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Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.
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Riñón/inmunología , Nefritis Lúpica/inmunología , Biomarcadores , Biopsia , Análisis por Conglomerados , Biología Computacional/métodos , Células Epiteliales/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunofenotipificación , Interferones/metabolismo , Riñón/metabolismo , Riñón/patología , Leucocitos/inmunología , Leucocitos/metabolismo , Nefritis Lúpica/genética , Nefritis Lúpica/metabolismo , Nefritis Lúpica/patología , Linfocitos/inmunología , Linfocitos/metabolismo , Anotación de Secuencia Molecular , Células Mieloides/inmunología , Células Mieloides/metabolismo , Análisis de la Célula Individual , TranscriptomaRESUMEN
To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90)+HLA-DRAhi sublining fibroblasts, IL1B+ pro-inflammatory monocytes, ITGAX+TBX21+ autoimmune-associated B cells and PDCD1+ peripheral helper T (TPH) cells and follicular helper T (TFH) cells. We defined distinct subsets of CD8+ T cells characterized by GZMK+, GZMB+, and GNLY+ phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1+HLA-DRAhi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.
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Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Perfilación de la Expresión Génica , Membrana Sinovial/metabolismo , Transcriptoma , Artritis Reumatoide/patología , Autoinmunidad/genética , Biomarcadores , Biología Computacional/métodos , Estudios Transversales , Citocinas/metabolismo , Fibroblastos/metabolismo , Citometría de Flujo , Expresión Génica , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Leucocitos/inmunología , Leucocitos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Transducción de Señal , Análisis de la Célula Individual/métodos , Membrana Sinovial/patología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Flujo de TrabajoRESUMEN
Structural variants (SVs) rearrange large segments of DNA1 and can have profound consequences in evolution and human disease2,3. As national biobanks, disease-association studies, and clinical genetic testing have grown increasingly reliant on genome sequencing, population references such as the Genome Aggregation Database (gnomAD)4 have become integral in the interpretation of single-nucleotide variants (SNVs)5. However, there are no reference maps of SVs from high-coverage genome sequencing comparable to those for SNVs. Here we present a reference of sequence-resolved SVs constructed from 14,891 genomes across diverse global populations (54% non-European) in gnomAD. We discovered a rich and complex landscape of 433,371 SVs, from which we estimate that SVs are responsible for 25-29% of all rare protein-truncating events per genome. We found strong correlations between natural selection against damaging SNVs and rare SVs that disrupt or duplicate protein-coding sequence, which suggests that genes that are highly intolerant to loss-of-function are also sensitive to increased dosage6. We also uncovered modest selection against noncoding SVs in cis-regulatory elements, although selection against protein-truncating SVs was stronger than all noncoding effects. Finally, we identified very large (over one megabase), rare SVs in 3.9% of samples, and estimate that 0.13% of individuals may carry an SV that meets the existing criteria for clinically important incidental findings7. This SV resource is freely distributed via the gnomAD browser8 and will have broad utility in population genetics, disease-association studies, and diagnostic screening.
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Enfermedad/genética , Variación Genética , Genética Médica/normas , Genética de Población/normas , Genoma Humano/genética , Femenino , Pruebas Genéticas , Técnicas de Genotipaje , Humanos , Masculino , Persona de Mediana Edad , Mutación , Polimorfismo de Nucleótido Simple/genética , Grupos Raciales/genética , Estándares de Referencia , Selección Genética , Secuenciación Completa del GenomaRESUMEN
Colonization by Lactobacillus in the female genital tract is thought to be critical for maintaining genital health. However, little is known about how genital microbiota influence host immune function and modulate disease susceptibility. We studied a cohort of asymptomatic young South African women and found that the majority of participants had genital communities with low Lactobacillus abundance and high ecological diversity. High-diversity communities strongly correlated with genital pro-inflammatory cytokine concentrations in both cross-sectional and longitudinal analyses. Transcriptional profiling suggested that genital antigen-presenting cells sense gram-negative bacterial products in situ via Toll-like receptor 4 signaling, contributing to genital inflammation through activation of the NF-κB signaling pathway and recruitment of lymphocytes by chemokine production. Our study proposes a mechanism by which cervicovaginal microbiota impact genital inflammation and thereby might affect a woman's reproductive health, including her risk of acquiring HIV.
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Interacciones Huésped-Patógeno/inmunología , Lactobacillus/inmunología , Vagina/inmunología , Vagina/microbiología , Adolescente , Adulto , África , Bacterias/genética , Bacterias/inmunología , Biodiversidad , Citocinas/inmunología , Femenino , Humanos , Lactobacillus/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia , Sudáfrica , Adulto JovenRESUMEN
Structural variants (SVs), including small insertion and deletion variants (indels), are challenging to detect through standard alignment-based variant calling methods. Sequence assembly offers a powerful approach to identifying SVs, but is difficult to apply at scale genome-wide for SV detection due to its computational complexity and the difficulty of extracting SVs from assembly contigs. We describe SvABA, an efficient and accurate method for detecting SVs from short-read sequencing data using genome-wide local assembly with low memory and computing requirements. We evaluated SvABA's performance on the NA12878 human genome and in simulated and real cancer genomes. SvABA demonstrates superior sensitivity and specificity across a large spectrum of SVs and substantially improves detection performance for variants in the 20-300 bp range, compared with existing methods. SvABA also identifies complex somatic rearrangements with chains of short (<1000 bp) templated-sequence insertions copied from distant genomic regions. We applied SvABA to 344 cancer genomes from 11 cancer types and found that short templated-sequence insertions occur in â¼4% of all somatic rearrangements. Finally, we demonstrate that SvABA can identify sites of viral integration and cancer driver alterations containing medium-sized (50-300 bp) SVs.
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Genoma Humano/genética , Variación Estructural del Genoma/genética , Genómica , Mutación INDEL/genética , Bases de Datos Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de Secuencia de ADN , Eliminación de Secuencia/genética , Programas Informáticos , Integración Viral/genéticaRESUMEN
Understanding the principles governing mammalian gene regulation has been hampered by the difficulty in measuring in vivo binding dynamics of large numbers of transcription factors (TF) to DNA. Here, we develop a high-throughput Chromatin ImmunoPrecipitation (HT-ChIP) method to systematically map protein-DNA interactions. HT-ChIP was applied to define the dynamics of DNA binding by 25 TFs and 4 chromatin marks at 4 time-points following pathogen stimulus of dendritic cells. Analyzing over 180,000 TF-DNA interactions we find that TFs vary substantially in their temporal binding landscapes. This data suggests a model for transcription regulation whereby TF networks are hierarchically organized into cell differentiation factors, factors that bind targets prior to stimulus to prime them for induction, and factors that regulate specific gene programs. Overlaying HT-ChIP data on gene-expression dynamics shows that many TF-DNA interactions are established prior to the stimuli, predominantly at immediate-early genes, and identified specific TF ensembles that coordinately regulate gene-induction.
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Inmunoprecipitación de Cromatina/métodos , Células Dendríticas/metabolismo , Regulación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Animales , ADN/genética , ADN/metabolismo , Ratones , Factores de Transcripción/metabolismoRESUMEN
Over 90% of the world's population is persistently infected with Epstein-Barr virus. While EBV does not cause disease in most individuals, it is the common cause of acute infectious mononucleosis (AIM) and has been associated with several cancers and autoimmune diseases, highlighting a need for a preventive vaccine. At present, very few primary, circulating EBV genomes have been sequenced directly from infected individuals. While low levels of diversity and low viral evolution rates have been predicted for double-stranded DNA (dsDNA) viruses, recent studies have demonstrated appreciable diversity in common dsDNA pathogens (e.g., cytomegalovirus). Here, we report 40 full-length EBV genome sequences obtained from matched oral wash and B cell fractions from a cohort of 10 AIM patients. Both intra- and interpatient diversity were observed across the length of the entire viral genome. Diversity was most pronounced in viral genes required for establishing latent infection and persistence, with appreciable levels of diversity also detected in structural genes, including envelope glycoproteins. Interestingly, intrapatient diversity declined significantly over time (P < 0.01), and this was particularly evident on comparison of viral genomes sequenced from B cell fractions in early primary infection and convalescence (P < 0.001). B cell-associated viral genomes were observed to converge, becoming nearly identical to the B95.8 reference genome over time (Spearman rank-order correlation test; r = -0.5589, P = 0.0264). The reduction in diversity was most marked in the EBV latency genes. In summary, our data suggest independent convergence of diverse viral genome sequences toward a reference-like strain within a relatively short period following primary EBV infection.IMPORTANCE Identification of viral proteins with low variability and high immunogenicity is important for the development of a protective vaccine. Knowledge of genome diversity within circulating viral populations is a key step in this process, as is the expansion of intrahost genomic variation during infection. We report full-length EBV genomes sequenced from the blood and oral wash of 10 individuals early in primary infection and during convalescence. Our data demonstrate considerable diversity within the pool of circulating EBV strains, as well as within individual patients. Overall viral diversity decreased from early to persistent infection, particularly in latently infected B cells, which serve as the viral reservoir. Reduction in B cell-associated viral genome diversity coincided with a convergence toward a reference-like EBV genotype. Greater convergence positively correlated with time after infection, suggesting that the reference-like genome is the result of selection.
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Infecciones por Virus de Epstein-Barr/virología , Variación Genética , Genoma Viral , Herpesvirus Humano 4/genética , Biología Computacional/métodos , Genómica/métodos , Genotipo , Herpesvirus Humano 4/clasificación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Sistemas de Lectura Abierta , FilogeniaRESUMEN
The MS2 system has been extensively used to visualize single mRNA molecules in live cells and follow their localization and behavior. In their Letter to the Editor recently published, Garcia and Parker suggest that use of the MS2 system may yield erroneous mRNA localization results due to the accumulation of 3' decay products. Here we cite published works and provide new data which demonstrate that this is not a phenomenon general to endogenously expressed MS2-tagged transcripts, and that some of the results obtained in their study could have arisen from artifacts of gene expression.
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Aptámeros de Nucleótidos/metabolismo , Proteínas Fúngicas/metabolismo , Unión Proteica , ARN Mensajero/metabolismoRESUMEN
MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified murine miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan precursor miRNA (pre-miRNA), consequential 5' heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28.
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Genes/genética , Genoma/genética , MicroARNs/genética , Animales , Línea Celular , Perfilación de la Expresión Génica , Humanos , Secuencias Invertidas Repetidas/genética , Ratones , MicroARNs/biosíntesis , MicroARNs/metabolismo , Ribonucleasa III/metabolismoRESUMEN
The comparison of related genomes has emerged as a powerful lens for genome interpretation. Here we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and locate constrained elements covering â¼4.2% of the genome. We use evolutionary signatures and comparisons with experimental data sets to suggest candidate functions for â¼60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements and more than 1,000 primate- and human-accelerated elements. Overlap with disease-associated variants indicates that our findings will be relevant for studies of human biology, health and disease.
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Evolución Molecular , Genoma Humano/genética , Genoma/genética , Mamíferos/genética , Animales , Enfermedad , Exones/genética , Genómica , Salud , Humanos , Anotación de Secuencia Molecular , Filogenia , ARN/clasificación , ARN/genética , Selección Genética/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.
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Transferencia de Gen Horizontal , Interacciones Huésped-Parásitos/genética , Oomicetos/genética , Saprolegnia/genética , Virulencia/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Molecular , Peces/genética , Peces/parasitología , Genoma , Oomicetos/clasificación , Oomicetos/patogenicidad , Filogenia , Plantas/parasitología , Saprolegnia/clasificación , Saprolegnia/patogenicidadRESUMEN
Phytophthora infestans is one of the most destructive plant pathogens of potato and tomato globally. The pathogen is closely related to four other Phytophthora species in the 1c clade including P. phaseoli, P. ipomoeae, P. mirabilis and P. andina that are important pathogens of other wild and domesticated hosts. P. andina is an interspecific hybrid between P. infestans and an unknown Phytophthora species. We have sequenced mitochondrial genomes of the sister species of P. infestans and examined the evolutionary relationships within the clade. Phylogenetic analysis indicates that the P. phaseoli mitochondrial lineage is basal within the clade. P. mirabilis and P. ipomoeae are sister lineages and share a common ancestor with the Ic mitochondrial lineage of P. andina. These lineages in turn are sister to the P. infestans and P. andina Ia mitochondrial lineages. The P. andina Ic lineage diverged much earlier than the P. andina Ia mitochondrial lineage and P. infestans. The presence of two mitochondrial lineages in P. andina supports the hybrid nature of this species. The ancestral state of the P. andina Ic lineage in the tree and its occurrence only in the Andean regions of Ecuador, Colombia and Peru suggests that the origin of this species hybrid in nature may occur there.
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Genoma Mitocondrial , Mitocondrias/genética , Filogenia , Phytophthora infestans/genética , Phytophthora/genética , Evolución Biológica , Quimera/microbiología , Colombia , ADN Mitocondrial/genética , Ecuador , Solanum lycopersicum/microbiología , Perú , Filogeografía , Phytophthora/clasificación , Phytophthora infestans/clasificación , Enfermedades de las Plantas/microbiología , Análisis de Secuencia de ADN , Solanum tuberosum/microbiologíaRESUMEN
Exceptionally accurate genome reference sequences have proven to be of great value to microbial researchers. Thus, to date, about 1800 bacterial genome assemblies have been "finished" at great expense with the aid of manual laboratory and computational processes that typically iterate over a period of months or even years. By applying a new laboratory design and new assembly algorithm to 16 samples, we demonstrate that assemblies exceeding finished quality can be obtained from whole-genome shotgun data and automated computation. Cost and time requirements are thus dramatically reduced.
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Bacterias/genética , Genoma Bacteriano , Biblioteca Genómica , Análisis de Secuencia de ADN/métodos , AlgoritmosRESUMEN
Eliminating the bacterial cloning step has been a major factor in the vastly improved efficiency of massively parallel sequencing approaches. However, this also has made it a technical challenge to produce the modern equivalent of the Fosmid- or BAC-end sequences that were crucial for assembling and analyzing complex genomes during the Sanger-based sequencing era. To close this technology gap, we developed Fosill, a method for converting Fosmids to Illumina-compatible jumping libraries. We constructed Fosmid libraries in vectors with Illumina primer sequences and specific nicking sites flanking the cloning site. Our family of pFosill vectors allows multiplex Fosmid cloning of end-tagged genomic fragments without physical size selection and is compatible with standard and multiplex paired-end Illumina sequencing. To excise the bulk of each cloned insert, we introduced two nicks in the vector, translated them into the inserts, and cleaved them. Recircularization of the vector via coligation of insert termini followed by inverse PCR generates a jumping library for paired-end sequencing with 101-base reads. The yield of unique Fosmid-sized jumps is sufficiently high, and the background of short, incorrectly spaced and chimeric artifacts sufficiently low, to enable applications such as mapping of structural variation and scaffolding of de novo assemblies. We demonstrate the power of Fosill to map genome rearrangements in a cancer cell line and identified three fusion genes that were corroborated by RNA-seq data. Our Fosill-powered assembly of the mouse genome has an N50 scaffold length of 17.0 Mb, rivaling the connectivity (16.9 Mb) of the Sanger-sequencing based draft assembly.
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Escherichia coli/genética , Vectores Genéticos/genética , Genoma Bacteriano , Genoma Fúngico , Biblioteca Genómica , Schizosaccharomyces/genética , Análisis de Secuencia de ADN/métodos , Animales , Reordenamiento Génico , Ratones , Ratones Endogámicos C57BLRESUMEN
The degree to which molecular epidemiology reveals information about the sources and transmission patterns of an outbreak depends on the resolution of the technology used and the samples studied. Isolates of Escherichia coli O104:H4 from the outbreak centered in Germany in May-July 2011, and the much smaller outbreak in southwest France in June 2011, were indistinguishable by standard tests. We report a molecular epidemiological analysis using multiplatform whole-genome sequencing and analysis of multiple isolates from the German and French outbreaks. Isolates from the German outbreak showed remarkably little diversity, with only two single nucleotide polymorphisms (SNPs) found in isolates from four individuals. Surprisingly, we found much greater diversity (19 SNPs) in isolates from seven individuals infected in the French outbreak. The German isolates form a clade within the more diverse French outbreak strains. Moreover, five isolates derived from a single infected individual from the French outbreak had extremely limited diversity. The striking difference in diversity between the German and French outbreak samples is consistent with several hypotheses, including a bottleneck that purged diversity in the German isolates, variation in mutation rates in the two E. coli outbreak populations, or uneven distribution of diversity in the seed populations that led to each outbreak.
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Brotes de Enfermedades/estadística & datos numéricos , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/genética , Europa (Continente)/epidemiología , Humanos , Modelos Genéticos , Filogenia , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
DNA methylation is essential for normal development and has been implicated in many pathologies including cancer. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine.
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Diferenciación Celular , Metilación de ADN , Genómica , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Animales , Células Cultivadas , Secuencia Conservada , Islas de CpG/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Genoma/genética , Histonas/genética , Histonas/metabolismo , Masculino , Ratones , Neuronas/citologíaRESUMEN
Massively parallel DNA sequencing technologies are revolutionizing genomics by making it possible to generate billions of relatively short (~100-base) sequence reads at very low cost. Whereas such data can be readily used for a wide range of biomedical applications, it has proven difficult to use them to generate high-quality de novo genome assemblies of large, repeat-rich vertebrate genomes. To date, the genome assemblies generated from such data have fallen far short of those obtained with the older (but much more expensive) capillary-based sequencing approach. Here, we report the development of an algorithm for genome assembly, ALLPATHS-LG, and its application to massively parallel DNA sequence data from the human and mouse genomes, generated on the Illumina platform. The resulting draft genome assemblies have good accuracy, short-range contiguity, long-range connectivity, and coverage of the genome. In particular, the base accuracy is high (≥99.95%) and the scaffold sizes (N50 size = 11.5 Mb for human and 7.2 Mb for mouse) approach those obtained with capillary-based sequencing. The combination of improved sequencing technology and improved computational methods should now make it possible to increase dramatically the de novo sequencing of large genomes. The ALLPATHS-LG program is available at http://www.broadinstitute.org/science/programs/genome-biology/crd.
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Algoritmos , Genómica/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Animales , Genoma/genética , Humanos , Internet , Ratones , Reproducibilidad de los ResultadosRESUMEN
Schizosaccharomyces pombe Sre1 is a membrane-bound transcription factor that controls adaptation to hypoxia. Like its mammalian homolog, sterol regulatory element-binding protein (SREBP), Sre1 activation requires release from the membrane. However, in fission yeast, this release occurs through a strikingly different mechanism that requires the Golgi Dsc E3 ubiquitin ligase complex and the proteasome. The mechanistic details of Sre1 cleavage, including the link between the Dsc E3 ligase complex and proteasome, are not well understood. Here, we present results of a genetic selection designed to identify additional components required for Sre1 cleavage. From the selection, we identified two new components of the fission yeast SREBP pathway: Dsc5 and Cdc48. The AAA (ATPase associated with diverse cellular activities) ATPase Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing protein, interact with known Dsc complex components and are required for SREBP cleavage. These findings provide a mechanistic link between the Dsc E3 ligase complex and the proteasome in SREBP cleavage and add to a growing list of similarities between the Dsc E3 ligase and membrane E3 ligases involved in endoplasmic reticulum-associated degradation.