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High-throughput technologies produce gene expression time-series data that need fast and specialized algorithms to be processed. While current methods already deal with different aspects, such as the non-stationarity of the process and the temporal correlation, they often fail to take into account the pairing among replicates. We propose PairGP, a non-stationary Gaussian process method to compare gene expression time-series across several conditions that can account for paired longitudinal study designs and can identify groups of conditions that have different gene expression dynamics. We demonstrate the method on both simulated data and previously unpublished RNA sequencing (RNA-seq) time-series with five conditions. The results show the advantage of modeling the pairing effect to better identify groups of conditions with different dynamics. The pairing effect model displays good capabilities of selecting the most probable grouping of conditions even in the presence of a high number of conditions. The developed method is of general application and can be applied to any gene expression time series dataset. The model can identify common replicate effects among the samples coming from the same biological replicates and model those as separate components. Learning the pairing effect as a separate component, not only allows us to exclude it from the model to get better estimates of the condition effects, but also to improve the precision of the model selection process. The pairing effect that was accounted before as noise, is now identified as a separate component, resulting in more accurate and explanatory models of the data.
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BACKGROUND: The use of cell-free DNA (cfDNA) as a noninvasive biomarker to detect allograft damage is expanding rapidly. However, quantifying the low fraction of donor-derived cfDNA (ddcfDNA) is challenging and requires a highly sensitive technique. ddcfDNA detection through unique donor single nucleotide polymorphisms (SNPs) is a recent new approach, however there are limited data in pediatric solid organ transplant (SOT) recipients. METHODS: We developed an assay using a combination of 61 SNPs to quantify the ddcfDNA accurately using a custom R script to model for both the patient and donor genotypes requiring only a single sample from the allograft recipient. Performance of the assay was validated using genomic DNA (gDNA), cfDNA and donor samples where available. RESULTS: The R "genotype-free" method gave results comparable to when using the known donor genotype. applicable to both related and unrelated pairs and can reliably measure ddcfDNA (limit of blank, below 0.12%; limit of detection, above 0.25%; limit of quantification 0.5% resulting in 84% accuracy). 159 pediatric SOT recipients (kidney, heart, and lung) were tested without the need for donor genotyping. Serial sampling was obtained from 82 patients. CONCLUSION: We have developed and validated a new assay to measure the fraction of ddcfDNA in the plasma of pediatric SOT recipients. Our method can be applicable in any donor-recipient pair without the need for donor genotyping and can provide results in 48 h at a low cost. Additional prospective studies are required to demonstrate its clinical validity in a large cohort of pediatric SOT recipients.
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Análisis Químico de la Sangre/métodos , Ácidos Nucleicos Libres de Células/sangre , Trasplante de Órganos , Biomarcadores/sangre , Ácidos Nucleicos Libres de Células/genética , Niño , Preescolar , Femenino , Fluorometría , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Límite de Detección , Masculino , Reacción en Cadena de la Polimerasa Multiplex , Polimorfismo de Nucleótido Simple , Donantes de Tejidos , Receptores de TrasplantesRESUMEN
BACKGROUND: Noninvasive prenatal diagnosis (NIPD) for monogenic disorders has a high uptake by families. Since 2013, our accredited public health service laboratory has offered NIPD for monogenic disorders, predominantly for de novo or paternally dominantly inherited mutations. Here we describe the extension of this service to include definitive NIPD for a recessive condition, cystic fibrosis (CF). METHODS: Definitive NIPD for CF was developed using next-generation sequencing. Validation was performed on 13 cases from 10 families before implementation. All cases referred for CF NIPD were reviewed to determine turnaround times, genotyping results, and pregnancy outcomes. RESULTS: Of 38 referrals, 36 received a result with a mean turnaround of 5.75 days (range, 3-11 days). Nine cases were initially inconclusive, with 3 reported unaffected because the low-risk paternal allele was inherited and 4 cases in which the high-risk paternal allele was inherited, receiving conclusive results following repeat testing. One case was inconclusive owing to a paternal recombination around the mutation site, and one case was uninformative because of no heterozygosity. Before 2016, 3 invasive referrals for CF were received annually compared with 38 for NIPD in the 24 months since offering a definitive NIPD service. CONCLUSIONS: Timely and accurate NIPD for definitive prenatal diagnosis of CF is possible in a public health service laboratory. The method detects recombinations, and the service is well-received as evidenced by the significant increase in referrals. The bioinformatic approach is gene agnostic and will be used to expand the range of conditions tested for.
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Fibrosis Quística/diagnóstico , Pruebas Prenatales no Invasivas/métodos , Ácidos Nucleicos Libres de Células/química , Ácidos Nucleicos Libres de Células/metabolismo , Femenino , Genotipo , Haplotipos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Polimorfismo de Nucleótido Simple , EmbarazoRESUMEN
Importance: Neuroinflammatory disorders are a range of severe neurological disorders causing brain and spinal inflammation and are now increasingly recognized in the pediatric population. They are often characterized by marked genotypic and phenotypic heterogeneity, complicating diagnostic work in clinical practice and molecular diagnosis. Objective: To develop and evaluate a next-generation sequencing panel targeting genes causing neuroinflammation or mimicking neuroinflammation. Design, Setting, and Participants: Cohort study in which a total of 257 genes associated with monogenic neuroinflammation and/or cerebral vasculopathy, including monogenic noninflammatory diseases mimicking these entities, were selected. A customized enrichment capture array, the neuroinflammation gene panel (NIP), was created. Targeted high-coverage sequencing was applied to DNA samples taken from eligible patients referred to Great Ormond Street Hospital in London, United Kingdom, between January 1, 2017, and January 30, 2019, because of onset of disease early in life, family history, and/or complex neuroinflammatory phenotypes. Main Outcomes and Measures: The main outcome was the percentage of individuals with definitive molecular diagnoses, variant classification, and clinical phenotyping of patients with pathogenic variants identified using the NIP panel. The NIP panel was initially validated in 16 patients with known genetic diagnoses. Results: The NIP was both sensitive (95%) and specific (100%) for detection of known mutations, including gene deletions, copy number variants, small insertions and deletions, and somatic mosaicism with allele fraction as low as 3%. Prospective testing of 60 patients (30 [50%] male; median [range] age, 9.8 [0.8-20] years) presenting with heterogeneous neuroinflammatory phenotypes revealed at least 1 class 5 (clearly pathogenic) variant in 9 of 60 patients (15%); 18 of 60 patients (30%) had at least 1 class 4 (likely pathogenic) variant. Overall, a definitive molecular diagnosis was established in 12 of 60 patients (20%). Conclusions and Relevance: The NIP was associated with molecular diagnosis in this cohort and complemented routine laboratory and radiological workup of patients with neuroinflammation. Unexpected genotype-phenotype associations in patients with pathogenic variants deviating from the classic phenotype were identified. Obtaining an accurate molecular diagnosis in a timely fashion informed patient management, including successful targeted treatment in some instances and early institution of hematopoietic stem cell transplantation in others.
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Encefalopatías/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inflamación/genética , Adolescente , Niño , Preescolar , Genotipo , Humanos , Lactante , Londres , Técnicas de Diagnóstico Molecular , Mutación , Fenotipo , Sensibilidad y Especificidad , Adulto JovenRESUMEN
We recently described a new neurodevelopmental syndrome (TAF1/MRXS33 intellectual disability syndrome) (MIM# 300966) caused by pathogenic variants involving the X-linked gene TAF1, which participates in RNA polymerase II transcription. The initial study reported eleven families, and the syndrome was defined as presenting early in life with hypotonia, facial dysmorphia, and developmental delay that evolved into intellectual disability (ID) and/or autism spectrum disorder (ASD). We have now identified an additional 27 families through a genotype-first approach. Familial segregation analysis, clinical phenotyping, and bioinformatics were capitalized on to assess potential variant pathogenicity, and molecular modelling was performed for those variants falling within structurally characterized domains of TAF1. A novel phenotypic clustering approach was also applied, in which the phenotypes of affected individuals were classified using 51 standardized Human Phenotype Ontology (HPO) terms. Phenotypes associated with TAF1 variants show considerable pleiotropy and clinical variability, but prominent among previously unreported effects were brain morphological abnormalities, seizures, hearing loss, and heart malformations. Our allelic series broadens the phenotypic spectrum of TAF1/MRXS33 intellectual disability syndrome and the range of TAF1 molecular defects in humans. It also illustrates the challenges for determining the pathogenicity of inherited missense variants, particularly for genes mapping to chromosome X. This article is protected by copyright. All rights reserved.
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Down syndrome (DS), trisomy of human chromosome 21 (Hsa21), results in a broad range of phenotypes. A recent study reported that DS cells show genome-wide transcriptional changes in which up- or down-regulated genes are clustered in gene expression dysregulation domains (GEDDs). GEDDs were also reported in fibroblasts derived from a DS mouse model duplicated for some Hsa21-orthologous genes, indicating cross-species conservation of this phenomenon. Here we investigate GEDDs using the Dp1Tyb mouse model of DS, which is duplicated for the entire Hsa21-orthologous region of mouse chromosome 16. Our statistical analysis shows that GEDDs are present both in DS cells and in Dp1Tyb mouse fibroblasts and hippocampus. However, we find that GEDDs do not depend on the DS genotype but occur whenever gene expression changes. We conclude that GEDDs are not a specific feature of DS but instead result from the clustering of co-regulated genes, a function of mammalian genome organisation.
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Síndrome de Down/genética , Fibroblastos/metabolismo , Expresión Génica/genética , Hipocampo/metabolismo , Animales , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Genoma , Genotipo , Ratones , Familia de Multigenes , FenotipoRESUMEN
PURPOSE: To develop a comprehensive next-generation sequencing panel assay that screens genes known to cause developmental eye disorders and inherited eye disease and to evaluate its diagnostic yield in a pediatric cohort with malformations of the globe, anterior segment anomalies, childhood glaucoma, or a combination thereof. DESIGN: Evaluation of diagnostic test. PARTICIPANTS: Two hundred seventy-seven children, 0 to 16 years of age, diagnosed with nonsyndromic or syndromic developmental eye defects without a genetic diagnosis. METHODS: We developed a new oculome panel using a custom-designed Agilent SureSelect QXT target capture method (Agilent Technologies, Santa Clara, CA) to capture and perform parallel high-throughput sequencing analysis of 429 genes associated with eye disorders. Bidirectional Sanger sequencing confirmed suspected pathogenic variants. MAIN OUTCOME MEASURES: Collated clinical details and oculome molecular genetic results. RESULTS: The oculome design covers 429 known eye disease genes; these are subdivided into 5 overlapping virtual subpanels for anterior segment developmental anomalies including glaucoma (ASDA; 59 genes), microphthalmia-anophthalmia-coloboma (MAC; 86 genes), congenital cataracts and lens-associated conditions (70 genes), retinal dystrophies (RET; 235 genes), and albinism (15 genes), as well as additional genes implicated in optic atrophy and complex strabismus (10 genes). Panel development and testing included analyzing 277 clinical samples and 3 positive control samples using Illumina sequencing platforms; more than 30× read depth was achieved for 99.5% of the targeted 1.77-Mb region. Bioinformatics analysis performed using a pipeline based on Freebayes and ExomeDepth to identify coding sequence and copy number variants, respectively, resulted in a definitive diagnosis in 68 of 277 samples, with variability in diagnostic yield between phenotypic subgroups: MAC, 8.2% (8 of 98 cases solved); ASDA, 24.8% (28 of 113 cases solved); other or syndromic, 37.5% (3 of 8 cases solved); RET, 42.8% (21 of 49 cases solved); and congenital cataracts and lens-associated conditions, 88.9% (8 of 9 cases solved). CONCLUSIONS: The oculome test diagnoses a comprehensive range of genetic conditions affecting the development of the eye, potentially replacing protracted and costly multidisciplinary assessments and allowing for faster targeted management. The oculome enabled molecular diagnosis of a significant number of cases in our sample cohort of varied ocular birth defects.
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Variaciones en el Número de Copia de ADN/genética , Anomalías del Ojo/diagnóstico , Anomalías del Ojo/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Técnicas de Diagnóstico Molecular , Mutación/genética , Proteoma/genética , Adolescente , Niño , Preescolar , Femenino , Genoma Humano , Humanos , Lactante , Recién Nacido , Masculino , LinajeRESUMEN
Interleukin (IL)-36α, IL-36ß, and IL-36γ are innate mediators of acute epithelial inflammation. We sought to demonstrate that these cytokines are also required for the pathogenesis of plaque psoriasis, a common and chronic skin disorder, caused by abnormal T helper 17 (TH17) cell activation. To investigate this possibility, we first defined the genes that are induced by IL-36 cytokines in primary human keratinocytes. This enabled us to demonstrate a significant IL-36 signature among the transcripts that are up-regulated in plaque psoriasis and the susceptibility loci associated with the disease in genome-wide studies. Next, we investigated the impact of in vivo and ex vivo IL-36 receptor blockade using a neutralizing antibody or a recombinant antagonist. Both inhibitors had marked anti-inflammatory effects on psoriatic skin, demonstrated by statistically significant reductions in IL-17 expression, keratinocyte activation, and leukocyte infiltration. Finally, we explored the potential safety profile associated with IL-36 blockade by phenotyping 12 individuals carrying knockout mutations of the IL-36 receptor gene. We found that normal immune function was broadly preserved in these individuals, suggesting that IL-36 signaling inhibition would not substantially compromise host defenses. These observations, which integrate the results of transcriptomics and model system analysis, pave the way for early-stage clinical trials of IL-36 antagonists.
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Interleucina-1/metabolismo , Psoriasis/metabolismo , Células Cultivadas , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucina-1/genética , Interleucina-17/metabolismo , Queratinocitos/metabolismo , Psoriasis/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Piel/metabolismoRESUMEN
BACKGROUND: Monogenic autoinflammatory diseases (AID) are a rapidly expanding group of genetically diverse but phenotypically overlapping systemic inflammatory disorders associated with dysregulated innate immunity. They cause significant morbidity, mortality and economic burden. Here, we aimed to develop and evaluate the clinical impact of a NGS targeted gene panel, the "Vasculitis and Inflammation Panel" (VIP) for AID and vasculitis. METHODS: The Agilent SureDesign tool was used to design 2 versions of VIP; VIP1 targeting 113 genes, and a later version, VIP2, targeting 166 genes. Captured and indexed libraries (QXT Target Enrichment System) prepared for 72 patients were sequenced as a multiplex of 16 samples on an Illumina MiSeq sequencer in 150bp paired-end mode. The cohort comprised 22 positive control DNA samples from patients with previously validated mutations in a variety of the genes; and 50 prospective samples from patients with suspected AID in whom previous Sanger based genetic screening had been non-diagnostic. RESULTS: VIP was sensitive and specific at detecting all the different types of known mutations in 22 positive controls, including gene deletion, small INDELS, and somatic mosaicism with allele fraction as low as 3%. Six/50 patients (12%) with unclassified AID had at least one class 5 (clearly pathogenic) variant; and 11/50 (22%) had at least one likely pathogenic variant (class 4). Overall, testing with VIP resulted in a firm or strongly suspected molecular diagnosis in 16/50 patients (32%). CONCLUSIONS: The high diagnostic yield and accuracy of this comprehensive targeted gene panel validate the use of broad NGS-based testing for patients with suspected AID.
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Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inflamación/genética , Vasculitis/genética , Anciano , Niño , Preescolar , ADN/genética , Femenino , Heterocigoto , Humanos , Masculino , Mutación/genética , Reproducibilidad de los ResultadosRESUMEN
RNA-binding proteins of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU-rich elements in the 3' untranslated region and promoting mRNA decay. Here we identified an indispensable role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal-zone B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene-expression program related to signaling, cell adhesion and locomotion; it achieved this in part by limiting expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RNA-binding proteins for maintaining cellular identity among closely related cell types.
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Linfocitos B/inmunología , Adhesión Celular/genética , Movimiento Celular/genética , Proteínas Nucleares/genética , Proteínas de Unión al ARN/genética , Animales , Factor 1 de Respuesta al Butirato , Adhesión Celular/inmunología , Movimiento Celular/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Factores Reguladores del Interferón/genética , Factores de Transcripción de Tipo Kruppel/genética , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Ratones , Proteínas Nucleares/inmunología , Fenotipo , Proteínas de Unión al ARN/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARN , Transducción de SeñalRESUMEN
The aryl hydrocarbon receptor (AhR), a transcription factor known for mediating xenobiotic toxicity, is expressed in B cells, which are known targets for environmental pollutants. However, it is unclear what the physiological functions of AhR in B cells are. We show here that expression of Ahr in B cells is up-regulated upon B-cell receptor (BCR) engagement and IL-4 treatment. Addition of a natural ligand of AhR, FICZ, induces AhR translocation to the nucleus and transcription of the AhR target gene Cyp1a1, showing that the AhR pathway is functional in B cells. AhR-deficient (Ahr-/-) B cells proliferate less than AhR-sufficient (Ahr+/+) cells following in vitro BCR stimulation and in vivo adoptive transfer models confirmed that Ahr-/- B cells are outcompeted by Ahr+/+ cells. Transcriptome comparison of AhR-deficient and AhR-sufficient B cells identified cyclin O (Ccno), a direct target of AhR, as a top candidate affected by AhR deficiency.
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Linfocitos B/fisiología , Proliferación Celular , Receptores de Hidrocarburo de Aril/metabolismo , Ciclinas/metabolismo , Citocromo P-450 CYP1A1/biosíntesis , Perfilación de la Expresión Génica , Interleucina-4/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Hidrocarburo de Aril/deficiencia , Transcripción GenéticaRESUMEN
The RNA-binding proteins Zfp36l1 and Zfp36l2 act redundantly to enforce the ß-selection checkpoint during thymopoiesis, yet their molecular targets remain largely unknown. In this study, we identify these targets on a genome-wide scale in primary mouse thymocytes and show that Zfp36l1/l2 regulate DNA damage response and cell cycle transcripts to ensure proper ß-selection. Double-negative 3 thymocytes lacking Zfp36l1/l2 share a gene expression profile with postselected double-negative 3b cells despite the absence of intracellular TCRß and reduced IL-7 signaling. Our findings show that in addition to controlling the timing of proliferation at ß-selection, posttranscriptional control by Zfp36l1/l2 limits DNA damage responses, which are known to promote thymocyte differentiation. Zfp36l1/l2 therefore act as posttranscriptional safeguards against chromosomal instability and replication stress by integrating pre-TCR and IL-7 signaling with DNA damage and cell cycle control.
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Ciclo Celular , Daño del ADN , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Timocitos/citología , Tristetraprolina/metabolismo , Animales , Factor 1 de Respuesta al Butirato , Ciclo Celular/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Fenotipo , Proteínas de Unión al ARN/genética , Timocitos/metabolismo , Tristetraprolina/deficiencia , Tristetraprolina/genéticaRESUMEN
Epithelia function as barriers against environmental insults and express the transcription factor aryl hydrocarbon receptor (AhR). However, AhR function in these tissues is unknown. Here we show that AhR regulates multiciliogenesis in both murine airway epithelia and in Xenopus laevis epidermis. In air-exposed airway epithelia, induction of factors required for multiciliogenesis, including cyclin O (Ccno) and Multicilin (Mcidas), is AhR dependent, and air exposure induces AhR binding to the Ccno promoter. Submersion and hypoxic conditions impede AhR-dependent Ccno induction. This is mediated by the persistence of Notch signalling, as Notch blockade renders multiciliogenesis and Ccno induction by AhR independent from air exposure. In contrast to Ccno induction, air exposure does not induce the canonical AhR target cytochrome P450 1a1 (Cyp1a1). Inversely, exposure to AhR ligands induces Cyp1a1 but not Ccno and impeded ciliogenesis. These data indicate that AhR involvement in detoxification of environmental pollutants may impede its physiological role, resulting in respiratory pathology.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ciclinas/biosíntesis , Ciclinas/genética , Receptores de Hidrocarburo de Aril/metabolismo , Mucosa Respiratoria/metabolismo , Contaminantes Atmosféricos/farmacocinética , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Células Cultivadas , Citocromo P-450 CYP1A1/biosíntesis , Epidermis/metabolismo , Regulación de la Expresión Génica , Inactivación Metabólica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/deficiencia , Receptores de Hidrocarburo de Aril/genética , Mucosa Respiratoria/citología , Proteínas de Xenopus/biosíntesis , Proteínas de Xenopus/deficiencia , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
MOTIVATION: Mechanistic models based on ordinary differential equations provide powerful and accurate means to describe the dynamics of molecular machinery which orchestrates gene regulation. When combined with appropriate statistical techniques, mechanistic models can be calibrated using experimental data and, in many cases, also the model structure can be inferred from time-course measurements. However, existing mechanistic models are limited in the sense that they rely on the assumption of static network structure and cannot be applied when transient phenomena affect, or rewire, the network structure. In the context of gene regulatory network inference, network rewiring results from the net impact of possible unobserved transient phenomena such as changes in signaling pathway activities or epigenome, which are generally difficult, but important, to account for. RESULTS: We introduce a novel method that can be used to infer dynamically evolving regulatory networks from time-course data. Our method is based on the notion that all mechanistic ordinary differential equation models can be coupled with a latent process that approximates the network structure rewiring process. We illustrate the performance of the method using simulated data and, further, we apply the method to study the regulatory interactions during T helper 17 (Th17) cell differentiation using time-course RNA sequencing data. The computational experiments with the real data show that our method is capable of capturing the experimentally verified rewiring effects of the core Th17 regulatory network. We predict Th17 lineage specific subnetworks that are activated sequentially and control the differentiation process in an overlapping manner. AVAILABILITY AND IMPLEMENTATION: An implementation of the method is available at http://research.ics.aalto.fi/csb/software/lem/ CONTACTS: jukka.intosalmi@aalto.fi or harri.lahdesmaki@aalto.fi.
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Redes Reguladoras de Genes , Algoritmos , Diferenciación Celular , Regulación de la Expresión Génica , Análisis de Secuencia de ARN , Transducción de SeñalRESUMEN
RNA-binding proteins (RBPs) facilitate post-transcriptional control of eukaryotic gene expression at multiple levels. The RBP tristetraprolin (TTP/Zfp36) is a signal-induced phosphorylated anti-inflammatory protein guiding unstable mRNAs of pro-inflammatory proteins for degradation and preventing translation. Using iCLIP, we have identified numerous mRNA targets bound by wild-type TTP and by a non-MK2-phosphorylatable TTP mutant (TTP-AA) in 1 h LPS-stimulated macrophages and correlated their interaction with TTP to changes at the level of mRNA abundance and translation in a transcriptome-wide manner. The close similarity of the transcriptomes of TTP-deficient and TTP-expressing macrophages upon short LPS stimulation suggested an effective inactivation of TTP by MK2, whereas retained RNA-binding capacity of TTP-AA to 3'UTRs caused profound changes in the transcriptome and translatome, altered NF-κB-activation and induced cell death. Increased TTP binding to the 3'UTR of feedback inhibitor mRNAs, such as Ier3, Dusp1 or Tnfaip3, in the absence of MK2-dependent TTP neutralization resulted in a strong reduction of their protein synthesis contributing to the deregulation of the NF-κB-signaling pathway. Taken together, our study uncovers a role of TTP as a suppressor of feedback inhibitors of inflammation and highlights the importance of fine-tuned TTP activity-regulation by MK2 in order to control the pro-inflammatory response.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Inflamación/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Supervivencia Celular , Reactivos de Enlaces Cruzados , Citocinas/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunoprecipitación , Inflamación/genética , Inflamación/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/inmunología , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por Sustrato , TranscriptomaRESUMEN
Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-µ at the pre-BCR checkpoint.
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Linfocitos B/citología , Proteínas Nucleares/fisiología , Proteínas de Unión al ARN/fisiología , Fase S/fisiología , Tristetraprolina/fisiología , Animales , Factor 1 de Respuesta al Butirato , Secuencia Conservada , Ciclinas/metabolismo , Fase G1/genética , Fase G1/fisiología , Regulación de la Expresión Génica , Cadenas mu de Inmunoglobulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Receptores de Células Precursoras de Linfocitos B , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Fase de Descanso del Ciclo Celular/genética , Fase de Descanso del Ciclo Celular/fisiología , Fase S/genética , Selección Genética , Transcripción Genética , Tristetraprolina/genética , Recombinación V(D)JRESUMEN
Uncontrolled Th17 cell activity is associated with cancer and autoimmune and inflammatory diseases. To validate the potential relevance of mouse models of targeting the Th17 pathway in human diseases we used RNA sequencing to compare the expression of coding and non-coding transcripts during the priming of Th17 cell differentiation in both human and mouse. In addition to already known targets, several transcripts not previously linked to Th17 cell polarization were found in both species. Moreover, a considerable number of human-specific long non-coding RNAs were identified that responded to cytokines stimulating Th17 cell differentiation. We integrated our transcriptomics data with known disease-associated polymorphisms and show that conserved regulation pinpoints genes that are relevant to Th17 cell-mediated human diseases and that can be modelled in mouse. Substantial differences observed in non-coding transcriptomes between the two species as well as increased overlap between Th17 cell-specific gene expression and disease-associated polymorphisms underline the need of parallel analysis of human and mouse models. Comprehensive analysis of genes regulated during Th17 cell priming and their classification to conserved and non-conserved between human and mouse facilitates translational research, pointing out which candidate targets identified in human are worth studying by using in vivo mouse models.
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Biomarcadores/metabolismo , Polimorfismo de Nucleótido Simple , Células Th17/inmunología , Células Th17/metabolismo , Transcriptoma , Animales , Células Cultivadas , Humanos , Recién Nacido , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: The differentiation of naive CD 4(+) helper T (Th) cells into effector Th17 cells is steered by extracellular cytokines that activate and control the lineage specific transcriptional program. While the inducing cytokine signals and core transcription factors driving the differentiation towards Th17 lineage are well known, detailed mechanistic interactions between the key components are poorly understood. RESULTS: We develop an integrative modeling framework which combines RNA sequencing data with mathematical modeling and enables us to construct a mechanistic model for the core Th17 regulatory network in a data-driven manner. CONCLUSIONS: Our results show significant evidence, for instance, for inhibitory mechanisms between the transcription factors and reveal a previously unknown dependency between the dosage of the inducing cytokine TGF ß and the expression of the master regulator of competing (induced) regulatory T cell lineage. Further, our experimental validation approves this dependency in Th17 polarizing conditions.
Asunto(s)
Diferenciación Celular/genética , Redes Reguladoras de Genes , Modelos Genéticos , Células Th17/citología , Citocinas/genética , Citocinas/metabolismo , Citocinas/fisiología , Análisis de Secuencia de ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
IL-22 is a cytokine that regulates tissue homeostasis at barrier surfaces. A variety of IL-22-producing cell types is known, but identification on the single-cell level remains difficult. Therefore, we generated a fate reporter mouse that would allow the identification of IL-22-producing cells and their fate mapping in vivo. To trace IL-22-expressing cells, a sequence encoding Cre recombinase was cloned into the Il22 locus, and IL22(Cre) mice were crossed with reporter mice expressing enhanced yellow fluorescence protein (eYFP) under control of the endogenous Rosa26 promoter. In IL22(Cre)R26R(eYFP) mice, the fluorescent reporter permanently labels cells that have switched on Il22 expression, irrespective of cytokine production. Despite a degree of underreporting, eYFP expression was detectable in nonimmune mice and restricted to group 3 innate lymphoid cells (ILC3) in the gut and γδ T cells in skin or lung. Upon skin challenge with imiquimod, eYFP(+) γδ and CD4 T cells expanded in the skin. Infection with Citrobacter rodentium initially was controlled by ILC3, followed by expansion of eYFP(+) CD4 T cells, which were induced in innate lymphoid follicles in the colon. No eYFP expression was detected in small intestinal Th17 cells, and they did not expand in the immune response. Colonic eYFP(+) CD4 T cells exhibited plasticity during infection with expression of additional cytokines, in contrast to ILC3, which remained largely stable. Single-cell quantitative PCR analysis of eYFP(+) CD4 T cells confirmed their heterogeneity, suggesting that IL-22 expression is not confined to particular subsets or a dedicated Th22 subset.
Asunto(s)
Homeostasis , Infecciones/metabolismo , Interleucinas/biosíntesis , Animales , Citrobacter rodentium/inmunología , Análisis por Conglomerados , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Infecciones por Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Orden Génico , Marcación de Gen , Genes Reporteros , Sitios Genéticos , Homocigoto , Infecciones/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Interleucinas/genética , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones , Ratones Transgénicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Interleucina-22RESUMEN
Environmental stimuli are known to contribute to psoriasis pathogenesis and that of other autoimmune diseases, but the mechanisms are largely unknown. Here we show that the aryl hydrocarbon receptor (AhR), a transcription factor that senses environmental stimuli, modulates pathology in psoriasis. AhR-activating ligands reduced inflammation in the lesional skin of psoriasis patients, whereas AhR antagonists increased inflammation. Similarly, AhR signaling via the endogenous ligand FICZ reduced the inflammatory response in the imiquimod-induced model of skin inflammation and AhR-deficient mice exhibited a substantial exacerbation of the disease, compared to AhR-sufficient controls. Nonhematopoietic cells, in particular keratinocytes, were responsible for this hyperinflammatory response, which involved upregulation of AP-1 family members of transcription factors. Thus, our data suggest a critical role for AhR in the regulation of inflammatory responses and open the possibility for novel therapeutic strategies in chronic inflammatory disorders.