RESUMEN
Upon fertilization, embryos undergo chromatin reprogramming and genome activation; however, the mechanisms that regulate these processes are poorly understood. Here, we generated a triple mutant for Nanog, Pou5f3, and Sox19b (NPS) in zebrafish and found that NPS pioneer chromatin opening at >50% of active enhancers. NPS regulate acetylation across core histones at enhancers and promoters, and their function in gene activation can be bypassed by recruiting histone acetyltransferase to individual genes. NPS pioneer chromatin opening individually, redundantly, or additively depending on sequence context, and we show that high nucleosome occupancy facilitates NPS pioneering activity. Nucleosome position varies based on the input of different transcription factors (TFs), providing a flexible platform to modulate pioneering activity. Altogether, our results illuminate the sequence of events during genome activation and offer a conceptual framework to understand how pioneer factors interpret the genome and integrate different TF inputs across cell types and developmental transitions.
Asunto(s)
Cromatina , Nucleosomas , Animales , Cromatina/genética , Genoma/genética , Histonas/genética , Histonas/metabolismo , Nucleosomas/genética , Factores de Transcripción SOX/genética , Factores de Transcripción SOX/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Maternally-loaded factors in the egg accumulate during oogenesis and are essential for the acquisition of oocyte and egg developmental competence to ensure the production of viable embryos. However, their molecular nature and functional importance remain poorly understood. Here, we present a collection of 9 recessive maternal-effect mutants identified in a zebrafish forward genetic screen that reveal unique molecular insights into the mechanisms controlling the vertebrate oocyte-to-embryo transition. Four genes, over easy, p33bjta, poached and black caviar, were found to control initial steps in yolk globule sizing and protein cleavage during oocyte maturation that act independently of nuclear maturation. The krang, kazukuram, p28tabj, and spotty genes play distinct roles in egg activation, including cortical granule biology, cytoplasmic segregation, the regulation of microtubule organizing center assembly and microtubule nucleation, and establishing the basic body plan. Furthermore, we cloned two of the mutant genes, identifying the over easy gene as a subunit of the Adaptor Protein complex 5, Ap5m1, which implicates it in regulating intracellular trafficking and yolk vesicle formation. The novel maternal protein Krang/Kiaa0513, highly conserved in metazoans, was discovered and linked to the function of cortical granules during egg activation. These mutant genes represent novel genetic entry points to decipher the molecular mechanisms functioning in the oocyte-to-embryo transition, fertility, and human disease. Additionally, our genetic adult screen not only contributes to the existing knowledge in the field but also sets the basis for future investigations. Thus, the identified maternal genes represent key players in the coordination and execution of events prior to fertilization.
Asunto(s)
Oocitos , Oogénesis , Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/genética , Oocitos/metabolismo , Oocitos/crecimiento & desarrollo , Oogénesis/genética , Femenino , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Regulación del Desarrollo de la Expresión Génica , Herencia Materna/genética , Mutación , Embrión no Mamífero , Desarrollo Embrionario/genéticaRESUMEN
The Balbiani body (Bb) is the first marker of polarity in vertebrate oocytes. The Bb is a conserved structure found in diverse animals including insects, fish, amphibians, and mammals. During early zebrafish oogenesis, the Bb assembles as a transient aggregate of mRNA, proteins, and membrane-bound organelles at the presumptive vegetal side of the oocyte. As the early oocyte develops, the Bb appears to grow slowly, until at the end of stage I of oogenesis it disassembles and deposits its cargo of localized mRNAs and proteins. In fish and frogs, this cargo includes the germ plasm as well as gene products required to specify dorsal tissues of the future embryo. We demonstrate that the Bb is a stable, solid structure that forms a size exclusion barrier similar to other biological hydrogels. Despite its central role in oocyte polarity, little is known about the mechanism behind the Bb's action. Analysis of the few known protein components of the Bb is insufficient to explain how the Bb assembles, translocates, and disassembles. We isolated Bbs from zebrafish oocytes and performed mass spectrometry to define the Bb proteome. We successfully identified 77 proteins associated with the Bb sample, including known Bb proteins and novel RNA-binding proteins. In particular, we identified Cirbpa and Cirbpb, which have both an RNA-binding domain and a predicted self-aggregation domain. In stage I oocytes, Cirbpa and Cirbpb localize to the Bb rather than the nucleus (as in somatic cells), indicating that they may have a specialized function in the germ line. Both the RNA-binding domain and the self-aggregation domain are sufficient to localize to the Bb, suggesting that Cirbpa and Cirbpb interact with more than just their mRNA targets within the Bb. We propose that Cirbp proteins crosslink mRNA cargo and proteinaceous components of the Bb as it grows. Beyond Cirbpa and Cirbpb, our proteomics dataset presents many candidates for further study, making it a valuable resource for building a comprehensive mechanism for Bb function at a protein level.
Asunto(s)
Proteínas de Pez Cebra , Pez Cebra , Animales , Polaridad Celular/genética , Mamíferos/metabolismo , Oocitos/metabolismo , Oogénesis/genética , Orgánulos/metabolismo , Proteómica , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Posttranscriptional regulation plays a crucial role in shaping gene expression. During the maternal-to-zygotic transition (MZT), thousands of maternal transcripts are regulated. However, how different cis-elements and trans-factors are integrated to determine mRNA stability remains poorly understood. Here, we show that most transcripts are under combinatorial regulation by multiple decay pathways during zebrafish MZT. By using a massively parallel reporter assay, we identified cis-regulatory sequences in the 3' UTR, including U-rich motifs that are associated with increased mRNA stability. In contrast, miR-430 target sequences, UAUUUAUU AU-rich elements (ARE), CCUC, and CUGC elements emerged as destabilizing motifs, with miR-430 and AREs causing mRNA deadenylation upon genome activation. We identified trans-factors by profiling RNA-protein interactions and found that poly(U)-binding proteins are preferentially associated with 3' UTR sequences and stabilizing motifs. We show that this activity is antagonized by C-rich motifs and correlated with protein binding. Finally, we integrated these regulatory motifs into a machine learning model that predicts reporter mRNA stability in vivo.
Asunto(s)
Regiones no Traducidas 3' , Regulación del Desarrollo de la Expresión Génica , Estabilidad del ARN/genética , Proteínas de Unión al ARN/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Aprendizaje Automático , Modelos Genéticos , Secuencias Reguladoras de Ácido Ribonucleico , Pez Cebra/embriología , Pez Cebra/genética , CigotoRESUMEN
SUMMARY: Experimental laboratory management and data-driven science require centralized software for sharing information, such as lab collections or genomic sequencing datasets. Although database servers such as PostgreSQL can store such information with multiple-user access, they lack user-friendly graphical and programmatic interfaces for easy data access and inputting. We developed LabxDB, a versatile open-source solution for organizing and sharing structured data. We provide several out-of-the-box databases for deployment in the cloud including simple mutant or plasmid collections and purchase-tracking databases. We also developed a high-throughput sequencing (HTS) database, LabxDB seq, dedicated to storage of hierarchical sample annotations. Scientists can import their own or publicly available HTS data into LabxDB seq to manage them from production to publication. Using LabxDB's programmatic access (REST API), annotations can be easily integrated into bioinformatics pipelines. LabxDB is modular, offering a flexible framework that scientists can leverage to build new database interfaces adapted to their needs. AVAILABILITY AND IMPLEMENTATION: LabxDB is available at https://gitlab.com/vejnar/labxdb and https://labxdb.vejnar.org for documentation. LabxDB is licensed under the terms of the Mozilla Public License 2.0. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Laboratorios , Programas Informáticos , Bases de Datos Factuales , Genómica , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Cellular transitions require dramatic changes in gene expression that are supported by regulated mRNA decay and new transcription. The maternal-to-zygotic transition is a conserved developmental progression during which thousands of maternal mRNAs are cleared by post-transcriptional mechanisms. Although some maternal mRNAs are targeted for degradation by microRNAs, this pathway does not fully explain mRNA clearance. We investigated how codon identity and translation affect mRNA stability during development and homeostasis. We show that the codon triplet contains translation-dependent regulatory information that influences transcript decay. Codon composition shapes maternal mRNA clearance during the maternal-to-zygotic transition in zebrafish, Xenopus, mouse, and Drosophila, and gene expression during homeostasis across human tissues. Some synonymous codons show consistent stabilizing or destabilizing effects, suggesting that amino acid composition influences mRNA stability. Codon composition affects both polyadenylation status and translation efficiency. Thus, the ribosome interprets two codes within the mRNA: the genetic code which specifies the amino acid sequence and a conserved "codon optimality code" that shapes mRNA stability and translation efficiency across vertebrates.
Asunto(s)
Codón , Regulación de la Expresión Génica , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Mensajero/genética , Cigoto/crecimiento & desarrollo , Animales , Drosophila , Humanos , Ratones , Ribosomas/metabolismo , Xenopus , Pez CebraRESUMEN
Gene expression is extensively regulated at the levels of mRNA stability, localization and translation. However, decoding functional RNA-regulatory features remains a limitation to understanding post-transcriptional regulation in vivo. Here, we developed RNA-element selection assay (RESA), a method that selects RNA elements on the basis of their activity in vivo and uses high-throughput sequencing to provide a quantitative measurement of their regulatory functions at near-nucleotide resolution. We implemented RESA to identify sequence elements modulating mRNA stability during zebrafish embryogenesis. RESA provides a sensitive and quantitative measure of microRNA activity in vivo and also identifies novel regulatory sequences. To uncover specific sequence requirements within regulatory elements, we developed a bisulfite-mediated nucleotide-conversion strategy for large-scale mutational analysis (RESA-bisulfite). Finally, we used the versatile RESA platform to map candidate protein-RNA interactions in vivo (RESA-CLIP).
Asunto(s)
Técnicas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Mensajero , Secuencias Reguladoras de Ácidos Nucleicos , Regiones no Traducidas 3' , Animales , Embrión no Mamífero , Inmunoprecipitación , Estabilidad del ARN , ARN Mensajero/genética , Sulfitos , Pez Cebra/embriologíaRESUMEN
The impact of the CRISPR-Cas biotechnological systems has recently broadened the genome editing toolbox available to different model organisms further with the addition of new efficient RNA-guided endonucleases. We have recently optimized CRISPR-Cpf1 (renamed Cas12a) system in zebrafish. We showed that (i) in the absence of Cpf1 protein, crRNAs are unstable and degraded in vivo, and CRISPR-Cpf1 RNP complexes efficiently mutagenize the zebrafish genome; and (ii) temperature modulates Cpf1 activity especially affecting AsCpf1, which experiences a reduced performance below 37⯰C. Here, we describe a step-by-step protocol on how to easily design and generate crRNAs in vitro, purify recombinant Cpf1 proteins, and assemble ribonucleoprotein complexes to carry out efficient mutagenesis in zebrafish in a constitutive and temperature-controlled manner. Finally, we explain how to induce Cpf1-mediated homology-directed repair using single-stranded DNA oligonucleotides. In summary, this protocol includes the steps to efficiently modify the zebrafish genome and other ectothermic organisms using the CRISPR-Cpf1 system.
Asunto(s)
Proteínas Bacterianas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Endonucleasas/genética , Edición Génica/métodos , Pez Cebra/genética , Animales , Clostridiales/genética , Reparación del ADN por Unión de Extremidades/genética , Genoma/genética , ARN Guía de Kinetoplastida/genética , Reparación del ADN por Recombinación/genéticaRESUMEN
Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo.
Asunto(s)
Secuencia Conservada , Evolución Molecular , Sistemas de Lectura Abierta/genética , ARN Mensajero/genética , Ribosomas/metabolismo , Pez Cebra/genética , Animales , Secuencia de Bases , Biología Computacional , Perfilación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Ensayos de Protección de Nucleasas , Oligopéptidos/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN/métodos , Pez Cebra/embriologíaRESUMEN
CRISPR-Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observed that guanine enrichment and adenine depletion increased sgRNA stability and activity, whereas differential sgRNA loading, nucleosome positioning and Cas9 off-target binding were not major determinants. We also identified sgRNAs truncated by one or two nucleotides and containing 5' mismatches as efficient alternatives to canonical sgRNAs. On the basis of these results, we created a predictive sgRNA-scoring algorithm, CRISPRscan, that effectively captures the sequence features affecting the activity of CRISPR-Cas9 in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos 3' UTR led to the generation of maternal-zygotic mutants, as well as increased viability and decreased somatic mutations. These results identify determinants that influence Cas9 activity and provide a framework for the design of highly efficient sgRNAs for genome targeting in vivo.
Asunto(s)
Algoritmos , Sistemas CRISPR-Cas , Marcación de Gen/métodos , ARN Guía de Kinetoplastida/genética , Regiones no Traducidas 3' , Adenina , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Embrión no Mamífero , Femenino , Regulación de la Expresión Génica , Ingeniería Genética/métodos , Genoma , Guanina , Mutación , Xenopus/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
In liver, most metabolic pathways are under circadian control, and hundreds of protein-encoding genes are thus transcribed in a cyclic fashion. Here we show that rhythmic transcription extends to the locus specifying miR-122, a highly abundant, hepatocyte-specific microRNA. Genetic loss-of-function and gain-of-function experiments have identified the orphan nuclear receptor REV-ERBalpha as the major circadian regulator of mir-122 transcription. Although due to its long half-life mature miR-122 accumulates at nearly constant rates throughout the day, this miRNA is tightly associated with control mechanisms governing circadian gene expression. Thus, the knockdown of miR-122 expression via an antisense oligonucleotide (ASO) strategy resulted in the up- and down-regulation of hundreds of mRNAs, of which a disproportionately high fraction accumulates in a circadian fashion. miR-122 has previously been linked to the regulation of cholesterol and lipid metabolism. The transcripts associated with these pathways indeed show the strongest time point-specific changes upon miR-122 depletion. The identification of Pparbeta/delta and the peroxisome proliferator-activated receptor alpha (PPARalpha) coactivator Smarcd1/Baf60a as novel miR-122 targets suggests an involvement of the circadian metabolic regulators of the PPAR family in miR-122-mediated metabolic control.
Asunto(s)
Ritmo Circadiano/fisiología , Regulación de la Expresión Génica , Hígado/metabolismo , MicroARNs/metabolismo , Animales , Ritmo Circadiano/genética , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Genoma/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Activados del Proliferador del Peroxisoma/metabolismo , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de TiempoRESUMEN
Arginine, a semiessential amino acid implicated in diverse cellular processes, is a substrate for two arginases-Arg1 and Arg2-having different expression patterns and functions. Although appropriately regulated Arg1 expression is critical for immune responses, this has not been documented for Arg2. We show that Arg2 is the dominant enzyme in dendritic cells (DCs) and is repressed by microRNA-155 (miR155) during their maturation. miR155 is known to be strongly induced in various mouse and human DC subsets in response to diverse maturation signals, and miR155-deficient DCs exhibit an impaired ability to induce Ag-specific T cell responses. By means of expression profiling studies, we identified Arg2 mRNA as a novel miR155 target in mouse DCs. Abnormally elevated levels of Arg2 expression and activity were observed in activated miR155-deficient DCs. Conversely, overexpression of miR155 inhibited Arg2 expression. Bioinformatic and functional analyses confirmed that Arg2 mRNA is a direct target of miR155. Finally, in vitro and in vivo functional assays using DCs exhibiting deregulated Arg2 expression indicated that Arg2-mediated arginine depletion in the extracellular milieu impairs T cell proliferation. These results indicate that miR155-induced repression of Arg2 expression is critical for the ability of DCs to drive T cell activation by controlling arginine availability in the extracellular environment.
Asunto(s)
Arginasa/biosíntesis , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/enzimología , Activación de Linfocitos/inmunología , MicroARNs/genética , Animales , Arginasa/antagonistas & inhibidores , Arginasa/genética , Arginina/metabolismo , Proliferación Celular , Células Dendríticas/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Células HEK293 , Humanos , Activación de Linfocitos/genética , Ratones , Ratones Noqueados , Procesamiento Postranscripcional del ARN , ARN Mensajero/genéticaRESUMEN
MicroRNAs (miRNAs) posttranscriptionally repress the expression of protein-coding genes. Based on the partial complementarity between miRNA and messenger RNA pairs with a mandatory so-called 'seed' sequence, many thousands of potential targets can be identified. Our open-source software library, miRmap, ranks these potential targets with a biologically meaningful criterion, the repression strength. MiRmap combines thermodynamic, evolutionary, probabilistic and sequence-based features, which cover features from TargetScan, PITA, PACMIT and miRanda. Our miRmap web application offers a user-friendly and feature-rich resource for browsing precomputed miRNA target predictions for model organisms, as well as for predicting and ranking targets for user-submitted sequences. MiRmap web integrates sorting, filtering and exporting of results from multiple queries, as well as providing programmatic access, and is available at http://mirmap.ezlab.org.
Asunto(s)
MicroARNs/metabolismo , ARN Mensajero/metabolismo , Programas Informáticos , Regiones no Traducidas 3' , Regulación de la Expresión Génica , Internet , MicroARNs/química , ARN Mensajero/química , Análisis de Secuencia de ARN , TermodinámicaRESUMEN
MicroRNAs (miRNAs) are regulatory noncoding RNAs that affect the production of a significant fraction of human mRNAs via post-transcriptional regulation. Interindividual variation of the miRNA expression levels is likely to influence the expression of miRNA target genes and may therefore contribute to phenotypic differences in humans, including susceptibility to common disorders. The extent to which miRNA levels are genetically controlled is largely unknown. In this report, we assayed the expression levels of miRNAs in primary fibroblasts from 180 European newborns of the GenCord project and performed association analysis to identify eQTLs (expression quantitative traits loci). We detected robust expression for 121 miRNAs out of 365 interrogated. We have identified significant cis- (10%) and trans- (11%) eQTLs. Furthermore, we detected one genomic locus (rs1522653) that influences the expression levels of five miRNAs, thus unraveling a novel mechanism for coregulation of miRNA expression.
Asunto(s)
Elementos de Facilitación Genéticos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , MicroARNs/metabolismo , Sitios de Carácter Cuantitativo/genética , Procesamiento Postranscripcional del ARN , Línea Celular , Europa (Continente) , Perfilación de la Expresión Génica , Variación Genética , Estudio de Asociación del Genoma Completo , Humanos , Recién Nacido , MicroARNs/genéticaRESUMEN
MicroRNAs, or miRNAs, post-transcriptionally repress the expression of protein-coding genes. The human genome encodes over 1000 miRNA genes that collectively target the majority of messenger RNAs (mRNAs). Base pairing of the so-called miRNA 'seed' region with mRNAs identifies many thousands of putative targets. Evaluating the strength of the resulting mRNA repression remains challenging, but is essential for a biologically informative ranking of potential miRNA targets. To address these challenges, predictors may use thermodynamic, evolutionary, probabilistic or sequence-based features. We developed an open-source software library, miRmap, which for the first time comprehensively covers all four approaches using 11 predictor features, 3 of which are novel. This allowed us to examine feature correlations and to compare their predictive power in an unbiased way using high-throughput experimental data from immunopurification, transcriptomics, proteomics and polysome fractionation experiments. Overall, target site accessibility appears to be the most predictive feature. Our novel feature based on PhyloP, which evaluates the significance of negative selection, is the best performing predictor in the evolutionary category. We combined all the features into an integrated model that almost doubles the predictive power of TargetScan. miRmap is freely available from http://cegg.unige.ch/mirmap.
Asunto(s)
MicroARNs/metabolismo , ARN Mensajero/metabolismo , Programas Informáticos , Emparejamiento Base , Evolución Molecular , Regulación de la Expresión Génica , Células HeLa , Humanos , MicroARNs/química , Probabilidad , ARN Mensajero/química , TermodinámicaRESUMEN
Post-transcriptional mRNA regulation shapes gene expression, yet how cis-elements and mRNA translation interface to regulate mRNA stability is poorly understood. We find that the strength of translation initiation, upstream open reading frame (uORF) content, codon optimality, AU-rich elements, microRNA binding sites, and open reading frame (ORF) length function combinatorially to regulate mRNA stability. Machine-learning analysis identifies ORF length as the most important conserved feature regulating mRNA decay. We find that Upf1 binds poorly translated and untranslated ORFs, which are associated with a higher decay rate, including mRNAs with uORFs and those with exposed ORFs after stop codons. Our study emphasizes Upf1's converging role in surveilling mRNAs with exposed ORFs that are poorly translated, such as mRNAs with long ORFs, ORF-like 3' UTRs, and mRNAs containing uORFs. We propose that Upf1 regulation of poorly/untranslated ORFs provides a unifying mechanism of surveillance in regulating mRNA stability and homeostasis in an exon-junction complex (EJC)-independent nonsense-mediated decay (NMD) pathway that we term ORF-mediated decay (OMD).
Asunto(s)
ARN Helicasas , Estabilidad del ARN , Transactivadores , Humanos , Regiones no Traducidas 3'/genética , Degradación de ARNm Mediada por Codón sin Sentido , Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas , ARN Helicasas/metabolismo , ARN Helicasas/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Transactivadores/metabolismo , Transactivadores/genética , Células HEK293RESUMEN
MicroRNAs (miRNAs) are small, noncoding RNAs that regulate target mRNAs by binding to their 3' untranslated regions. There is growing evidence that microRNA-155 (miR155) modulates gene expression in various cell types of the immune system and is a prominent player in the regulation of innate and adaptive immune responses. To define the role of miR155 in dendritic cells (DCs) we performed a detailed analysis of its expression and function in human and mouse DCs. A strong increase in miR155 expression was found to be a general and evolutionarily conserved feature associated with the activation of DCs by diverse maturation stimuli in all DC subtypes tested. Analysis of miR155-deficient DCs demonstrated that miR155 induction is required for efficient DC maturation and is critical for the ability of DCs to promote antigen-specific T-cell activation. Expression-profiling studies performed with miR155(-/-) DCs and DCs overexpressing miR155, combined with functional assays, revealed that the mRNA encoding the transcription factor c-Fos is a direct target of miR155. Finally, all of the phenotypic and functional defects exhibited by miR155(-/-) DCs could be reproduced by deregulated c-Fos expression. These results indicate that silencing of c-Fos expression by miR155 is a conserved process that is required for DC maturation and function.
Asunto(s)
Células Dendríticas/fisiología , Silenciador del Gen/inmunología , MicroARNs/inmunología , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Células Dendríticas/citología , Evolución Molecular , Humanos , Ratones , Ratones Mutantes , MicroARNs/genética , Monocitos/citología , ARN Mensajero/genética , ARN Mensajero/inmunologíaRESUMEN
Sertoli cells (SCs) are the central, essential coordinators of spermatogenesis, without which germ cell development cannot occur. We previously showed that Dicer, an RNaseIII endonuclease required for microRNA (miRNA) biogenesis, is absolutely essential for Sertoli cells to mature, survive, and ultimately sustain germ cell development. Here, using isotope-coded protein labeling, a technique for protein relative quantification by mass spectrometry, we investigated the impact of Sertoli cell-Dicer and subsequent miRNA loss on the testicular proteome. We found that, a large proportion of proteins (50 out of 130) are up-regulated by more that 1.3-fold in testes lacking Sertoli cell-Dicer, yet that this protein up-regulation is mild, never exceeding a 2-fold change, and is not preceeded by alterations of the corresponding mRNAs. Of note, the expression levels of six proteins of interest were further validated using the Absolute Quantification (AQUA) peptide technology. Furthermore, through 3'UTR luciferase assays we identified one up-regulated protein, SOD-1, a Cu/Zn superoxide dismutase whose overexpression has been linked to enhanced cell death through apoptosis, as a likely direct target of three Sertoli cell-expressed miRNAs, miR-125a-3p, miR-872 and miR-24. Altogether, our study, which is one of the few in vivo analyses of miRNA effects on protein output, suggests that, at least in our system, miRNAs play a significant role in translation control.
Asunto(s)
Proteoma/metabolismo , Ribonucleasa III/deficiencia , Células de Sertoli/metabolismo , Testículo/metabolismo , Regiones no Traducidas 3' , Animales , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Regiones Promotoras Genéticas , Interferencia de ARN , Ribonucleasa III/genética , Eliminación de Secuencia , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Espectrometría de Masas en Tándem , Testículo/patología , Transcripción Genética , Regulación hacia ArribaRESUMEN
Nanoscale chromatin organization regulates gene expression. Although chromatin is notably reprogrammed during zygotic genome activation (ZGA), the organization of chromatin regulatory factors during this universal process remains unclear. In this work, we developed chromatin expansion microscopy (ChromExM) to visualize chromatin, transcription, and transcription factors in vivo. ChromExM of embryos during ZGA revealed how the pioneer factor Nanog interacts with nucleosomes and RNA polymerase II (Pol II), providing direct visualization of transcriptional elongation as string-like nanostructures. Blocking elongation led to more Pol II particles clustered around Nanog, with Pol II stalled at promoters and Nanog-bound enhancers. This led to a new model termed "kiss and kick", in which enhancer-promoter contacts are transient and released by transcriptional elongation. Our results demonstrate that ChromExM is broadly applicable to study nanoscale nuclear organization.
Asunto(s)
Cromatina , Microscopía Fluorescente , Transcripción Genética , Cigoto , Cromatina/química , Nucleosomas/química , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Microscopía Fluorescente/métodos , Animales , Pez Cebra , Embrión no Mamífero , Cigoto/metabolismo , Proteína Homeótica Nanog/química , Proteína Homeótica Nanog/metabolismoRESUMEN
Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains have recently been found to contain cryptic open-reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here, we show that two putative lincRNAs (linc-mipep, also called lnc-rps25, and linc-wrb) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes - evolutionarily newer cell types that develop postnatally in humans. These findings reveal a key missing link in the evolution of vertebrate brain cell development and illustrate a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease.