RESUMEN
The NF-κB protein RelB controls dendritic cell (DC) maturation and may be targeted therapeutically to manipulate T cell responses in disease. Here we report that RelB promoted DC activation not as the expected RelB-p52 effector of the noncanonical NF-κB pathway, but as a RelB-p50 dimer regulated by canonical IκBs, IκBα and IκBÉ. IκB control of RelB minimized spontaneous maturation but enabled rapid pathogen-responsive maturation. Computational modeling of the NF-κB signaling module identified control points of this unexpected cell type-specific regulation. Fibroblasts that we engineered accordingly showed DC-like RelB control. Canonical pathway control of RelB regulated pathogen-responsive gene expression programs. This work illustrates the potential utility of systems analyses in guiding the development of combination therapeutics for modulating DC-dependent T cell responses.
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Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Activación de Linfocitos , FN-kappa B/metabolismo , Factor de Transcripción ReIB/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Quinasa I-kappa B/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , Multimerización de Proteína , Transducción de Señal , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 9/metabolismo , Factor de Transcripción ReIB/genéticaRESUMEN
The rapid emergence of SARS-CoV-2 variants raised public health questions concerning the capability of diagnostic tests to detect new strains, the efficacy of vaccines, and how to map the geographical distribution of variants to understand transmission patterns and loads on healthcare resources. Next-generation sequencing (NGS) is the primary method for detecting and tracing new variants, but it is expensive, and it can take weeks before sequence data are available in public repositories. This article describes a customizable reverse transcription PCR (RT-PCR)-based genotyping approach which is significantly less expensive, accelerates reporting, and can be implemented in any lab that performs RT-PCR. Specific single-nucleotide polymorphisms (SNPs) and indels were identified which had high positive-percent agreement (PPA) and negative-percent agreement (NPA) compared to NGS for the major genotypes that circulated through September 11, 2021. Using a 48-marker panel, testing on 1,031 retrospective SARS-CoV-2 positive samples yielded a PPA and NPA ranging from 96.3 to 100% and 99.2 to 100%, respectively, for the top 10 most prevalent World Health Organization (WHO) lineages during that time. The effect of reducing the quantity of panel markers was explored, and a 16-marker panel was determined to be nearly as effective as the 48-marker panel at lineage assignment. Responding to the emergence of Omicron, a genotyping panel was developed which distinguishes Delta and Omicron using four highly specific SNPs. The results demonstrate the utility of the condensed panel to rapidly track the growing prevalence of Omicron across the US in December 2021 and January 2022.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Humanos , Técnicas de Amplificación de Ácido Nucleico , Estudios Retrospectivos , SARS-CoV-2/genéticaRESUMEN
The spliceosome catalyzes the removal of introns from pre-messenger RNA (mRNA) and subsequent pairing of exons with remarkable fidelity. Some exons are known to be skipped or included in the mature mRNA in a cell type- or context-dependent manner (cassette exons), thereby contributing to the diversification of the human proteome. Interestingly, splicing is initiated (and sometimes completed) co-transcriptionally. Here, we develop a kinetic mathematical modeling framework to investigate alternative co-transcriptional splicing (CTS) and, specifically, the control of cassette exons' inclusion. We show that when splicing is co-transcriptional, default splice patterns of exon inclusion are more likely than when splicing is post-transcriptional, and that certain exons are more likely to be regulatable (i.e. cassette exons) than others, based on the exon-intron structure context. For such regulatable exons, transcriptional elongation rates may affect splicing outcomes. Within the CTS paradigm, we examine previously described hypotheses of co-operativity between splice sites of short introns (i.e. 'intron definition') or across short exons (i.e. 'exon definition'), and find that models encoding these faithfully recapitulate observations in the fly and human genomes, respectively.
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Empalme Alternativo/genética , Redes Reguladoras de Genes/genética , Modelos Teóricos , Precursores del ARN/genética , Sitios de Empalme de ARN/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Secuencia de Bases , Estructuras Cromosómicas/genética , Biología Computacional , Drosophila melanogaster/genética , Genes , Humanos , Cadenas de Markov , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN/métodos , Empalmosomas/metabolismo , Transcripción Genética/genéticaRESUMEN
Targeted deletion of BAFF causes severe deficiency of splenic B cells. BAFF-R is commonly thought to signal to nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase dependent noncanonical NF-κB RelB. However, RelB-deficient mice have normal B-cell numbers. Recent studies showed that BAFF also signals to the canonical NF-κB pathway, and we found that both RelB and cRel are persistently activated, suggesting BAFF signaling coordinates both pathways to ensure robust B-cell development. Indeed, we report now that combined loss of these 2 NF-κB family members leads to impaired BAFF-mediated survival and development in vitro. Although single deletion of RelB and cRel was dispensable for normal B-cell development, double knockout mice displayed an early B-cell developmental blockade and decreased mature B cells. Despite disorganized splenic architecture in Relb(-/-)cRel(-/-) mice, generation of mixed-mouse chimeras established the developmental phenotype to be B-cell intrinsic. Together, our results indicate that BAFF signals coordinate both RelB and cRel activities to ensure survival during peripheral B-cell maturation.
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Linfocitos B/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , Transducción de Señal/fisiología , Factor de Transcripción ReIB/metabolismo , Animales , Factor Activador de Células B/genética , Factor Activador de Células B/metabolismo , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/metabolismo , Linfocitos B/citología , Supervivencia Celular/genética , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-ret/genética , Factor de Transcripción ReIB/genéticaRESUMEN
When messenger RNA splicing occurs co-transcriptionally, the potential for kinetic control based on transcription dynamics is widely recognized. Indeed, perturbation studies have reported that when transcription kinetics are perturbed genetically or pharmacologically splice patterns may change. However, whether kinetic control is contributing to the control of splicing within the normal range of physiological conditions remains unknown. We examined if the kinetic determinants for co-transcriptional splicing (CTS) might be reflected in the structure and expression patterns of the genome and epigenome. To identify and then quantitatively relate multiple, simultaneous CTS determinants, we constructed a scalable mathematical model of the kinetic interplay of RNA synthesis and CTS and parameterized it with diverse next generation sequencing (NGS) data. We thus found a variety of CTS determinants encoded in vertebrate genomes and epigenomes, and that these combine variously for different groups of genes such as housekeeping versus regulated genes. Together, our findings indicate that the kinetic basis of splicing is functionally and physiologically relevant, and may meaningfully inform the analysis of genomic and epigenomic data to provide insights that are missed when relying on statistical approaches alone.
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Modelos Genéticos , Empalme del ARN , Transcripción Genética , Animales , Simulación por Computador , Epigénesis Genética , Genes Esenciales , Genómica , Humanos , Cinética , Ratones , ARN Polimerasa II/metabolismo , ARN Mensajero/biosíntesis , Análisis de Secuencia de ARNRESUMEN
Understanding the functions of multi-cellular organs in terms of the molecular networks within each cell is an important step in the quest to predict phenotype from genotype. B-lymphocyte population dynamics, which are predictive of immune response and vaccine effectiveness, are determined by individual cells undergoing division or death seemingly stochastically. Based on tracking single-cell time-lapse trajectories of hundreds of B cells, single-cell transcriptome, and immunofluorescence analyses, we constructed an agent-based multi-modular computational model to simulate lymphocyte population dynamics in terms of the molecular networks that control NF-κB signaling, the cell cycle, and apoptosis. Combining modeling and experimentation, we found that NF-κB cRel enforces the execution of a cellular decision between mutually exclusive fates by promoting survival in growing cells. But as cRel deficiency causes growing B cells to die at similar rates to non-growing cells, our analysis reveals that the phenomenological decision model of wild-type cells is rooted in a biased race of cell fates. We show that a multi-scale modeling approach allows for the prediction of dynamic organ-level physiology in terms of intra-cellular molecular networks.
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Linfocitos B/citología , División Celular , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Animales , Apoptosis , Linfocitos B/metabolismo , Proliferación Celular , Ratones , Modelos Moleculares , Análisis de Secuencia de ARN , Transducción de Señal , Bazo/citologíaRESUMEN
The transcription factor NF-κB is a regulator of inflammatory and adaptive immune responses, yet only IκBα was shown to limit NF-κB activation and inflammatory responses. We investigated another negative feedback regulator, IκBε, in the regulation of B cell proliferation and survival. Loss of IκBε resulted in increased B cell proliferation and survival in response to both antigenic and innate stimulation. NF-κB activity was elevated during late-phase activation, but the dimer composition was stimulus specific. In response to IgM, cRel dimers were elevated in IκBε-deficient cells, yet in response to LPS, RelA dimers also were elevated. The corresponding dimer-specific sequences were found in the promoters of hyperactivated genes. Using a mathematical model of the NF-κB-signaling system in B cells, we demonstrated that kinetic considerations of IκB kinase-signaling input and IκBε's interactions with RelA- and cRel-specific dimers could account for this stimulus specificity. cRel is known to be the key regulator of B cell expansion. We found that the RelA-specific phenotype in LPS-stimulated cells was physiologically relevant: unbiased transcriptome profiling revealed that the inflammatory cytokine IL-6 was hyperactivated in IκBε(-/-) B cells. When IL-6R was blocked, LPS-responsive IκBε(-/-) B cell proliferation was reduced to near wild-type levels. Our results provide novel evidence for a critical role for immune-response functions of IκBε in B cells; it regulates proliferative capacity via at least two mechanisms involving cRel- and RelA-containing NF-κB dimers. This study illustrates the importance of kinetic considerations in understanding the functional specificity of negative-feedback regulators.
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Linfocitos B/inmunología , Proliferación Celular , Quinasa I-kappa B/inmunología , Proteínas Proto-Oncogénicas c-rel/inmunología , Factor de Transcripción ReIA/inmunología , Algoritmos , Animales , Linfocitos B/metabolismo , Western Blotting , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Retroalimentación Fisiológica/efectos de los fármacos , Citometría de Flujo , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-6/metabolismo , Cinética , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Inmunológicos , Multimerización de Proteína/inmunología , Proteínas Proto-Oncogénicas c-rel/química , Proteínas Proto-Oncogénicas c-rel/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Factor de Transcripción ReIA/química , Factor de Transcripción ReIA/metabolismo , Transcriptoma/efectos de los fármacos , Transcriptoma/inmunologíaRESUMEN
RNA-seq technologies are now replacing microarrays for profiling gene expression. Here we describe a robust RNA-seq strategy for multiplex analysis of RNA samples based on deep sequencing. First, an oligo-dT linked to an adaptor sequence is used to prime cDNA synthesis. Upon solid phase selection, second strand synthesis is initiated using a random primer linked to another adaptor sequence. Finally, the library is released from the beads and amplified using a bar-coded primer together with a common primer. This method, referred to as Multiplex Analysis of PolyA-linked Sequences (MAPS), preserves strand information, permits rapid identification of potentially new polyadenylation sites, and profiles gene expression in a highly cost effective manner. We have applied this technology to determine the transcriptome response to knockdown of the RNA binding protein TLS, and compared the result to current microarray technology, demonstrating the ability of MAPS to robustly detect regulated gene expression.
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Regiones no Traducidas 3'/fisiología , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Poli A/metabolismo , ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Regiones no Traducidas 3'/genética , Células HeLa , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Poli A/genética , Poliadenilación , ARN/genética , Proteína FUS de Unión a ARN/genética , TranscriptomaRESUMEN
Pompe disease, an autosomal recessive disorder caused by deficient lysosomal acid α-glucosidase (GAA), is characterized by accumulation of intra-lysosomal glycogen in skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant, the most frequent GAA pathogenic mutation in people of Southern Han Chinese ancestry, causes infantile-onset Pompe disease (IOPD), presenting neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, respiratory failure, and infantile mortality. We applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a Gaaem1935C>A knock-in mouse model and a myoblast cell line carrying the Gaa c.1935C>A mutation. Herein we describe the molecular, biochemical, histological, physiological, and behavioral characterization of 3-month-old homozygous Gaaem1935C>A mice. Homozygous Gaaem1935C>A knock-in mice exhibited normal Gaa mRNA expression levels relative to wild-type mice, had near-abolished GAA enzymatic activity, markedly increased tissue glycogen storage, and concomitantly impaired autophagy. Three-month-old mice demonstrated skeletal muscle weakness and hypertrophic cardiomyopathy but no premature mortality. The Gaaem1935C>A knock-in mouse model recapitulates multiple salient aspects of human IOPD caused by the GAA c.1935C>A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes.
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Cardiomiopatía Hipertrófica , Enfermedad del Almacenamiento de Glucógeno Tipo II , alfa-Glucosidasas , Animales , Humanos , Lactante , Ratones , alfa-Glucosidasas/genética , alfa-Glucosidasas/metabolismo , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Modelos Animales de Enfermedad , Glucano 1,4-alfa-Glucosidasa , Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/patología , Músculo Esquelético/metabolismoRESUMEN
Infantile-onset Pompe Disease (IOPD), caused by mutations in lysosomal acid alpha-glucosidase (Gaa), manifests rapidly progressive fatal cardiac and skeletal myopathy incompletely attenuated by synthetic GAA intravenous infusions. The currently available murine model does not fully simulate human IOPD, displaying skeletal myopathy with late-onset hypertrophic cardiomyopathy. Bearing a Cre-LoxP induced exonic disruption of the murine Gaa gene, this model is also not amenable to genome-editing based therapeutic approaches. We report the early onset of severe hypertrophic cardiomyopathy in a novel murine IOPD model generated utilizing CRISPR-Cas9 homology-directed recombination to harbor the orthologous Gaa mutation c.1826dupA (p.Y609*), which causes human IOPD. We demonstrate the dual sgRNA approach with a single-stranded oligonucleotide donor is highly specific for the Gaac.1826 locus without genomic off-target effects or rearrangements. Cardiac and skeletal muscle were deficient in Gaa mRNA and enzymatic activity and accumulated high levels of glycogen. The mice demonstrated skeletal muscle weakness but did not experience early mortality. Altogether, these results demonstrate that the CRISPR-Cas9 generated Gaac.1826dupA murine model recapitulates hypertrophic cardiomyopathy and skeletal muscle weakness of human IOPD, indicating its utility for evaluation of novel therapeutics.
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Cardiomiopatía Hipertrófica/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Debilidad Muscular/genética , alfa-Glucosidasas/genética , Edad de Inicio , Animales , Sistemas CRISPR-Cas/genética , Cardiomiopatía Hipertrófica/patología , Modelos Animales de Enfermedad , Femenino , Técnicas de Sustitución del Gen , Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/complicaciones , Humanos , Lactante , Masculino , Ratones , Ratones Transgénicos , Debilidad Muscular/patología , Debilidad Muscular/fisiopatología , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Miocardio/patología , ARN Guía de Kinetoplastida/genética , alfa-Glucosidasas/metabolismoRESUMEN
Nuclear factor κB (NF-κB) RelA is the potent transcriptional activator of inflammatory response genes. We stringently defined a list of direct RelA target genes by integrating physical (chromatin immunoprecipitation sequencing [ChIP-seq]) and functional (RNA sequencing [RNA-seq] in knockouts) datasets. We then dissected each gene's regulatory strategy by testing RelA variants in a primary-cell genetic-complementation assay. All endogenous target genes require RelA to make DNA-base-specific contacts, and none are activatable by the DNA binding domain alone. However, endogenous target genes differ widely in how they employ the two transactivation domains. Through model-aided analysis of the dynamic time-course data, we reveal the gene-specific synergy and redundancy of TA1 and TA2. Given that post-translational modifications control TA1 activity and intrinsic affinity for coactivators determines TA2 activity, the differential TA logics suggests context-dependent versus context-independent control of endogenous RelA-target genes. Although some inflammatory initiators appear to require co-stimulatory TA1 activation, inflammatory resolvers are a part of the NF-κB RelA core response.
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Inflamación/genética , Factor de Transcripción ReIA/metabolismo , Activación Transcripcional/genética , Animales , Secuencia de Bases , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Lógica , Ratones Endogámicos C57BL , Modelos Biológicos , Dominios Proteicos , Factor de Necrosis Tumoral alfaRESUMEN
INTRODUCTION: The emergence and mass utilization of high-throughput (HT) technologies, including sequencing technologies (genomics) and mass spectrometry (proteomics, metabolomics, lipids), has allowed geneticists, biologists, and biostatisticians to bridge the gap between genotype and phenotype on a massive scale. These new technologies have brought rapid advances in our understanding of cell biology, evolutionary history, microbial environments, and are increasingly providing new insights and applications towards clinical care and personalized medicine. Areas covered: The very success of this industry also translates into daunting big data challenges for researchers and institutions that extend beyond the traditional academic focus of algorithms and tools. The main obstacles revolve around analysis provenance, data management of massive datasets, ease of use of software, interpretability and reproducibility of results. Expert commentary: The authors review the challenges associated with implementing bioinformatics best practices in a large-scale setting, and highlight the opportunity for establishing bioinformatics pipelines that incorporate data tracking and auditing, enabling greater consistency and reproducibility for basic research, translational or clinical settings.
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Biología Computacional , Investigación Genética , Genómica , Biología Computacional/instrumentación , Biología Computacional/métodos , Biología Computacional/tendencias , Genómica/instrumentación , Genómica/métodos , Genómica/tendenciasRESUMEN
The regenerative capacity of the injured CNS in adult mammals is severely limited, yet axons in the peripheral nervous system (PNS) regrow, albeit to a limited extent, after injury. We reasoned that coordinate regulation of gene expression in injured neurons involving multiple pathways was central to PNS regenerative capacity. To provide a framework for revealing pathways involved in PNS axon regrowth after injury, we applied a comprehensive systems biology approach, starting with gene expression profiling of dorsal root ganglia (DRGs) combined with multi-level bioinformatic analyses and experimental validation of network predictions. We used this rubric to identify a drug that accelerates DRG neurite outgrowth in vitro and optic nerve outgrowth in vivo by inducing elements of the identified network. The work provides a functional genomics foundation for understanding neural repair and proof of the power of such approaches in tackling complex problems in nervous system biology.
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Axones/fisiología , Ganglios Espinales/citología , Regeneración Nerviosa/fisiología , Neuronas/citología , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Animales , Animales Recién Nacidos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Canales Iónicos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos , Regeneración Nerviosa/genética , Transferasas de Grupos Nitrogenados/genética , Transferasas de Grupos Nitrogenados/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
IMPORTANCE: Previous studies have indicated a heritable component of the etiology of neurodegenerative diseases such as Alzheimer disease (AD), frontotemporal dementia (FTD), and progressive supranuclear palsy (PSP). However, few have examined the contribution of low-frequency coding variants on a genome-wide level. OBJECTIVE: To identify low-frequency coding variants that affect susceptibility to AD, FTD, and PSP. DESIGN, SETTING, AND PARTICIPANTS: We used the Illumina HumanExome BeadChip array to genotype a large number of variants (most of which are low-frequency coding variants) in a cohort of patients with neurodegenerative disease (224 with AD, 168 with FTD, and 48 with PSP) and in 224 control individuals without dementia enrolled between 2005-2012 from multiple centers participating in the Genetic Investigation in Frontotemporal Dementia and Alzheimer's Disease (GIFT) Study. An additional multiancestral replication cohort of 240 patients with AD and 240 controls without dementia was used to validate suggestive findings. Variant-level association testing and gene-based testing were performed. MAIN OUTCOMES AND MEASURES: Statistical association of genetic variants with clinical diagnosis of AD, FTD, and PSP. RESULTS: Genetic variants typed by the exome array explained 44%, 53%, and 57% of the total phenotypic variance of AD, FTD, and PSP, respectively. An association with the known AD gene ABCA7 was replicated in several ancestries (discovery P=.0049, European P=.041, African American P=.043, and Asian P=.027), suggesting that exonic variants within this gene modify AD susceptibility. In addition, 2 suggestive candidate genes, DYSF (P=5.53×10(-5)) and PAXIP1 (P=2.26×10(-4)), were highlighted in patients with AD and differentially expressed in AD brain. Corroborating evidence from other exome array studies and gene expression data points toward potential involvement of these genes in the pathogenesis of AD. CONCLUSIONS AND RELEVANCE: Low-frequency coding variants with intermediate effect size may account for a significant fraction of the genetic susceptibility to AD and FTD. Furthermore, we found evidence that coding variants in the known susceptibility gene ABCA7, as well as candidate genes DYSF and PAXIP1, confer risk for AD.
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Enfermedad de Alzheimer/genética , Exoma , Demencia Frontotemporal/genética , Predisposición Genética a la Enfermedad , Parálisis Supranuclear Progresiva/genética , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/diagnóstico , Femenino , Demencia Frontotemporal/diagnóstico , Pruebas Genéticas/métodos , Variación Genética/genética , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , RiesgoRESUMEN
BAFF, an activator of the noncanonical NFκB pathway, provides critical survival signals during B cell maturation and contributes to B cell proliferation. We found that the NFκB family member RelB is required ex vivo for B cell maturation, but cRel is required for proliferation. Combined molecular network modeling and experimentation revealed Nfkb2 p100 as a pathway switch; at moderate p100 synthesis rates in maturing B cells, BAFF fully utilizes p100 to generate the RelB:p52 dimer, whereas at high synthesis rates, p100 assembles into multimeric IκBsome complexes, which BAFF neutralizes in order to potentiate cRel activity and B cell expansion. Indeed, moderation of p100 expression or disruption of IκBsome assembly circumvented the BAFF requirement for full B cell expansion. Our studies emphasize the importance of p100 in determining distinct NFκB network states during B cell biology, which causes BAFF to have context-dependent functional consequences.
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Factor Activador de Células B/metabolismo , Linfocitos B/metabolismo , Proliferación Celular , FN-kappa B/metabolismo , Transducción de Señal , Animales , Factor Activador de Células B/genética , Linfocitos B/citología , Linfocitos B/fisiología , Diferenciación Celular , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Ratones , Modelos Biológicos , FN-kappa B/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
Understanding human-specific patterns of brain gene expression and regulation can provide key insights into human brain evolution and speciation. Here, we use next-generation sequencing, and Illumina and Affymetrix microarray platforms, to compare the transcriptome of human, chimpanzee, and macaque telencephalon. Our analysis reveals a predominance of genes differentially expressed within human frontal lobe and a striking increase in transcriptional complexity specific to the human lineage in the frontal lobe. In contrast, caudate nucleus gene expression is highly conserved. We also identify gene coexpression signatures related to either neuronal processes or neuropsychiatric diseases, including a human-specific module with CLOCK as its hub gene and another module enriched for neuronal morphological processes and genes coexpressed with FOXP2, a gene important for language evolution. These data demonstrate that transcriptional networks have undergone evolutionary remodeling even within a given brain region, providing a window through which to view the foundation of uniquely human cognitive capacities.
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Encéfalo/anatomía & histología , Encéfalo/metabolismo , Expresión Génica/fisiología , Factores de Transcripción/metabolismo , Animales , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Evolución Molecular , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/genética , Humanos , Macaca , Trastornos Mentales/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Pan troglodytes , Factores de Transcripción/genéticaRESUMEN
Genome-wide transcriptional profiling was used to characterize the molecular underpinnings of neocortical organization in rhesus macaque, including cortical areal specialization and laminar cell-type diversity. Microarray analysis of individual cortical layers across sensorimotor and association cortices identified robust and specific molecular signatures for individual cortical layers and areas, prominently involving genes associated with specialized neuronal function. Overall, transcriptome-based relationships were related to spatial proximity, being strongest between neighboring cortical areas and between proximal layers. Primary visual cortex (V1) displayed the most distinctive gene expression compared to other cortical regions in rhesus and human, both in the specialized layer 4 as well as other layers. Laminar patterns were more similar between macaque and human compared to mouse, as was the unique V1 profile that was not observed in mouse. These data provide a unique resource detailing neocortical transcription patterns in a nonhuman primate with great similarity in gene expression to human.
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Macaca mulatta/anatomía & histología , Neocórtex/citología , Neocórtex/metabolismo , Transcriptoma/fisiología , Análisis de Varianza , Animales , Femenino , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Análisis por Micromatrices , Microdisección , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas/fisiología , Neuronas , Análisis de Componente Principal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
An enigmatic feature of age-related neurodegenerative diseases is that they seldom, if ever, are fully manifested in nonhuman species under natural conditions. The neurodegenerative tauopathies are typified by the intracellular aggregation of hyperphosphorylated microtubule-associated protein tau (MAPT) and the dysfunction and death of affected neurons. We document the first case of tauopathy with paired helical filaments in an aged chimpanzee (Pan troglodytes). Pathologic forms of tau in neuronal somata, neuropil threads, and plaque-like clusters of neurites were histologically identified throughout the neocortex and, to a lesser degree, in allocortical and subcortical structures. Ultrastructurally, the neurofibrillary tangles consisted of tau-immunoreactive paired helical filaments with a diameter and helical periodicity indistinguishable from those seen in Alzheimer's disease. A moderate degree of Abeta deposition was present in the cerebral vasculature and, less frequently, in senile plaques. Sequencing of the exons and flanking intronic regions in the genomic MAPT locus disclosed no mutations that are associated with the known human hereditary tauopathies, nor any polymorphisms of obvious functional significance. Although the lesion profile in this chimpanzee differed somewhat from that in Alzheimer's disease, the copresence of paired helical filaments and Abeta-amyloidosis indicates that the molecular mechanisms for the pathogenesis of the two canonical Alzheimer lesions--neurofibrillary tangles and senile plaques--are present in aged chimpanzees.