at the level of bifurcations. It is meant to be fully integrated into the scanpy ecosystem for seamless analysis of trajectories from single-cell data of various modalities (e.g. RNA and ATAC). AVAILABILITY AND IMPLEMENTATION: scFates is released as open-source software under the BSD 3-Clause 'New' License and is available from the Python Package Index at https://pypi.org/project/scFates/. The source code is available on GitHub at https://github.com/LouisFaure/scFates/. Code reproduction and tutorials on published datasets are available on GitHub at https://github.com/LouisFaure/scFates_notebooks. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Ecosistema , Programas InformáticosRESUMEN
RNA abundance is a powerful indicator of the state of individual cells. Single-cell RNA sequencing can reveal RNA abundance with high quantitative accuracy, sensitivity and throughput1. However, this approach captures only a static snapshot at a point in time, posing a challenge for the analysis of time-resolved phenomena such as embryogenesis or tissue regeneration. Here we show that RNA velocity-the time derivative of the gene expression state-can be directly estimated by distinguishing between unspliced and spliced mRNAs in common single-cell RNA sequencing protocols. RNA velocity is a high-dimensional vector that predicts the future state of individual cells on a timescale of hours. We validate its accuracy in the neural crest lineage, demonstrate its use on multiple published datasets and technical platforms, reveal the branching lineage tree of the developing mouse hippocampus, and examine the kinetics of transcription in human embryonic brain. We expect RNA velocity to greatly aid the analysis of developmental lineages and cellular dynamics, particularly in humans.
Asunto(s)
Encéfalo/citología , Cresta Neural/metabolismo , Neuronas/citología , Empalme del ARN/genética , ARN/análisis , ARN/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Linaje de la Célula/genética , Células Cromafines/citología , Células Cromafines/metabolismo , Conjuntos de Datos como Asunto , Femenino , Ácido Glutámico/metabolismo , Hipocampo/citología , Hipocampo/embriología , Hipocampo/metabolismo , Cinética , Masculino , Ratones , Cresta Neural/citología , Neuronas/metabolismo , Reproducibilidad de los Resultados , Factores de Tiempo , Transcripción Genética/genéticaRESUMEN
NUT midline carcinoma (NMC), a subtype of squamous cell cancer, is one of the most aggressive human solid malignancies known. NMC is driven by the creation of a translocation oncoprotein, BRD4-NUT, which blocks differentiation and drives growth of NMC cells. BRD4-NUT forms distinctive nuclear foci in patient tumors, which we found correlate with â¼100 unprecedented, hyperacetylated expanses of chromatin that reach up to 2 Mb in size. These "megadomains" appear to be the result of aberrant, feed-forward loops of acetylation and binding of acetylated histones that drive transcription of underlying DNA in NMC patient cells and naïve cells induced to express BRD4-NUT. Megadomain locations are typically cell lineage-specific; however, the cMYC and TP63 regions are targeted in all NMCs tested and play functional roles in tumor growth. Megadomains appear to originate from select pre-existing enhancers that progressively broaden but are ultimately delimited by topologically associating domain (TAD) boundaries. Therefore, our findings establish a basis for understanding the powerful role played by large-scale chromatin organization in normal and aberrant lineage-specific gene transcription.
Asunto(s)
Carcinoma de Células Escamosas/fisiopatología , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Elementos de Facilitación Genéticos , Humanos , Proteínas de Neoplasias , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Estructura Terciaria de Proteína , Factores de Transcripción/genéticaRESUMEN
BACKGROUND & AIMS: Myeloid cells are key regulators of cirrhosis, a major cause of mortality worldwide. Because stromal cells can modulate the functionality of myeloid cells in vitro, targeting stromal-myeloid interactions has become an attractive potential therapeutic strategy. We aimed to investigate how human liver stromal cells impact myeloid cell properties and to understand the utility of a stromal-myeloid coculture system to study these interactions in the context of cirrhosis. METHODS: Single-cell RNA-sequencing analyses of non-cirrhotic (n = 7) and cirrhotic (n = 5) human liver tissue were correlated to the bulk RNA-sequencing results of in vitro cocultured human CD14+ and primary liver stromal cells. Complimentary mechanistic experiments and flow cytometric analysis were performed on human liver stromal-myeloid coculture systems. RESULTS: We found that stromal-myeloid coculture reduces the frequency CD14+ cell subsets transcriptionally similar to liver macrophages, showing that stromal cells inhibit the maturation of monocytes into macrophages. Stromal cells also influenced in vitro macrophage differentiation by skewing away from cirrhosis-linked CD9+ scar-associated macrophage-like cells and towards CD163+ Kupffer cell-like macrophages. We identify IL-6 production as a mechanism by which stromal cells limit CD9+ macrophage differentiation and find that local IL-6 levels are decreased in early-stage human liver disease compared to healthy liver tissue, suggesting a protective role for local IL-6 in the healthy liver. CONCLUSIONS: Our work reveals an unanticipated role for liver stromal cells in impeding the maturation and altering the differentiation of macrophages and should prompt investigations into the role of local IL-6 production in the pathogenesis of liver disease. These studies provide a framework for investigating macrophage-stromal interactions during cirrhosis. LAY SUMMARY: The impact of human liver stromal cells on myeloid cell maturation and differentiation in liver disease is incompletely understood. In this study, we present a mechanistic analysis using a primary in vitro human liver stromal-myeloid coculture system that is translated to liver disease using single-cell RNA sequencing analysis of cirrhotic and non-cirrhotic human liver tissue. Our work supports a role for stromal cell contact in restricting macrophage maturation and for stromal-derived IL-6 in limiting the differentiation of a cirrhotic macrophage subset.
Asunto(s)
Interleucina-6 , Hepatopatías , Diferenciación Celular , Humanos , Cirrosis Hepática/etiología , Hepatopatías/patología , Macrófagos/patología , Monocitos/patología , ARN , Células del Estroma/patologíaRESUMEN
Single-cell RNA sequencing is often applied in study designs that include multiple individuals, conditions or tissues. To identify recurrent cell subpopulations in such heterogeneous collections, we developed Conos, an approach that relies on multiple plausible inter-sample mappings to construct a global graph connecting all measured cells. The graph enables identification of recurrent cell clusters and propagation of information between datasets in multi-sample or atlas-scale collections.
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Médula Ósea/metabolismo , Biología Computacional/métodos , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de la Célula Individual/métodos , HumanosRESUMEN
RNA velocity has enabled the recovery of directed dynamic information from single-cell transcriptomics by connecting measurements to the underlying kinetics of gene expression. This approach has opened up new ways of studying cellular dynamics. Here, we review the current state of RNA velocity modeling approaches, discuss various examples illustrating limitations and potential pitfalls, and provide guidance on how the ensuing challenges may be addressed. We then outline future directions on how to generalize the concept of RNA velocity to a wider variety of biological systems and modalities.
Asunto(s)
ARN , Transcriptoma , Cinética , ARN/genéticaRESUMEN
Heterochromatin protein 1 (HP1a) has conserved roles in gene silencing and heterochromatin and is also implicated in transcription, DNA replication, and repair. Here we identify chromatin-associated protein and RNA interactions of HP1a by BioTAP-XL mass spectrometry and sequencing from Drosophila S2 cells, embryos, larvae, and adults. Our results reveal an extensive list of known and novel HP1a-interacting proteins, of which we selected three for validation. A strong novel interactor, dADD1 (Drosophila ADD1) (CG8290), is highly enriched in heterochromatin, harbors an ADD domain similar to human ATRX, displays selective binding to H3K9me2 and H3K9me3, and is a classic genetic suppressor of position-effect variegation. Unexpectedly, a second hit, HIPP1 (HP1 and insulator partner protein-1) (CG3680), is strongly connected to CP190-related complexes localized at putative insulator sequences throughout the genome in addition to its colocalization with HP1a in heterochromatin. A third interactor, the histone methyltransferase MES-4, is also enriched in heterochromatin. In addition to these protein-protein interactions, we found that HP1a selectively associated with a broad set of RNAs transcribed from repetitive regions. We propose that this rich network of previously undiscovered interactions will define how HP1a complexes perform their diverse functions in cells and developing organisms.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Heterocromatina/metabolismo , ARN/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Proteínas Portadoras/genética , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Estadios del Ciclo de Vida/fisiología , Unión Proteica , ARN/genética , Análisis de Secuencia de ARN , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genéticaRESUMEN
Characterization of intratumoral heterogeneity is critical to cancer therapy, as the presence of phenotypically diverse cell populations commonly fuels relapse and resistance to treatment. Although genetic variation is a well-studied source of intratumoral heterogeneity, the functional impact of most genetic alterations remains unclear. Even less understood is the relative importance of other factors influencing heterogeneity, such as epigenetic state or tumor microenvironment. To investigate the relationship between genetic and transcriptional heterogeneity in a context of cancer progression, we devised a computational approach called HoneyBADGER to identify copy number variation and loss of heterozygosity in individual cells from single-cell RNA-sequencing data. By integrating allele and normalized expression information, HoneyBADGER is able to identify and infer the presence of subclone-specific alterations in individual cells and reconstruct the underlying subclonal architecture. By examining several tumor types, we show that HoneyBADGER is effective at identifying deletions, amplifications, and copy-neutral loss-of-heterozygosity events and is capable of robustly identifying subclonal focal alterations as small as 10 megabases. We further apply HoneyBADGER to analyze single cells from a progressive multiple myeloma patient to identify major genetic subclones that exhibit distinct transcriptional signatures relevant to cancer progression. Other prominent transcriptional subpopulations within these tumors did not line up with the genetic subclonal structure and were likely driven by alternative, nonclonal mechanisms. These results highlight the need for integrative analysis to understand the molecular and phenotypic heterogeneity in cancer.
Asunto(s)
Heterogeneidad Genética , Mieloma Múltiple/genética , Neoplasias/genética , Transcripción Genética , Alelos , Biología Computacional , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mieloma Múltiple/patología , Mutación , Neoplasias/patología , Polimorfismo de Nucleótido Simple , Análisis de la Célula Individual/métodosRESUMEN
Neurogenesis in the adult brain, a powerful mechanism for neuronal plasticity and brain repair, is altered by aging and pathological conditions, including metabolic disorders. The search for mechanisms and therapeutic solutions to alter neurogenesis requires understanding of cell kinetics within neurogenic niches using a high-throughput quantitative approach. The challenge is in the dynamic nature of the process and multiple cell types involved, each having several potential modes of division or cell fate. Here we show that cell kinetics can be revealed through a combination of the BrdU/EdU pulse-chase, based on the circadian pattern of DNA replication, and a differential equations model that describes time-dependent cell densities. The model is validated through the analysis of cell kinetics in the cerebellar neurogenic niche of normal young adult male zebrafish, with cells quantified in 2D (sections), and with neuronal fate and reactivation of stem cells confirmed in 3D whole-brain images (CLARITY). We then reveal complex alterations in cell kinetics associated with accelerated aging due to chronic high caloric intake. Low activity of neuronal stem cells in this condition persists 2 months after reverting to normal diet, and is accompanied by overproduction of transient amplifying cells, their accelerated cell death, and slow migration of postmitotic progeny. This combined experimental and mathematical approach should allow for relatively high-throughput analysis of early signs of pathological and age-related changes in neurogenesis, evaluation of specific therapeutic targets, and drug efficacy.SIGNIFICANCE STATEMENT Understanding normal cell kinetics of adult neurogenesis and the type of cells affected by a pathological process is needed to develop effective prophylactic and therapeutic measures directed at specific cell targets. Complex time-dependent mechanisms involved in the kinetics of multiple cell types require a combination of experimental and mathematical modeling approaches. This study demonstrates such a combined approach by comparing normal neurogenesis with that altered by diet-induced accelerated aging in adult zebrafish.
Asunto(s)
Envejecimiento Prematuro/patología , Dieta/efectos adversos , Ingestión de Energía , Neurogénesis/fisiología , Nicho de Células Madre/fisiología , Pez Cebra/fisiología , Animales , Encéfalo/diagnóstico por imagen , División Celular , Ritmo Circadiano , Replicación del ADN , Hiperfagia/patología , Cinética , Imagen por Resonancia Magnética , Masculino , Mitosis , Modelos Teóricos , Células-Madre NeuralesRESUMEN
Intra-tumoral genetic heterogeneity has been characterized across cancers by genome sequencing of bulk tumors, including chronic lymphocytic leukemia (CLL). In order to more accurately identify subclones, define phylogenetic relationships, and probe genotype-phenotype relationships, we developed methods for targeted mutation detection in DNA and RNA isolated from thousands of single cells from five CLL samples. By clearly resolving phylogenic relationships, we uncovered mutated LCP1 and WNK1 as novel CLL drivers, supported by functional evidence demonstrating their impact on CLL pathways. Integrative analysis of somatic mutations with transcriptional states prompts the idea that convergent evolution generates phenotypically similar cells in distinct genetic branches, thus creating a cohesive expression profile in each CLL sample despite the presence of genetic heterogeneity. Our study highlights the potential for single-cell RNA-based targeted analysis to sensitively determine transcriptional and mutational profiles of individual cancer cells, leading to increased understanding of driving events in malignancy.
Asunto(s)
Biomarcadores de Tumor/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Mutación , Análisis de Secuencia de ADN/métodos , Análisis de la Célula Individual/métodos , Adulto , Estudios de Casos y Controles , Evolución Molecular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Transcripción GenéticaRESUMEN
Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.
Asunto(s)
Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Cromatina/genética , Cromatina/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Animales , Línea Celular , Centrómero/genética , Centrómero/metabolismo , Cromatina/química , Ensamble y Desensamble de Cromatina/genética , Replicación del ADN/genética , Elementos de Facilitación Genéticos/genética , Epigénesis Genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Anotación de Secuencia Molecular , Lámina Nuclear/metabolismo , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas/genética , Especificidad de la EspecieRESUMEN
To investigate the mechanism that drives dramatic mistargeting of active chromatin in NUT midline carcinoma (NMC), we have identified protein interactions unique to the BRD4-NUT fusion oncoprotein compared with wild-type BRD4. Using cross-linking, affinity purification, and mass spectrometry, we identified the EP300 acetyltransferase as uniquely associated with BRD4 through the NUT fusion in both NMC and non-NMC cell types. We also discovered ZNF532 associated with BRD4-NUT in NMC patient cells but not detectable in 293T cells. EP300 and ZNF532 are both implicated in feed-forward regulatory loops leading to propagation of the oncogenic chromatin complex in BRD4-NUT patient cells. Adding key functional significance to our biochemical findings, we independently discovered a ZNF532-NUT translocation fusion in a newly diagnosed NMC patient. ChIP sequencing of the major players NUT, ZNF532, BRD4, EP300, and H3K27ac revealed the formation of ZNF532-NUT-associated hyperacetylated megadomains, distinctly localized but otherwise analogous to those found in BRD4-NUT patient cells. Our results support a model in which NMC is dependent on ectopic NUT-mediated interactions between EP300 and components of BRD4 regulatory complexes, leading to a cascade of misregulation.