RESUMEN
Murine regulatory T (Treg) cells in tissues promote tissue homeostasis and regeneration. We sought to identify features that characterize human Treg cells with these functions in healthy tissues. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells.
Asunto(s)
Cromatina/inmunología , Linfocitos T Reguladores/inmunología , Cicatrización de Heridas/inmunología , Adulto , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Diferenciación Celular/inmunología , Línea Celular , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/inmunología , Células HaCaT , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Receptores CCR8/inmunología , Células T Auxiliares Foliculares/inmunologíaRESUMEN
Regulatory T cells (Treg cells) perform two distinct functions: they maintain self-tolerance, and they support organ homeostasis by differentiating into specialized tissue Treg cells. We found that epigenetic modifications defined the molecular characteristics of tissue Treg cells. Tagmentation-based whole-genome bisulfite sequencing revealed more than 11,000 regions that were methylated differentially in pairwise comparisons of tissue Treg cell populations and lymphoid T cells. Similarities in the epigenetic landscape led to the identification of a common tissue Treg cell population that was present in many organs and was characterized by gain and loss of DNA methylation that included many gene sites associated with the TH2 subset of helper T cells, such as the gene encoding cytokine IL-33 receptor ST2, as well as the production of tissue-regenerative factors. Furthermore, the ST2-expressing population was dependent on the transcriptional regulator BATF and could be expanded by IL-33. Thus, tissue Treg cells integrate multiple waves of epigenetic reprogramming that define their tissue-restricted specialization.
Asunto(s)
Metilación de ADN , Estudio de Asociación del Genoma Completo , Linfocitos T Reguladores/metabolismo , Animales , Biomarcadores , Análisis por Conglomerados , Biología Computacional/métodos , Islas de CpG , Epigénesis Genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunofenotipificación , Ratones , Ratones Transgénicos , Anotación de Secuencia Molecular , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Regiones Promotoras Genéticas , Células Th2/metabolismo , Sitio de Iniciación de la Transcripción , TranscriptomaRESUMEN
This corrects the article DOI: 10.1038/ni.3799.
RESUMEN
Specialized regulatory T (Treg) cells accumulate and perform homeostatic and regenerative functions in nonlymphoid tissues. Whether common precursors for nonlymphoid-tissue Treg cells exist and how they differentiate remain elusive. Using transcription factor nuclear factor, interleukin 3 regulated (Nfil3) reporter mice and single-cell RNA-sequencing (scRNA-seq), we identified two precursor stages of interleukin 33 (IL-33) receptor ST2-expressing nonlymphoid tissue Treg cells, which resided in the spleen and lymph nodes. Global chromatin profiling of nonlymphoid tissue Treg cells and the two precursor stages revealed a stepwise acquisition of chromatin accessibility and reprogramming toward the nonlymphoid-tissue Treg cell phenotype. Mechanistically, we identified and validated the transcription factor Batf as the driver of the molecular tissue program in the precursors. Understanding this tissue development program will help to harness regenerative properties of tissue Treg cells for therapy.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Ganglios Linfáticos/inmunología , Bazo/inmunología , Linfocitos T Reguladores/citología , Traslado Adoptivo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Diferenciación Celular/genética , Cromatina/metabolismo , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ratones , Especificidad de Órganos/inmunología , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Linfocitos T Reguladores/metabolismoRESUMEN
Patients with the neurological disorder HSAN-I suffer frequent infections, attributed to a lack of pain sensation and failure to seek care for minor injuries. Whether protective CD8+ T cells are affected in HSAN-I patients remains unknown. Here, we report that HSAN-I-associated mutations in serine palmitoyltransferase subunit SPTLC2 dampened human T cell responses. Antigen stimulation and inflammation induced SPTLC2 expression, and murine T-cell-specific ablation of Sptlc2 impaired antiviral-T-cell expansion and effector function. Sptlc2 deficiency reduced sphingolipid biosynthetic flux and led to prolonged activation of the mechanistic target of rapamycin complex 1 (mTORC1), endoplasmic reticulum (ER) stress, and CD8+ T cell death. Protective CD8+ T cell responses in HSAN-I patient PBMCs and Sptlc2-deficient mice were restored by supplementing with sphingolipids and pharmacologically inhibiting ER stress-induced cell death. Therefore, SPTLC2 underpins protective immunity by translating extracellular stimuli into intracellular anabolic signals and antagonizes ER stress to promote T cell metabolic fitness.
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Linfocitos T CD8-positivos/inmunología , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Serina C-Palmitoiltransferasa/genética , Animales , Proliferación Celular , Células Cultivadas , Citocinas/biosíntesis , Estrés del Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/inmunología , Femenino , Humanos , Coriomeningitis Linfocítica/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Transducción de Señal/inmunología , Esfingolípidos/biosíntesisRESUMEN
Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.
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5-Metilcitosina , Citosina/análogos & derivados , Glucólisis , Mitocondrias , Metástasis de la Neoplasia , Fosforilación Oxidativa , ARN Mitocondrial , 5-Metilcitosina/biosíntesis , 5-Metilcitosina/metabolismo , Antígenos CD36 , Supervivencia Celular , Citosina/metabolismo , Progresión de la Enfermedad , Glucólisis/efectos de los fármacos , Humanos , Metilación/efectos de los fármacos , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Fosforilación Oxidativa/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismoRESUMEN
R loops are three-stranded nucleic acid structures consisting of an RNA:DNA heteroduplex and a "looped-out" nontemplate strand. As aberrant formation and persistence of R loops block transcription elongation and cause DNA damage, mechanisms that resolve R loops are essential for genome stability. Here we show that the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase DDX21 efficiently unwinds R loops and that depletion of DDX21 leads to accumulation of cellular R loops and DNA damage. Significantly, the activity of DDX21 is regulated by acetylation. Acetylation by CBP inhibits DDX21 activity, while deacetylation by SIRT7 augments helicase activity and overcomes R-loop-mediated stalling of RNA polymerases. Knockdown of SIRT7 leads to the same phenotype as depletion of DDX21 (i.e., increased formation of R loops and DNA double-strand breaks), indicating that SIRT7 and DDX21 cooperate to prevent R-loop accumulation, thus safeguarding genome integrity. Moreover, DDX21 resolves estrogen-induced R loops on estrogen-responsive genes in breast cancer cells, which prevents the blocking of transcription elongation on these genes.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Inestabilidad Genómica/genética , Conformación de Ácido Nucleico , Sirtuinas/metabolismo , Acetilación , ARN Helicasas DEAD-box/genética , ADN/química , ADN/genética , Daño del ADN/genética , ADN Helicasas/metabolismo , Reparación del ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Activación Enzimática , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Células MCF-7 , Sirtuinas/genéticaRESUMEN
Quantification of DNA methylation in neoplastic cells is crucial both from mechanistic and diagnostic perspectives. However, such measurements are prone to different experimental biases. Polymerase chain reaction (PCR) bias results in an unequal recovery of methylated and unmethylated alleles at the sample preparation step. Post-PCR biases get introduced additionally by the readout processes. Correcting the biases is more practicable than optimising experimental conditions, as demonstrated previously. However, utilisation of our earlier developed algorithm strongly necessitates automation. Here, we present two R packages: rBiasCorrection, the core algorithms to correct biases; and BiasCorrector, its web-based graphical user interface frontend. The software detects and analyses experimental biases in calibration DNA samples at a single base resolution by using cubic polynomial and hyperbolic regression. The correction coefficients from the best regression type are employed to compensate for the bias. Three common technologies-bisulphite pyrosequencing, next-generation sequencing and oligonucleotide microarrays-were used to comprehensively test BiasCorrector. We demonstrate the accuracy of BiasCorrector's performance and reveal technology-specific PCR- and post-PCR biases. BiasCorrector effectively eliminates biases regardless of their nature, locus, the number of interrogated methylation sites and the detection method, thus representing a user-friendly tool for producing accurate epigenetic results.
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Algoritmos , Metilación de ADN , Neoplasias/genética , Reacción en Cadena de la Polimerasa/normas , Análisis de Secuencia de ADN/normas , Programas Informáticos , Sesgo , Islas de CpG , Humanos , TecnologíaRESUMEN
The Epstein-Barr virus (EBV) genome encodes several hundred transcripts. We have used ribosome profiling to characterize viral translation in infected cells and map new translation initiation sites. We show here that EBV transcripts are translated with highly variable efficiency, owing to variable transcription and translation rates, variable ribosome recruitment to the leader region and coverage by monosomes versus polysomes. Some transcripts were hardly translated, others mainly carried monosomes, showed ribosome accumulation in leader regions and most likely represent non-coding RNAs. A similar process was visible for a subset of lytic genes including the key transactivators BZLF1 and BRLF1 in cells infected with weakly replicating EBV strains. This suggests that ribosome trapping, particularly in the leader region, represents a new checkpoint for the repression of lytic replication. We could identify 25 upstream open reading frames (uORFs) located upstream of coding transcripts that displayed 5' leader ribosome trapping, six of which were located in the leader region shared by many latent transcripts. These uORFs repressed viral translation and are likely to play an important role in the regulation of EBV translation.
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Linfocitos B/metabolismo , Herpesvirus Humano 4/genética , Biosíntesis de Proteínas , Ribosomas/metabolismo , Linfocitos B/citología , Linfocitos B/virología , Células Cultivadas , Regulación Viral de la Expresión Génica , Genoma Viral/genética , Herpesvirus Humano 4/fisiología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Mutación , Sistemas de Lectura Abierta/genética , Ribosomas/genéticaRESUMEN
Supplementation of micronutrients like folate is a double-edged sword in terms of their ambivalent role in cell metabolism. Although several epidemiological studies support a protective role of folate in carcinogenesis, there are also data arguing for an opposite effect. To address this issue in the context of human papillomavirus (HPV)-induced transformation, the molecular events of different folate availability on human keratinocytes immortalized by HPV16 E6 and E7 oncoproteins were examined. Several sublines were established: Control (4.5 µM folate), folate deficient (0.002 µM folate), and repleted cells (4.5 µM folate). Cells were analyzed in terms of oncogene expression, DNA damage and repair, karyotype changes, whole-genome sequencing, and transcriptomics. Here we show that folate depletion irreversibly induces DNA damage, impairment of DNA repair fidelity, and unique chromosomal alterations. Repleted cells additionally underwent growth advantage and enhanced clonogenicity, while the above mentioned impaired molecular properties became even more pronounced. Overall, it appears that a period of folate deficiency followed by repletion can shape immortalized cells toward an anomalous phenotype, thereby potentially contributing to carcinogenesis. These observations should elicit questions and inquiries for broader additional studies regarding folate fortification programs, especially in developing countries with micronutrient deficiencies and high HPV prevalence.
Asunto(s)
Deficiencia de Ácido Fólico/genética , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/genética , Transcripción Genética , Carcinogénesis/genética , Daño del ADN/ética , Reparación del ADN/genética , Ácido Fólico/genética , Deficiencia de Ácido Fólico/patología , Deficiencia de Ácido Fólico/virología , Genómica , Papillomavirus Humano 16/patogenicidad , Humanos , Queratinocitos/virología , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/virología , Proteínas Represoras/genéticaRESUMEN
Foamy viruses are unconventional and complex retroviruses distinct from the other members of the Retroviridae family. Currently, no disease has been firmly linked to persistent foamy virus infection of their cognate host including simians, bovines, felines, and equines or upon zoonotic transmission of different simian foamy viruses to humans. Bovine and simian foamy viruses have been recently shown to encode a RNA polymerase-III-driven micro RNA cluster which likely modulates and regulates host-virus interactions at different levels. Using sub-genomic bovine foamy virus micro RNA expression plasmids and dual luciferase reporter assays as readout, the requirements for expression and processing of the bovine foamy virus micro RNAs have been analyzed. Here, we report that the minimal BFV micro RNA cassette is significantly weaker than a U6 promoter-based construct and strongly suppressed by flanking sequences. The primary micro RNA sequence can be manipulated and chimerized as long as the dumbbell-like folding of the primary micro RNA is maintained. Since more subtle changes are associated with reduced functionality, the overall structure and shape, but possibly individual elements and residues also, are important for the expression and processing of the bovine foamy virus micro RNAs.
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Regulación Viral de la Expresión Génica , Secuencias Invertidas Repetidas , MicroARNs/química , MicroARNs/genética , ARN Viral , Infecciones por Retroviridae/virología , Spumavirus/genética , Animales , Bovinos , Línea Celular , Cricetinae , Perros , Orden Génico , Genes Reporteros , Genoma Viral , Humanos , Conformación de Ácido Nucleico , Regiones Promotoras GenéticasRESUMEN
BACKGROUND AND PURPOSE: Circulating microRNAs (miRNAs) are emerging biomarkers for stroke because of their high stability in the bloodstream and association with pathophysiologic conditions. However, the circulating whole-genome miRNAs (miRNome) has not been characterized comprehensively in the acute phase of stroke. METHODS: We profiled the circulating miRNome in mouse models of acute ischemic and hemorrhagic stroke by next-generation sequencing. Stroke models were compared with sham-operated and naive mice to identify deregulated circulating miRNAs. Top-ranked miRNAs were validated and further characterized by quantitative reverse transcription polymerase chain reaction. RESULTS: We discovered 24 circulating miRNAs with an altered abundance in the circulation 3 hours after ischemia, whereas the circulating miRNome was not altered after intracerebral hemorrhage compared with sham-operated mice. Among the upregulated miRNAs in ischemia, the top-listed miR-1264/1298/448 cluster was strongly dependent on reperfusion in different ischemia models. A time course experiment revealed that the miR-1264/1298/448 cluster peaked in the circulation around 3 hours after reperfusion and gradually decreased thereafter. CONCLUSIONS: Alteration of the miRNome in the circulation is associated with cerebral ischemia/reperfusion, but not hemorrhage, suggesting a potential to serve as biomarkers for reperfusion in the acute phase. The pathophysiological role of reperfusion-inducible miR-1264/1298/448 cluster, which is located on chromosome X within the introns of the serotonin receptor HTR2C, requires further investigation.
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Isquemia Encefálica/genética , MicroARNs/sangre , Familia de Multigenes , Daño por Reperfusión/genética , Accidente Cerebrovascular/genética , Animales , Biomarcadores/análisis , Hemorragia Cerebral/genética , Estudio de Asociación del Genoma Completo , Masculino , Ratones , Ratones Endogámicos C57BL , Reperfusión , Regulación hacia ArribaRESUMEN
The armadillo repeat protein ARVCF is a component of adherens junctions. Similar to related proteins, such as p120-catenin and ß-catenin, with known signaling functions, localization studies indicate a cytoplasmic and a nuclear pool of ARVCF. We find that ARVCF interacts with different proteins involved in mRNA-processing: the splicing factor SRSF1 (SF2/ASF), the RNA helicase p68 (DDX5), and the heterogeneous nuclear ribonucleoprotein hnRNP H2. All three proteins bind to ARVCF in an RNA-independent manner. Furthermore, ARVCF occurs in large RNA-containing complexes that contain both spliced and unspliced mRNAs of housekeeping genes. By domain analysis, we show that interactions occur via the ARVCF C terminus. Overexpression of ARVCF, p68, SRSF1, and hnRNP H2 induces a significant increase in splicing activity of a reporter mRNA. Upon depletion of ARVCF followed by RNA sequence analysis, several alternatively spliced transcripts are significantly changed. Therefore, we conclude that nuclear ARVCF influences splicing of pre-mRNAs. We hypothesize that ARVCF is involved in alternative splicing, generating proteomic diversity, and its deregulation may contribute to diseased states, such as cancer and neurological disorders.
Asunto(s)
Empalme Alternativo , Proteínas del Dominio Armadillo/metabolismo , Moléculas de Adhesión Celular/metabolismo , ARN Helicasas DEAD-box/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas del Dominio Armadillo/genética , Sitios de Unión/genética , Western Blotting , Células CACO-2 , Moléculas de Adhesión Celular/genética , ARN Helicasas DEAD-box/genética , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Humanos , Microscopía Confocal , Mutación , Proteínas Nucleares/genética , Fosfoproteínas/genética , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Precursores del ARN/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Empalme Serina-Arginina , Técnicas del Sistema de Dos HíbridosRESUMEN
Promiscuity of pattern recognition receptors, such as receptor for advanced glycation end products (RAGE), allows for a complex regulatory network controlling inflammation. Scavenging of RAGE ligands by soluble RAGE treatment is effective in reducing delayed-type hypersensitivity (DTH), even in RAGE(-/-) mice by 50% (p < 0.001). This has led to the hypothesis that molecules scavenged by soluble RAGE bind to receptors other than RAGE. This study identifies CD166/ALCAM (ALCAM) as a close structural and functional homolog of RAGE, and it shows that binding of S100B to CD166/ALCAM induces dose- and time-dependent expression of members of the NF-κB family in wild type (WT) and RAGE(-/-) mouse endothelial cells. Blocking CD166/ALCAM expression using small interfering RNA completely inhibited S100B-induced NF-κB activation in RAGE(-/-), but not in WT cells. The in vivo significance of these observations was demonstrated by attenuation of DTH in WT and RAGE(-/-) animals pretreated with CD166/ALCAM small interfering RNA by 50% and 40%, respectively (p < 0.001). Experiments in ALCAM(-/-) animals displayed an only slight reduction of 16% in DTH, explained by compensatory reciprocal upregulation of RAGE in animals devoid of CD166/ALCAM, and vice versa. Consistently, ALCAM(-/-) mice, but not WT mice treated with RAGE small interfering RNA show a 35% reduction in DTH, and ALCAM(-/-) RAGE(-/-) double-knockout mice show a 27% reduction in DTH reaction. Thus, S100B is a proinflammatory cytokine bridging RAGE and CD166/ALCAM downstream effector mechanisms, both being compensatory upregulated after genetic deletion of its counterpart.
Asunto(s)
Molécula de Adhesión Celular del Leucocito Activado/fisiología , Antígenos CD/fisiología , Glicoproteínas/fisiología , Hipersensibilidad Tardía/inmunología , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/fisiología , Factores de Crecimiento Nervioso/fisiología , Péptidos/fisiología , Proteínas S100/fisiología , Antígeno AC133 , Molécula de Adhesión Celular del Leucocito Activado/química , Animales , Antígenos CD/química , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/química , Humanos , Hipersensibilidad Tardía/metabolismo , Hipersensibilidad Tardía/prevención & control , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Crecimiento Nervioso/biosíntesis , Factores de Crecimiento Nervioso/química , Péptidos/antagonistas & inhibidores , Péptidos/química , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/biosíntesis , Proteínas S100/química , Relación Estructura-Actividad , Regulación hacia Arriba/inmunologíaRESUMEN
MOTIVATION: Alternative splicing is central for cellular processes and substantially increases transcriptome and proteome diversity. Aberrant splicing events often have pathological consequences and are associated with various diseases and cancer types. The emergence of next-generation RNA sequencing (RNA-seq) provides an exciting new technology to analyse alternative splicing on a large scale. However, algorithms that enable the analysis of alternative splicing from short-read sequencing are not fully established yet and there are still no standard solutions available for a variety of data analysis tasks. RESULTS: We present a new method and software to predict genes that are differentially spliced between two different conditions using RNA-seq data. Our method uses geometric angles between the high dimensional vectors of exon read counts. With this, differential splicing can be detected even if the splicing events are composed of higher complexity and involve previously unknown splicing patterns. We applied our approach to two case studies including neuroblastoma tumour data with favourable and unfavourable clinical courses. We show the validity of our predictions as well as the applicability of our method in the context of patient clustering. We verified our predictions by several methods including simulated experiments and complementary in silico analyses. We found a significant number of exons with specific regulatory splicing factor motifs for predicted genes and a substantial number of publications linking those genes to alternative splicing. Furthermore, we could successfully exploit splicing information to cluster tissues and patients. Finally, we found additional evidence of splicing diversity for many predicted genes in normalized read coverage plots and in reads that span exon-exon junctions. AVAILABILITY: SplicingCompass is licensed under the GNU GPL and freely available as a package in the statistical language R at http://www.ichip.de/software/SplicingCompass.html
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Empalme Alternativo , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Algoritmos , Exones , Humanos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Sitios de Empalme de ARNRESUMEN
CD8 T lymphocytes are classically viewed as cytotoxic T cells. Whether human CD8 T cells can, in parallel, induce a tissue regeneration program is poorly understood. Here, antigen-specific assay systems revealed that human CD8 T cells not only mediated cytotoxicity but also promoted tissue remodeling. Activated CD8 T cells could produce the epidermal growth factor receptor (EGFR)-ligand amphiregulin (AREG) and sensitize epithelial cells for enhanced regeneration potential. Blocking the EGFR or the effector cytokines IFN-γ and TNF could inhibit tissue remodeling. This regenerative program enhanced tumor spheroid and stem cell-mediated organoid growth. Using single-cell gene expression analysis, we identified an AREG+, tissue-resident CD8 T cell population in skin and adipose tissue from patients undergoing abdominal wall or abdominoplasty surgery. These tissue-resident CD8 T cells showed a strong TCR clonal relation to blood PD1+TIGIT+ CD8 T cells with tissue remodeling abilities. These findings may help to understand the complex CD8 biology in tumors and could become relevant for the design of therapeutic T cell products.
Asunto(s)
Linfocitos T CD8-positivos , Linfocitos T Citotóxicos , Humanos , Receptores ErbB , Tejido Adiposo , Ciclo CelularRESUMEN
Cancer cells showing low apoptotic effects following oxidative stress-induced DNA damage are mainly affected by growth arrest. Thus, recent studies focus on improving anti-cancer therapies by increasing apoptosis sensitivity. We aimed at identifying a universal molecule as potential target to enhance oxidative stress-based anti-cancer therapy through a switch from cell cycle arrest to apoptosis. A cDNA microarray was performed with hydrogen peroxide-treated oesophageal squamous epithelial cancer cells TE7. This cell line showed checkpoint activation via p21(WAF1) , but low apoptotic response following DNA damage. The potential target molecule was chosen depended on the following demands: it should regulate DNA damage response, cell cycle and apoptosis. As the transcription factor ATF2 is implicated in all these processes, we focused on this protein. We investigated checkpoint activation via ATF2. Indeed, ATF2 knockdown revealed ATF2-triggered p21(WAF1) protein expression, suggesting p21(WAF1) transactivation through ATF2. Using chromatin immunoprecipitation (ChIP), we identified a hitherto unknown ATF2-binding sequence in the p21(WAF1) promoter. p-ATF2 was found to interact with p-c-Jun, creating the AP-1 complex. Moreover, ATF2 knockdown led to c-Jun downregulation. This suggests ATF2-driven induction of c-Jun expression, thereby enhancing ATF2 transcriptional activity via c-Jun-ATF2 heterodimerization. Notably, downregulation of ATF2 caused a switch from cell cycle arrest to reinforced apoptosis, presumably via p21(WAF1) downregulation, confirming the importance of ATF2 in the establishment of cell cycle arrest. 1-Chloro-2,4-dinitrobenzene also led to ATF2-dependent G2/M arrest, suggesting that this is a general feature induced by oxidative stress. As ATF2 knockdown also increased apoptosis, we propose ATF2 as a target for combined oxidative stress-based anti-cancer therapies.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Apoptosis , Técnicas de Silenciamiento del Gen , Estrés Oxidativo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Sitios de Unión , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Modelos Biológicos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismoRESUMEN
Use of false cell lines remains a major problem in biological research. Short tandem repeat (STR) profiling represents the gold standard technique for cell line authentication. However, mismatch repair (MMR)-deficient cell lines are characterized by microsatellite instability, which could force allelic drifts in combination with a selective outgrowth of otherwise persisting side lines, and, thus, are likely to be misclassified by STR profiling. On the basis of the high-throughput Luminex platform, we developed a 24-plex single nucleotide polymorphism profiling assay, called multiplex cell authentication (MCA), for determining authentication of human cell lines. MCA was evaluated by analyzing a collection of 436 human cell lines from the German Collection of Microorganisms and Cell Cultures, previously characterized by eight-loci STR profiling. Both assays showed a very high degree of concordance and similar average matching probabilities (~1 × 10(-8) for STR profiling and ~1 × 10(-9) for MCA). MCA enabled the detection of less than 3% of contaminating human cells. By analyzing MMR-deficient cell lines, evidence was obtained for a higher robustness of the MCA compared to STR profiling. In conclusion, MCA could complement routine cell line authentication and replace the standard authentication STR technique in case of MSI cell lines.
Asunto(s)
Técnicas de Genotipaje/normas , Polimorfismo de Nucleótido Simple , Técnicas de Cultivo de Célula , Línea Celular , Reparación de la Incompatibilidad de ADN/genética , Sitios Genéticos , Humanos , Límite de Detección , Inestabilidad de Microsatélites , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: APOBEC3 (A3) proteins restrict viral replication by cytidine deamination of viral DNA genomes and impairing reverse transcription and integration. To escape this restriction, lentiviruses have evolved the viral infectivity factor (Vif), which binds A3 proteins and targets them for proteolytic degradation. In contrast, foamy viruses (FVs) encode Bet proteins that allow replication in the presence of A3, apparently by A3 binding and/or sequestration, thus preventing A3 packaging into virions and subsequent restriction. Due to a long-lasting FV-host coevolution, Bet proteins mainly counteract restriction by A3s from their cognate or highly related host species. RESULTS: Through bioinformatics, we identified conserved motifs in Bet, all localized in the bel2 exon. In line with the localization of these conserved motifs within bel2, this part of feline FV (FFV) Bet has been shown to be essential for feline A3 (feA3) inactivation and feA3 protein binding. To study the function of the Bet motifs in detail, we analyzed the ability of targeted deletion, substitution, and chimeric FFV-PFV (prototype FV) Bet mutants to physically bind and/or inactivate feA3. Binding of Bet to feA3Z2b is sensitive to mutations in the first three conserved motifs and N- and C-terminal deletions and substitutions across almost the complete bel2 coding sequence. In contrast, the Bel1 (also designated Tas) domain of Bet is dispensable for basal feA3Z2b inactivation and binding but mainly increases the steady state level of Bet. Studies with PFV Bel1 and full-length FFV Bel2 chimeras confirmed the importance of Bel2 for A3 inactivation indicating that Bel1 is dispensable for basal feA3Z2b inactivation and binding but increases Bet stability. Moreover, the bel1/tas exon may be required for expression of a fully functional Bet protein from a spliced transcript. CONCLUSIONS: We show that the Bel2 domain of FV Bet is essential for the inactivation of APOBEC3 cytidine deaminase restriction factors. The Bel1/Tas domain increases protein stability and can be exchanged by related sequence. Since feA3 binding and inactivation by Bet are highly correlated, the data support the view that FV Bet prevents A3-mediated restriction of viral replication by creating strong complexes with these proteins.
Asunto(s)
Citosina Desaminasa/inmunología , Citosina Desaminasa/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas de los Retroviridae/inmunología , Proteínas de los Retroviridae/metabolismo , Spumavirus/fisiología , Animales , Gatos , Línea Celular , Unión Proteica , Spumavirus/inmunologíaRESUMEN
Mapping of proteins involved in normal eye functions is a prerequisite to identify pathological changes during eye disease processes. We therefore analysed the proteome of human vitreous by applying in-depth proteomic screening technologies. For ethical reasons human vitreous samples were obtained by vitrectomy from "surrogate normal patients" with epiretinal gliosis that is considered to constitute only negligible pathological vitreoretinal changes. We applied different protein prefractionation strategies including liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both and compared the number of identified proteins obtained by the respective method. Liquid phase isoelectric focussing followed by SDS gel electrophoresis increased the number of identified proteins by a factor of five compared to the analysis of crude unseparated human vitreous. Depending on the prefractionation method proteins were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a UPLC system coupled online to an LTQ Orbitrap XL mass spectrometer. The obtained mass spectra were searched against the SwissProt database using the Mascot search engine. Bioinformatics tools were used to annotate known biological functions to the detected proteins. Following this strategy we examined the vitreous proteomes of three individuals and identified 1111 unique proteins. Besides structural, transport and binding proteins, we detected 261 proteins with known enzymatic activity, 51 proteases, 35 protease inhibitors, 35 members of complement and coagulation cascades, 15 peptide hormones, 5 growth factors, 11 cytokines, 47 receptors, 30 proteins of visual perception, 91 proteins involved in apoptosis regulation and 265 proteins with signalling activity. This highly complex mixture strikingly differs from the human plasma proteome. Thus human vitreous fluid seems to be a unique body fluid. 262 unique proteins were detected which are present in all three patient samples indicating that these might represent the constitutive protein pattern of human vitreous. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies on pathological vitreous proteome changes.