RESUMEN
5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression in vivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression.
RESUMEN
DNA single-strand breaks (SSBs) are among the most common lesions in the genome, arising spontaneously and as intermediates of many DNA transactions. Nevertheless, in contrast to double-strand breaks (DSBs), their distribution in the genome has hardly been addressed in a meaningful way. We now present a technique based on genome-wide ligation of 3'-OH ends followed by sequencing (GLOE-Seq) and an associated computational pipeline designed for capturing SSBs but versatile enough to be applied to any lesion convertible into a free 3'-OH terminus. We demonstrate its applicability to mapping of Okazaki fragments without prior size selection and provide insight into the relative contributions of DNA ligase 1 and ligase 3 to Okazaki fragment maturation in human cells. In addition, our analysis reveals biases and asymmetries in the distribution of spontaneous SSBs in yeast and human chromatin, distinct from the patterns of DSBs.
Asunto(s)
Mapeo Cromosómico/métodos , Replicación del ADN/genética , Análisis de Secuencia de ADN/métodos , Cromatina , ADN/genética , Roturas del ADN de Cadena Simple , Daño del ADN/genética , ADN Ligasa (ATP)/genética , Reparación del ADN/genética , Genoma/genética , Humanos , Nucleótidos , Saccharomyces cerevisiae/genéticaRESUMEN
Primordial germ cells (PGCs) are the precursors of germ cells, which migrate to the genital ridge during early development. Relatively little is known about PGCs after their migration. We studied this post-migratory stage using microscopy and sequencing techniques, and found that many PGC-specific genes, including genes known to induce PGC fate in the mouse, are only activated several days after migration. At this same time point, PGC nuclei become extremely gyrated, displaying general broad opening of chromatin and high levels of intergenic transcription. This is accompanied by changes in nuage morphology, expression of large loci (PGC-expressed non-coding RNA loci, PERLs) that are enriched for retro-transposons and piRNAs, and a rise in piRNA biogenesis signatures. Interestingly, no nuclear Piwi protein could be detected at any time point, indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post-migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation.
Asunto(s)
Núcleo Celular/genética , Células Germinativas/metabolismo , Transcripción Genética , Pez Cebra/genética , Animales , Núcleo Celular/ultraestructura , Elementos Transponibles de ADN/genética , ADN Intergénico/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Fertilización , Regulación del Desarrollo de la Expresión Génica , Sitios Genéticos , Células Germinativas/ultraestructura , Mutación/genética , ARN Interferente Pequeño/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Regulación hacia Arriba/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Cigoto/metabolismoRESUMEN
The identity of embryonic gastric epithelial progenitors is unknown. We used single-cell RNA-sequencing, genetic lineage tracing and organoid assays to assess whether Axin2- and Lgr5-expressing cells are gastric progenitors in the developing mouse stomach. We show that Axin2+ cells represent a transient population of embryonic epithelial cells in the forestomach. Lgr5+ cells generate both glandular corpus and squamous forestomach organoids ex vivo Only Lgr5+ progenitors give rise to zymogenic cells in culture. Modulating the activity of the WNT, BMP and Notch pathways in vivo and ex vivo, we found that WNTs are essential for the maintenance of Lgr5+ epithelial cells. Notch prevents differentiation of the embryonic epithelial cells along all secretory lineages and hence ensures their maintenance. Whereas WNTs promote differentiation of the embryonic progenitors along the zymogenic cell lineage, BMPs enhance their differentiation along the parietal lineage. In contrast, WNTs and BMPs are required to suppress differentiation of embryonic gastric epithelium along the pit cell lineage. Thus, coordinated action of the WNT, BMP and Notch pathways controls cell fate determination in the embryonic gastric epithelium.
Asunto(s)
Linaje de la Célula/fisiología , Células Epiteliales/metabolismo , Mucosa Gástrica/metabolismo , Transducción de Señal/fisiología , Células Madre/metabolismo , Estómago/fisiología , Animales , Diferenciación Celular/fisiología , Células Epiteliales/fisiología , Femenino , Mucosa Gástrica/fisiología , Ratones , Organoides/metabolismo , Organoides/fisiología , Células Madre/fisiologíaRESUMEN
Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.
Asunto(s)
Drosophila melanogaster/genética , Genoma , Animales , Mapeo Cromosómico , Cromosomas Artificiales Bacterianos , Biología Computacional , Mapeo Contig , Secuenciación de Nucleótidos de Alto Rendimiento , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Cromosomas Politénicos , Mapeo RestrictivoRESUMEN
Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.
Asunto(s)
Histonas/metabolismo , Linfoma no Hodgkin/genética , Mutación/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genoma Humano/genética , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Pérdida de Heterocigocidad/genética , Linfoma Folicular/enzimología , Linfoma Folicular/genética , Linfoma de Células B Grandes Difuso/enzimología , Linfoma de Células B Grandes Difuso/genética , Linfoma no Hodgkin/enzimología , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Factores de Transcripción MEF2 , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
Diffuse large B-cell lymphoma (DLBCL) accounts for 30% to 40% of newly diagnosed lymphomas and has an overall cure rate of approximately 60%. Previously, we observed FOXO1 mutations in non-Hodgkin lymphoma patient samples. To explore the effects of FOXO1 mutations, we assessed FOXO1 status in 279 DLBCL patient samples and 22 DLBCL-derived cell lines. FOXO1 mutations were found in 8.6% (24/279) of DLBCL cases: 92.3% (24/26) of mutations were in the first exon, 46.2% (12/26) were recurrent mutations affecting the N-terminal region, and another 38.5% (10/26) affected the Forkhead DNA binding domain. Recurrent mutations in the N-terminal region resulted in diminished T24 phosphorylation, loss of interaction with 14-3-3, and nuclear retention. FOXO1 mutation was associated with decreased overall survival in patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (P = .037), independent of cell of origin (COO) and the revised International Prognostic Index (R-IPI). This association was particularly evident (P = .003) in patients in the low-risk R-IPI categories. The independent relationship of mutations in FOXO1 to survival, transcending the prognostic influence of the R-IPI and COO, indicates that FOXO1 mutation is a novel prognostic factor that plays an important role in DLBCL pathogenesis.
Asunto(s)
Factores de Transcripción Forkhead/genética , Linfoma de Células B Grandes Difuso/genética , Mutación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Análisis Mutacional de ADN , Femenino , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/fisiología , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Linfoma de Células B Grandes Difuso/diagnóstico , Linfoma de Células B Grandes Difuso/patología , Masculino , Persona de Mediana Edad , Modelos Moleculares , Mutación/fisiología , Pronóstico , Adulto JovenRESUMEN
Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer composed of at least 2 molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease. Here we provide a whole-genome-sequencing-based perspective of DLBCL mutational complexity by characterizing 40 de novo DLBCL cases and 13 DLBCL cell lines and combining these data with DNA copy number analysis and RNA-seq from an extended cohort of 96 cases. Our analysis identified widespread genomic rearrangements including evidence for chromothripsis as well as the presence of known and novel fusion transcripts. We uncovered new gene targets of recurrent somatic point mutations and genes that are targeted by focal somatic deletions in this disease. We highlight the recurrence of germinal center B-cell-restricted mutations affecting genes that encode the S1P receptor and 2 small GTPases (GNA13 and GNAI2) that together converge on regulation of B-cell homing. We further analyzed our data to approximate the relative temporal order in which some recurrent mutations were acquired and demonstrate that ongoing acquisition of mutations and intratumoral clonal heterogeneity are common features of DLBCL. This study further improves our understanding of the processes and pathways involved in lymphomagenesis, and some of the pathways mutated here may indicate new avenues for therapeutic intervention.
Asunto(s)
Biomarcadores de Tumor/química , Biomarcadores de Tumor/genética , Variaciones en el Número de Copia de ADN/genética , Genoma Humano , Linfoma de Células B Grandes Difuso/genética , Mutación/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13/química , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Recent sequencing efforts have described the mutational landscape of the pediatric brain tumor medulloblastoma. Although MLL2 is among the most frequent somatic single nucleotide variants (SNV), the clinical and biological significance of these mutations remains uncharacterized. Through targeted re-sequencing, we identified mutations of MLL2 in 8 % (14/175) of MBs, the majority of which were loss of function. Notably, we also report mutations affecting the MLL2-binding partner KDM6A, in 4 % (7/175) of tumors. While MLL2 mutations were independent of age, gender, histological subtype, M-stage or molecular subgroup, KDM6A mutations were most commonly identified in Group 4 MBs, and were mutually exclusive with MLL2 mutations. Immunohistochemical staining for H3K4me3 and H3K27me3, the chromatin effectors of MLL2 and KDM6A activity, respectively, demonstrated alterations of the histone code in 24 % (53/220) of MBs across all subgroups. Correlating these MLL2- and KDM6A-driven histone marks with prognosis, we identified populations of MB with improved (K4+/K27-) and dismal (K4-/K27-) outcomes, observed primarily within Group 3 and 4 MBs. Group 3 and 4 MBs demonstrate somatic copy number aberrations, and transcriptional profiles that converge on modifiers of H3K27-methylation (EZH2, KDM6A, KDM6B), leading to silencing of PRC2-target genes. As PRC2-mediated aberrant methylation of H3K27 has recently been targeted for therapy in other diseases, it represents an actionable target for a substantial percentage of medulloblastoma patients with aggressive forms of the disease.
Asunto(s)
Neoplasias Cerebelosas , Predisposición Genética a la Enfermedad/genética , N-Metiltransferasa de Histona-Lisina/genética , Lisina/genética , Meduloblastoma , Secuencia de Bases , Neoplasias Cerebelosas/clasificación , Neoplasias Cerebelosas/diagnóstico , Neoplasias Cerebelosas/genética , Estudios de Cohortes , Proteínas de Unión al ADN/genética , Femenino , Genoma , Histona Demetilasas/genética , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/clasificación , Humanos , Masculino , Meduloblastoma/clasificación , Meduloblastoma/diagnóstico , Meduloblastoma/genética , Metilación , Mutación/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Enhancers are important cis-regulatory elements controlling cell-type specific expression patterns of genes. Furthermore, combinations of enhancers and minimal promoters are utilized to construct small, artificial promoters for gene delivery vectors. Large-scale functional screening methodology to construct genomic maps of enhancer activities has been successfully established in cultured cell lines, however, not yet applied to terminally differentiated cells and tissues in a living animal. Here, we transposed the Self-Transcribing Active Regulatory Region Sequencing (STARR-seq) technique to the mouse brain using adeno-associated-viruses (AAV) for the delivery of a highly complex screening library tiling entire genomic regions and covering in total 3 Mb of the mouse genome. We identified 483 sequences with enhancer activity, including sequences that were not predicted by DNA accessibility or histone marks. Characterizing the expression patterns of fluorescent reporters controlled by nine candidate sequences, we observed differential expression patterns also in sparse cell types. Together, our study provides an entry point for the unbiased study of enhancer activities in organisms during health and disease.
Asunto(s)
Elementos de Facilitación Genéticos , Genómica , Animales , Ratones , Genómica/métodos , Mapeo Cromosómico/métodos , Regiones Promotoras Genéticas , EncéfaloRESUMEN
Sciara coprophila (Diptera, Nematocera) constitutes a classic model to analyze unusual chromosome behavior such as the somatic elimination of paternal X chromosomes, the elimination of the whole paternal, plus non-disjunction of the maternal X chromosome at male meiosis. The molecular organization of the heterochromatin in S. coprophila is mostly unknown except for the ribosomal DNA located in the X chromosome pericentromeric heterochromatin. The characterization of the centromeric regions, thus, is an essential and required step for the establishment of S. coprophila as a model system to study fundamental mechanisms of chromosome segregation. To accomplish such a study, heterochromatic sections of the X chromosome centromeric region from salivary glands polytene chromosomes were microdissected and microcloned. Here, we report the identification and characterization of two tandem repeated DNA sequences from the pericentromeric region of the X chromosome, a pericentromeric RTE element and an AT-rich centromeric satellite. These sequences will be important tools for the cloning of S. coprophila centromeric heterochromatin using libraries of large genomic clones.
Asunto(s)
Centrómero/química , ADN/química , Dípteros/genética , Heterocromatina/química , Larva/genética , Cromosomas Politénicos/química , Secuencias Repetidas en Tándem/genética , Cromosoma X/química , Animales , Centrómero/genética , Mapeo Cromosómico , ADN/genética , Heterocromatina/genética , Hibridación Fluorescente in Situ , Masculino , Meiosis/genética , Datos de Secuencia Molecular , Filogenia , Cromosomas Politénicos/genética , Glándulas Salivales/química , Glándulas Salivales/citología , Fijación del Tejido , Cromosoma X/genéticaRESUMEN
The non-recombining Y chromosome is expected to degenerate over evolutionary time, however, gene gain is a common feature of Y chromosomes of mammals and Drosophila. Here, we report that a large palindrome containing interchromosomal segmental duplications is located in the vicinity of the first amplicon detected in the Y chromosome of D. melanogaster. The recent appearance of such amplicons suggests that duplications to the Y chromosome, followed by the amplification of the segmental duplications, are a mechanism for the continuing evolution of Drosophila Y chromosomes.
Asunto(s)
Drosophila melanogaster/genética , Duplicación de Gen , Genes de Insecto , Secuencias Invertidas Repetidas , Cromosoma Y , Animales , Evolución Molecular , Modelos Genéticos , Datos de Secuencia MolecularRESUMEN
AIMS: The mammalian gut is the largest endocrine organ. Dozens of hormones secreted by enteroendocrine cells regulate a variety of physiological functions of the gut but also of the pancreas and brain. Here, we examined the role of the helix-loop-helix transcription factor ID2 during the differentiation of intestinal stem cells along the enteroendocrine lineage. METHODS: To assess the functions of ID2 in the adult mouse small intestine, we used single-cell RNA sequencing, genetically modified mice, and organoid assays. RESULTS: We found that in the adult intestinal epithelium Id2 is predominantly expressed in enterochromaffin and peptidergic enteroendocrine cells. Consistently, the loss of Id2 leads to the reduction of Chromogranin A-positive enteroendocrine cells. In contrast, the numbers of tuft cells are increased in Id2 mutant small intestine. Moreover, ablation of Id2 elevates the numbers of Serotonin+ enterochromaffin cells and Ghrelin+ X-cells in the posterior part of the small intestine. Finally, ID2 acts downstream of BMP signalling during the differentiation of Glucagon-like peptide-1+ L-cells and Cholecystokinin+ I-cells towards Neurotensin+ PYY+ N-cells. CONCLUSION: ID2 plays an important role in cell fate decisions in the adult small intestine. First, ID2 is essential for establishing a differentiation gradient for enterochromaffin and X-cells along the anterior-posterior axis of the gut. Next, ID2 is necessary for the differentiation of N-cells thus ensuring a differentiation gradient along the crypt-villi axis. Finally, ID2 suppresses the commitment of secretory intestinal epithelial progenitors towards tuft cell lineage and thus controls host immune response to commensal and parasitic microbiota.
Asunto(s)
Diferenciación Celular , Células Enteroendocrinas , Proteína 2 Inhibidora de la Diferenciación/genética , Factores de Transcripción , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Mucosa Intestinal , Intestino Delgado/citología , Mamíferos , Ratones , Factores de Transcripción/genéticaRESUMEN
The centromeric and telomeric heterochromatin of eukaryotic chromosomes is mainly composed of middle-repetitive elements, such as transposable elements and tandemly repeated DNA sequences. Because of this repetitive nature, Whole Genome Shotgun Projects have failed in sequencing these regions. We describe a novel kind of transposon-based approach for sequencing highly repetitive DNA sequences in BAC clones. The key to this strategy relies on physical mapping the precise position of the transposon insertion, which enables the correct assembly of the repeated DNA. We have applied this strategy to a clone from the centromeric region of the Y chromosome of Drosophila melanogaster. The analysis of the complete sequence of this clone has allowed us to prove that this centromeric region evolved from a telomere, possibly after a pericentric inversion of an ancestral telocentric chromosome. Our results confirm that the use of transposon-mediated sequencing, including positional mapping information, improves current finishing strategies. The strategy we describe could be a universal approach to resolving the heterochromatic regions of eukaryotic genomes.
Asunto(s)
Centrómero/química , Drosophila melanogaster/genética , Evolución Molecular , Análisis de Secuencia de ADN/métodos , Telómero/química , Cromosoma Y/química , Animales , Cromosomas Artificiales Bacterianos , Clonación Molecular , ADN/química , Elementos Transponibles de ADN , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling responses that ultimately reach remote organs, including the brain. Using a mouse model of orthopedic surgery, we have previously demonstrated hippocampal metabolic, structural, and functional changes associated with cognitive impairment. However, the nature of the underlying signals responsible for such periphery-to-brain communication remains hitherto elusive. Here we present the first exploratory study that tests the hypothesis of extracellular vesicles (EVs) as potential mediators carrying information from the injured tissue to the distal organs including the brain. The primary goal was to investigate whether the cargo of circulating EVs after surgery can undergo quantitative changes that could potentially trigger phenotypic modifications in the target tissues. EVs were isolated from the serum of the mice subjected to a tibia surgery after 6, 24, and 72 h, and the proteome and miRNAome were investigated using mass spectrometry and RNA-seq approaches. We found substantial differential expression of proteins and miRNAs starting at 6 h post-surgery and peaking at 24 h. Interestingly, one of the up-regulated proteins at 24 h was α-synuclein, a pathogenic hallmark of certain neurodegenerative syndromes. Analysis of miRNA target mRNA and corresponding biological pathways indicate the potential of post-surgery EVs to modify the extracellular matrix of the recipient cells and regulate metabolic processes including fatty acid metabolism. We conclude that surgery alters the cargo of circulating EVs in the blood, and our results suggest EVs as potential systemic signal carriers mediating remote effects of surgery on the brain.
Asunto(s)
Biomarcadores , Vesículas Extracelulares/metabolismo , Heridas y Lesiones/metabolismo , Animales , Fraccionamiento Químico , Cromatografía Liquida , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Vesículas Extracelulares/ultraestructura , Ratones , MicroARNs/genética , Proteoma , Proteómica/métodos , ARN Mensajero/genética , Procedimientos Quirúrgicos Operativos/efectos adversos , Espectrometría de Masas en Tándem , Heridas y Lesiones/sangre , Heridas y Lesiones/etiologíaRESUMEN
RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. The very prominent secondary small interfering RNAs, also called 22G-RNAs, bear a 5' triphosphate group after loading into an Argonaute protein. This creates a technical issue, since 5'PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5' pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison. â¢We show that treatment of small RNA samples with RppH prior to sequencing library preparation increases the cloning efficiency of 5' triphosphorylated small RNAs;â¢RppH treatment is a valid alternative to TAP treatment.
RESUMEN
Forced transcription factor expression can transdifferentiate somatic cells into other specialised cell types or reprogram them into induced pluripotent stem cells (iPSCs) with variable efficiency. To better understand the heterogeneity of these processes, we used single-cell RNA sequencing to follow the transdifferentation of murine pre-B cells into macrophages as well as their reprogramming into iPSCs. Even in these highly efficient systems, there was substantial variation in the speed and path of fate conversion. We predicted and validated that these differences are inversely coupled and arise in the starting cell population, with Mychigh large pre-BII cells transdifferentiating slowly but reprogramming efficiently and Myclow small pre-BII cells transdifferentiating rapidly but failing to reprogram. Strikingly, differences in Myc activity predict the efficiency of reprogramming across a wide range of somatic cell types. These results illustrate how single cell expression and computational analyses can identify the origins of heterogeneity in cell fate conversion processes.
Asunto(s)
Linaje de la Célula , Transdiferenciación Celular , Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Células Precursoras de Linfocitos B/citología , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , RNA-Seq , Transducción de Señal , Análisis de la Célula Individual , TranscriptomaRESUMEN
A variety of single-cell RNA preparation procedures have been described. So far, protocols require fresh material, which hinders complex study designs. We describe a sample preservation method that maintains transcripts in viable single cells, allowing one to disconnect time and place of sampling from subsequent processing steps. We sequence single-cell transcriptomes from >1000 fresh and cryopreserved cells using 3'-end and full-length RNA preparation methods. Our results confirm that the conservation process did not alter transcriptional profiles. This substantially broadens the scope of applications in single-cell transcriptomics and could lead to a paradigm shift in future study designs.
Asunto(s)
Criopreservación , Perfilación de la Expresión Génica , Análisis de la Célula Individual , Transcriptoma , Animales , Línea Celular , Análisis por Conglomerados , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica/métodos , Xenoinjertos , Humanos , Ratones , Neoplasias/genética , Neoplasias/patología , ARN/genética , Estabilidad del ARN , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Análisis de la Célula Individual/métodosRESUMEN
Highly proliferative Lgr5+ stem cells maintain the intestinal epithelium and are thought to be largely homogeneous. Although quiescent intestinal stem cell (ISC) populations have been described, the identity and features of such a population remain controversial. Here we report unanticipated heterogeneity within the Lgr5+ ISC pool. We found that expression of the RNA-binding protein Mex3a labels a slowly cycling subpopulation of Lgr5+ ISCs that contribute to all intestinal lineages with distinct kinetics. Single-cell transcriptome profiling revealed that Lgr5+ cells adopt two discrete states, one of which is defined by a Mex3a expression program and relatively low levels of proliferation genes. During homeostasis, Mex3a+ cells continually shift into the rapidly dividing, self-renewing ISC pool. Chemotherapy and radiation preferentially target rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, helping to promote regeneration of the intestinal epithelium following toxic insults. Thus, Mex3a defines a reserve-like ISC population within the Lgr5+ compartment.
Asunto(s)
Proliferación Celular/fisiología , Mucosa Intestinal/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/metabolismo , Animales , Mucosa Intestinal/citología , Ratones , Ratones Transgénicos , Proteínas de Unión al ARN/genética , Receptores Acoplados a Proteínas G/genética , Células Madre/citologíaRESUMEN
Dysregulated adipocyte physiology leads to imbalanced energy storage, obesity, and associated diseases, imposing a costly burden on current health care. Cannabinoid receptor type-1 (CB1) plays a crucial role in controlling energy metabolism through central and peripheral mechanisms. In this work, adipocyte-specific inducible deletion of the CB1 gene (Ati-CB1-KO) was sufficient to protect adult mice from diet-induced obesity and associated metabolic alterations and to reverse the phenotype in already obese mice. Compared with controls, Ati-CB1-KO mice showed decreased body weight, reduced total adiposity, improved insulin sensitivity, enhanced energy expenditure, and fat depot-specific cellular remodeling toward lowered energy storage capacity and browning of white adipocytes. These changes were associated with an increase in alternatively activated macrophages concomitant with enhanced sympathetic tone in adipose tissue. Remarkably, these alterations preceded the appearance of differences in body weight, highlighting the causal relation between the loss of CB1 and the triggering of metabolic reprogramming in adipose tissues. Finally, the lean phenotype of Ati-CB1-KO mice and the increase in alternatively activated macrophages in adipose tissue were also present at thermoneutral conditions. Our data provide compelling evidence for a crosstalk among adipocytes, immune cells, and the sympathetic nervous system (SNS), wherein CB1 plays a key regulatory role.