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1.
Cell ; 175(7): 1972-1988.e16, 2018 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-30550791

RESUMEN

In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.


Asunto(s)
Modelos Inmunológicos , Neoplasias Experimentales/inmunología , Organoides/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Animales , Antígeno B7-H1/inmunología , Técnicas de Cocultivo , Femenino , Humanos , Inmunoterapia , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Organoides/patología
2.
Nature ; 588(7839): 670-675, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33238290

RESUMEN

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.


Asunto(s)
COVID-19/virología , Pulmón/citología , Modelos Biológicos , Organoides/citología , Organoides/virología , SARS-CoV-2/fisiología , Técnicas de Cultivo de Tejidos , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , COVID-19/metabolismo , COVID-19/patología , Diferenciación Celular , División Celular , Células Clonales/citología , Células Clonales/metabolismo , Células Clonales/virología , Humanos , Técnicas In Vitro , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Integrina alfa6/análisis , Integrina beta4/análisis , Queratina-5/análisis , Organoides/metabolismo , Neumonía Viral/metabolismo , Neumonía Viral/patología , Neumonía Viral/virología , SARS-CoV-2/crecimiento & desarrollo , Análisis de la Célula Individual , Receptor de TWEAK/análisis
3.
Nature ; 545(7653): 238-242, 2017 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-28467820

RESUMEN

The canonical Wnt/ß-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling ß-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/ß-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.


Asunto(s)
Autorrenovación de las Células , Intestinos/citología , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/citología , Células Madre/metabolismo , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Animales , Linaje de la Célula , Proliferación Celular , Femenino , Humanos , Ligandos , Masculino , Ratones , Organoides/citología , Organoides/crecimiento & desarrollo , Análisis de la Célula Individual , Nicho de Células Madre , Transcriptoma , Ubiquitina-Proteína Ligasas/metabolismo , beta Catenina/metabolismo
4.
Blood ; 133(10): 1119-1129, 2019 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-30591526

RESUMEN

Follicular lymphoma (FL) is a low-grade B-cell malignancy that transforms into a highly aggressive and lethal disease at a rate of 2% per year. Perfect isolation of the malignant B-cell population from a surgical biopsy is a significant challenge, masking important FL biology, such as immune checkpoint coexpression patterns. To resolve the underlying transcriptional networks of follicular B-cell lymphomas, we analyzed the transcriptomes of 34 188 cells derived from 6 primary FL tumors. For each tumor, we identified normal immune subpopulations and malignant B cells, based on gene expression. We used multicolor flow cytometry analysis of the same tumors to confirm our assignments of cellular lineages and validate our predictions of expressed proteins. Comparison of gene expression between matched malignant and normal B cells from the same patient revealed tumor-specific features. Malignant B cells exhibited restricted immunoglobulin (Ig) light chain expression (either Igκ or Igλ), as well the expected upregulation of the BCL2 gene, but also downregulation of the FCER2, CD52, and major histocompatibility complex class II genes. By analyzing thousands of individual cells per patient tumor, we identified the mosaic of malignant B-cell subclones that coexist within a FL and examined the characteristics of tumor-infiltrating T cells. We identified genes coexpressed with immune checkpoint molecules, such as CEBPA and B2M in regulatory T (Treg) cells, providing a better understanding of the gene networks involved in immune regulation. In summary, parallel measurement of single-cell expression in thousands of tumor cells and tumor-infiltrating lymphocytes can be used to obtain a systems-level view of the tumor microenvironment and identify new avenues for therapeutic development.


Asunto(s)
Linfoma de Células B/genética , Linfoma Folicular/genética , Linfocitos T Reguladores/citología , Biopsia , Proteínas Potenciadoras de Unión a CCAAT/genética , Linfocitos T CD4-Positivos/citología , Antígeno CD52/genética , Linaje de la Célula , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/citología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Sistema Inmunológico , Inmunoglobulina G , Lectinas Tipo C/genética , Leucocitos Mononucleares/citología , Linfoma de Células B/sangre , Linfoma Folicular/sangre , Tonsila Palatina/metabolismo , Receptores de IgE/genética , Análisis de Secuencia de ARN , Transcriptoma , Microambiente Tumoral , Microglobulina beta-2/genética
5.
Mol Cell ; 47(5): 734-45, 2012 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-22819322

RESUMEN

C. elegans 21U-RNAs are equivalent to the piRNAs discovered in other metazoans and have important roles in gametogenesis and transposon control. The biogenesis and molecular function of 21U-RNAs and piRNAs are poorly understood. Here, we demonstrate that transcription of each 21U-RNA is regulated separately through a conserved upstream DNA motif. We use genomic analysis to show that this motif is associated with low nucleosome occupancy, a characteristic of many promoters that drive expression of protein-coding genes, and that RNA polymerase II is localized to this nucleosome-depleted region. We establish that the most conserved 8-mer sequence in the upstream region of 21U-RNAs, CTGTTTCA, is absolutely required for their individual expression. Furthermore, we demonstrate that the 8-mer is specifically recognized by Forkhead family (FKH) transcription factors and that 21U-RNA expression is diminished in several FKH mutants. Our results suggest that thousands of small noncoding transcription units are regulated by FKH proteins.


Asunto(s)
Caenorhabditis elegans/genética , Factores de Transcripción Forkhead/metabolismo , Regiones Promotoras Genéticas/genética , ARN de Helminto/genética , ARN Interferente Pequeño/genética , Animales , Secuencia de Bases , Perfilación de la Expresión Génica , Nucleosomas/genética , ARN Polimerasa II/metabolismo
6.
Genes Dev ; 26(4): 338-43, 2012 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-22302877

RESUMEN

Long noncoding RNAs (lncRNAs) regulate diverse processes, yet a potential role for lncRNAs in maintaining the undifferentiated state in somatic tissue progenitor cells remains uncharacterized. We used transcriptome sequencing and tiling arrays to compare lncRNA expression in epidermal progenitor populations versus differentiating cells. We identified ANCR (anti-differentiation ncRNA) as an 855-base-pair lncRNA down-regulated during differentiation. Depleting ANCR in progenitor-containing populations, without any other stimuli, led to rapid differentiation gene induction. In epidermis, ANCR loss abolished the normal exclusion of differentiation from the progenitor-containing compartment. The ANCR lncRNA is thus required to enforce the undifferentiated cell state within epidermis.


Asunto(s)
Diferenciación Celular , Queratinocitos/citología , ARN no Traducido/metabolismo , Células Madre/citología , Células Cultivadas , Células Epidérmicas , Regulación del Desarrollo de la Expresión Génica , Interferencia de ARN , ARN Largo no Codificante , ARN no Traducido/genética , Transcriptoma
7.
Nature ; 493(7431): 231-5, 2013 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-23201690

RESUMEN

Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high messenger RNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a range of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25-nucleotide 'TINCR box' motif that is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyse TINCR binding capacity to approximately 9,400 human recombinant proteins revealed direct binding of TINCR RNA to the staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay, however, did not have differentiation effects. Instead, the TINCR-STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression.


Asunto(s)
Diferenciación Celular/genética , Células Epidérmicas , Epidermis/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Secuencia de Bases , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Proteínas Filagrina , Regulación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Queratinocitos , Mutación , Motivos de Nucleótidos/genética , Unión Proteica , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Enfermedades de la Piel/genética
8.
Proc Natl Acad Sci U S A ; 108(38): 15804-9, 2011 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-21911408

RESUMEN

Variations in microRNA (miRNA) gene and/or target repertoire are likely to be key drivers of phenotypic differences between species. To better understand these changes, we developed a computational method that identifies signatures of species-specific target site gain and loss associated with miRNA acquisition. Interestingly, several of the miRNAs implicated in mouse 3' UTR evolution derive from a single rapidly expanded rodent-specific miRNA cluster. Located in the intron of Sfmbt2, a maternally imprinted polycomb gene, these miRNAs (referred to as the Sfmbt2 cluster) are expressed in both embryonic stem cells and the placenta. One abundant miRNA from the cluster, miR-467a, functionally overlaps with the mir-290-295 cluster in promoting growth and survival of mouse embryonic stem cells. Predicted novel targets of the remaining cluster members are enriched in pathways regulating cell survival. Two relevant species-specific target candidates, Lats2 and Dedd2, were validated in cultured cells. We suggest that the rapid evolution of the Sfmbt2 cluster may be a result of intersex conflict for growth regulation in early mammalian development and could provide a general model for the genomic response to acquisition of miRNAs and similar regulatory factors.


Asunto(s)
Genoma/genética , Ratones/genética , MicroARNs/genética , Familia de Multigenes , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Northern Blotting , Supervivencia Celular/genética , Células Cultivadas , Mapeo Cromosómico , ARN Helicasas DEAD-box/genética , Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica , Humanos , Ratones Noqueados , MicroARNs/clasificación , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Interferencia de ARN , Proteínas Represoras , Ribonucleasa III/genética , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/genética
9.
PLoS Genet ; 7(5): e1002054, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21573140

RESUMEN

MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target.


Asunto(s)
Células Madre Embrionarias/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Secuencia de Bases , Caspasa 2/genética , Caspasa 2/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Doxorrubicina/farmacología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/efectos de la radiación , Rayos gamma , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal
10.
Cell Rep ; 33(3): 108273, 2020 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-33086071

RESUMEN

The mammary epithelial cell (MEC) system is a bilayered ductal epithelium of luminal and basal cells, maintained by a lineage of stem and progenitor populations. Here, we used integrated single-cell transcriptomics and chromatin accessibility analysis to reconstruct the cell types of the mouse MEC system and their underlying gene regulatory features in an unbiased manner. We define differentiation states within the secretory type of luminal cells, which forms a continuous spectrum of general luminal progenitor and lactation-committed progenitor cells. By integrating single-cell transcriptomics and chromatin accessibility landscapes, we identify cis- and trans-regulatory elements that are differentially activated in the specific epithelial cell types and our newly defined luminal differentiation states. Our work provides a resource to reveal cis/trans-regulatory elements associated with MEC identity and differentiation that will serve as a reference to determine how the chromatin accessibility landscape changes during breast cancer.


Asunto(s)
Cromatina/genética , Células Epiteliales/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular/genética , Linaje de la Célula , Proliferación Celular/genética , Cromatina/fisiología , Biología Computacional/métodos , Células Epiteliales/fisiología , Epitelio/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de la Célula Individual/métodos , Células Madre/metabolismo , Transcriptoma
11.
bioRxiv ; 2020 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-32743583

RESUMEN

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5 + basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5 + cells contained a distinct ITGA6 + ITGB4 + mitotic population whose proliferation segregated to a TNFRSF12A hi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12A hi subset of FACS-purified ITGA6 + ITGB4 + basal cells from human lung or derivative organoids. In vivo, TNFRSF12A + cells comprised ~10% of KRT5 + basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 apical-out organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.

12.
Nat Biotechnol ; 37(12): 1458-1465, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31792411

RESUMEN

Identifying the causes of human diseases requires deconvolution of abnormal molecular phenotypes spanning DNA accessibility, gene expression and protein abundance1-3. We present a single-cell framework that integrates highly multiplexed protein quantification, transcriptome profiling and analysis of chromatin accessibility. Using this approach, we establish a normal epigenetic baseline for healthy blood development, which we then use to deconvolve aberrant molecular features within blood from patients with mixed-phenotype acute leukemia4,5. Despite widespread epigenetic heterogeneity within the patient cohort, we observe common malignant signatures across patients as well as patient-specific regulatory features that are shared across phenotypic compartments of individual patients. Integrative analysis of transcriptomic and chromatin-accessibility maps identified 91,601 putative peak-to-gene linkages and transcription factors that regulate leukemia-specific genes, such as RUNX1-linked regulatory elements proximal to the marker gene CD69. These results demonstrate how integrative, multiomic analysis of single cells within the framework of normal development can reveal both distinct and shared molecular mechanisms of disease from patient samples.


Asunto(s)
Cromatina/genética , Leucemia Bifenotípica Aguda/genética , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Células de la Médula Ósea/citología , Cromatina/química , Análisis por Conglomerados , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Epigénesis Genética/genética , Epigenómica/métodos , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Secuencias Reguladoras de Ácidos Nucleicos/genética
13.
Nat Biotechnol ; 37(8): 925-936, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31375813

RESUMEN

Understanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here, we assess the performance of a massively parallel droplet-based method for mapping transposase-accessible chromatin in single cells using sequencing (scATAC-seq). We apply scATAC-seq to obtain chromatin profiles of more than 200,000 single cells in human blood and basal cell carcinoma. In blood, application of scATAC-seq enables marker-free identification of cell type-specific cis- and trans-regulatory elements, mapping of disease-associated enhancer activity and reconstruction of trajectories of cellular differentiation. In basal cell carcinoma, application of scATAC-seq reveals regulatory networks in malignant, stromal and immune cells in the tumor microenvironment. Analysis of scATAC-seq profiles from serial tumor biopsies before and after programmed cell death protein 1 blockade identifies chromatin regulators of therapy-responsive T cell subsets and reveals a shared regulatory program that governs intratumoral CD8+ T cell exhaustion and CD4+ T follicular helper cell development. We anticipate that scATAC-seq will enable the unbiased discovery of gene regulatory factors across diverse biological systems.


Asunto(s)
Células de la Médula Ósea/metabolismo , Cromatina/química , Análisis de la Célula Individual/métodos , Linfocitos T/metabolismo , Línea Celular , Simulación por Computador , Regulación de la Expresión Génica , Hematopoyesis , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucocitos Mononucleares , Factores de Transcripción/metabolismo
14.
Nat Commun ; 8: 14049, 2017 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-28091601

RESUMEN

Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3' mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.


Asunto(s)
Leucocitos Mononucleares/metabolismo , Transcriptoma , Línea Celular , Femenino , Humanos , Leucocitos Mononucleares/química , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de la Célula Individual
15.
Cell Stem Cell ; 21(1): 78-90.e6, 2017 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-28686870

RESUMEN

Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.


Asunto(s)
Antígenos de Diferenciación/metabolismo , Células Enteroendocrinas/metabolismo , Mucosa Intestinal/lesiones , Mucosa Intestinal/metabolismo , Yeyuno/lesiones , Yeyuno/metabolismo , Células Madre/metabolismo , Animales , Antígenos de Diferenciación/genética , Células Enteroendocrinas/patología , Regulación de la Expresión Génica , Mucosa Intestinal/patología , Yeyuno/patología , Ratones , Ratones Transgénicos , Células Madre/patología
16.
Nat Biotechnol ; 34(3): 303-11, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26829319

RESUMEN

Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.


Asunto(s)
Haplotipos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , ADN/genética , Genoma Humano , Variación Estructural del Genoma , Células Germinativas , Humanos , Conformación de Ácido Nucleico , Proteínas de Fusión Oncogénica/genética , Polimorfismo de Nucleótido Simple
17.
Sci Data ; 3: 160025, 2016 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-27271295

RESUMEN

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly.


Asunto(s)
Benchmarking , Genoma Humano , Exoma , Genómica , Humanos , Mutación INDEL
18.
BMC Bioinformatics ; 5: 40, 2004 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-15096276

RESUMEN

BACKGROUND: We present Pegasys--a flexible, modular and customizable software system that facilitates the execution and data integration from heterogeneous biological sequence analysis tools. RESULTS: The Pegasys system includes numerous tools for pair-wise and multiple sequence alignment, ab initio gene prediction, RNA gene detection, masking repetitive sequences in genomic DNA as well as filters for database formatting and processing raw output from various analysis tools. We introduce a novel data structure for creating workflows of sequence analyses and a unified data model to store its results. The software allows users to dynamically create analysis workflows at run-time by manipulating a graphical user interface. All non-serial dependent analyses are executed in parallel on a compute cluster for efficiency of data generation. The uniform data model and backend relational database management system of Pegasys allow for results of heterogeneous programs included in the workflow to be integrated and exported into General Feature Format for further analyses in GFF-dependent tools, or GAME XML for import into the Apollo genome editor. The modularity of the design allows for new tools to be added to the system with little programmer overhead. The database application programming interface allows programmatic access to the data stored in the backend through SQL queries. CONCLUSIONS: The Pegasys system enables biologists and bioinformaticians to create and manage sequence analysis workflows. The software is released under the Open Source GNU General Public License. All source code and documentation is available for download at http://bioinformatics.ubc.ca/pegasys/.


Asunto(s)
Alineación de Secuencia/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Biología Computacional/métodos , Biología Computacional/tendencias , Gráficos por Computador , ADN/genética , Bases de Datos Genéticas , Teoría del Juego , Heterogeneidad Genética , Humanos , Lenguajes de Programación , Alineación de Secuencia/tendencias , Programas Informáticos/tendencias , Diseño de Software , Interfaz Usuario-Computador
19.
Nat Struct Mol Biol ; 21(7): 585-90, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24929436

RESUMEN

Long noncoding RNAs (lncRNAs) are important regulators of cell fate, yet little is known about mechanisms controlling lncRNA expression. Here we show that transcription is quantitatively different for lncRNAs and mRNAs--as revealed by deficiency of Dicer (Dcr), a key RNase that generates microRNAs (miRNAs). Dcr loss in mouse embryonic stem cells led unexpectedly to decreased levels of hundreds of lncRNAs. The canonical Dgcr8-Dcr-miRNA pathway is required for robust lncRNA transcriptional initiation and elongation. Computational and genetic epistasis analyses demonstrated that Dcr activation of the oncogenic transcription factor cMyc is partly responsible for lncRNA expression. A quantitative metric of mRNA-lncRNA decoupling revealed that Dcr and cMyc differentially regulate lncRNAs versus mRNAs in diverse cell types and in vivo. Thus, numerous lncRNAs may be modulated as a class in development and disease, notably where Dcr and cMyc act.


Asunto(s)
ARN Helicasas DEAD-box/fisiología , MicroARNs/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , ARN Largo no Codificante/genética , Ribonucleasa III/fisiología , Transcripción Genética , Animales , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Regulación hacia Abajo , Técnicas de Inactivación de Genes , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Transcriptoma
20.
Nat Genet ; 43(9): 854-9, 2011 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-21857679

RESUMEN

MicroRNAs (miRNAs) are short, highly conserved noncoding RNA molecules that repress gene expression in a sequence-dependent manner. We performed single-cell measurements using quantitative fluorescence microscopy and flow cytometry to monitor a target gene's protein expression in the presence and absence of regulation by miRNA. We find that although the average level of repression is modest, in agreement with previous population-based measurements, the repression among individual cells varies dramatically. In particular, we show that regulation by miRNAs establishes a threshold level of target mRNA below which protein production is highly repressed. Near this threshold, protein expression responds sensitively to target mRNA input, consistent with a mathematical model of molecular titration. These results show that miRNAs can act both as a switch and as a fine-tuner of gene expression.


Asunto(s)
Regulación de la Expresión Génica , Marcación de Gen , MicroARNs/genética , Sitios de Unión , Citometría de Flujo , Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Luciferasas/genética , Microscopía Fluorescente
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