RESUMEN
cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated. This is independent of Linx transcription and independent of any effect on Tsix, the antisense regulator of Xist that shares the same TAD as Linx. Unlike Tsix, LinxP is well conserved across mammals, suggesting an ancestral mechanism for random monoallelic Xist regulation. When introduced in the same TAD as Xist, LinxP switches from a silencer to an enhancer. Our study uncovers an unsuspected regulatory axis for X chromosome inactivation and a class of cis-regulatory effects that may exploit TAD partitioning to modulate developmental decisions.
Asunto(s)
Secuencia Conservada/genética , ARN Largo no Codificante/genética , Cromosoma X/genética , Animales , Línea Celular , Elementos de Facilitación Genéticos/genética , Ratones , Regiones Promotoras Genéticas/genética , ARN sin Sentido/genética , Elementos Silenciadores Transcripcionales/genética , Transcripción Genética/genéticaRESUMEN
Xist represents a paradigm for the function of long non-coding RNA in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Several proteins that bind to Xist RNA have recently been identified, including the transcriptional repressor SPEN1-3, the loss of which has been associated with deficient XCI at multiple loci2-6. Here we show in mice that SPEN is a key orchestrator of XCI in vivo and we elucidate its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and in embryonic stem cells. SPEN is dispensable for maintenance of XCI in neural progenitors, although it significantly decreases the expression of genes that escape XCI. We show that SPEN is immediately recruited to the X chromosome upon the upregulation of Xist, and is targeted to enhancers and promoters of active genes. SPEN rapidly disengages from chromatin upon gene silencing, suggesting that active transcription is required to tether SPEN to chromatin. We define the SPOC domain as a major effector of the gene-silencing function of SPEN, and show that tethering SPOC to Xist RNA is sufficient to mediate gene silencing. We identify the protein partners of SPOC, including NCoR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in the regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for the initiation of XCI, bridging Xist RNA with the transcription machinery-as well as with nucleosome remodellers and histone deacetylases-at active enhancers and promoters.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Silenciador del Gen , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Inactivación del Cromosoma X/genética , Cromosoma X/genética , Animales , Blastocisto/citología , Blastocisto/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/química , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos/genética , Femenino , Histona Desacetilasas/metabolismo , Masculino , Metilación , Ratones , Regiones Promotoras Genéticas/genética , Dominios Proteicos , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/químicaRESUMEN
The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation that belongs to the DDX3 subfamily. The purified Ded1 protein is an ATP-dependent RNA-binding protein and an RNA-dependent ATPase, but it was previously found to lack substrate specificity and enzymatic regulation. Here we demonstrate through yeast genetics, yeast extract pull-down experiments, in situ localization, and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 in the presence of SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results provide a new understanding of the role of Ded1 during translation.
Asunto(s)
ARN Helicasas DEAD-box , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Partícula de Reconocimiento de Señal , Partícula de Reconocimiento de Señal/metabolismo , Partícula de Reconocimiento de Señal/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Transporte de ProteínasRESUMEN
During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long non-coding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2-dependent H3K27me3 and SETD8-dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the involvement of H4K20me1 in initiating gene silencing. To follow XCI dynamics in living cells, we developed a genetically encoded, H3K27me3-specific intracellular antibody or H3K27me3-mintbody. By combining live-cell imaging of H3K27me3, H4K20me1, the X chromosome and Xist RNA, with ChIP-seq analysis we uncover concurrent accumulation of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a Xist B and C repeat mutant, which still shows gene silencing on the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin.
Asunto(s)
Histonas , ARN Largo no Codificante , Animales , Femenino , Silenciador del Gen , Histonas/genética , Histonas/metabolismo , Placenta/metabolismo , Embarazo , ARN Largo no Codificante/genética , Cromosoma X/genética , Inactivación del Cromosoma X/genéticaRESUMEN
The DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5' 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrometry analyses, genetic experiments, sucrose gradients, in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs.
Asunto(s)
Núcleo Celular/enzimología , Citoplasma/enzimología , ARN Helicasas DEAD-box/metabolismo , Caperuzas de ARN/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Adenosina Trifosfatasas/metabolismo , ARN Helicasas DEAD-box/genética , Guanosina/análogos & derivados , Guanosina/metabolismo , Biosíntesis de Proteínas , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Subnuclear compartmentalization has been proposed to play an important role in gene regulation by segregating active and inactive parts of the genome in distinct physical and biochemical environments. During X chromosome inactivation (XCI), the noncoding Xist RNA coats the X chromosome, triggers gene silencing and forms a dense body of heterochromatin from which the transcription machinery appears to be excluded. Phase separation has been proposed to be involved in XCI, and might explain the exclusion of the transcription machinery by preventing its diffusion into the Xist-coated territory. Here, using quantitative fluorescence microscopy and single-particle tracking, we show that RNA polymerase II (RNAPII) freely accesses the Xist territory during the initiation of XCI. Instead, the apparent depletion of RNAPII is due to the loss of its chromatin stably bound fraction. These findings indicate that initial exclusion of RNAPII from the inactive X reflects the absence of actively transcribing RNAPII, rather than a consequence of putative physical compartmentalization of the inactive X heterochromatin domain.
Asunto(s)
ARN Polimerasa II , ARN Largo no Codificante , ARN Polimerasa II/metabolismo , Heterocromatina , Cromosoma X/genética , Cromosoma X/metabolismo , Inactivación del Cromosoma X , Cromatina , ARN no Traducido/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
Beta-propeller proteins function in catalysis, protein-protein interaction, cell cycle regulation, and innate immunity. The galactose-binding protein (GBP) from the plasma of the horseshoe crab, Carcinoscorpius rotundicauda, is a beta-propeller protein that functions in antimicrobial defense. Studies have shown that upon binding to Gram-negative bacterial lipopolysaccharide (LPS), GBP interacts with C-reactive protein (CRP) to form a pathogen-recognition complex, which helps to eliminate invading microbes. However, the molecular basis of interactions between GBP and LPS and how it interplays with CRP remain largely unknown. By homology modeling, we showed that GBP contains six beta-propeller/Tectonin domains. Ligand docking indicated that Tectonin domains 6 to 1 likely contain the LPS binding sites. Protein-protein interaction studies demonstrated that Tectonin domain 4 interacts most strongly with CRP. Hydrogen-deuterium exchange mass spectrometry mapped distinct sites of GBP that interact with LPS and with CRP, consistent with in silico predictions. Furthermore, infection condition (lowered Ca(2+) level) increases GBP-CRP affinity by 1000-fold. Resupplementing the system with a physiological level of Ca(2+) did not reverse the protein-protein affinity to the basal state, suggesting that the infection-induced complex had undergone irreversible conformational change. We propose that GBP serves as a bridging molecule, participating in molecular interactions, GBP-LPS and GBP-CRP, to form a stable pathogen-recognition complex. The interaction interfaces in these two partners suggest that Tectonin domains can differentiate self/nonself, crucial to frontline defense against infection. In addition, GBP shares architectural and functional homologies to a human protein, hTectonin, suggesting its evolutionarily conservation for approximately 500 million years, from horseshoe crab to human.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de la Membrana/química , Proteínas de Transporte de Monosacáridos/química , Proteínas de Unión Periplasmáticas/química , Secuencia de Aminoácidos , Animales , Evolución Biológica , Secuencia Conservada , Cangrejos Herradura , Interacciones Huésped-Patógeno , Ligandos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/metabolismo , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Técnicas del Sistema de Dos HíbridosRESUMEN
This paper reports our attempts to characterize transgene integration sites in transgenic mouse lines generated by the microinjection of large (from 30 to 145 kb) pig DNA fragments encompassing a mammary specific gene, the whey acidic protein gene (WAP). Among the various methods used, the thermal asymmetric interlaced (TAIL-) PCR method allowed us (1) to analyze transgene/genomic borders and internal concatamer junctions for eleven transgenic lines, (2) to obtain sequence information for seven borders, (3) to place three transgenes in the mouse genome, and (4) to obtain sequence data for seven transgene junctions in concatamers. Finally, we characterized various rearrangements in the borders and the inner parts of the transgene. The possibility of such complex rearrangements should be carefully considered when transgenic animals are produced with large genomic DNA fragments.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Cromosomas/ultraestructura , Ratones Transgénicos/genética , Recombinación Genética , Animales , Secuencia de Bases , Cromosomas/genética , ADN Recombinante/administración & dosificación , ADN Recombinante/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Microinyecciones , Proteínas de la Leche/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Homología de Secuencia de Ácido Nucleico , Porcinos/genéticaRESUMEN
Previous experiments revealed that DHH1, a RNA helicase involved in the regulation of mRNA stability and translation, complemented the phenotype of a Saccharomyces cerevisiae mutant affected in the expression of genes coding for monocarboxylic-acids transporters, JEN1 and ADY2 (Paiva S, Althoff S, Casal M, Leao C. FEMS Microbiol Lett, 1999, 170:301-306). In wild type cells, JEN1 expression had been shown to be undetectable in the presence of glucose or formic acid, and induced in the presence of lactate. In this work, we show that JEN1 mRNA accumulates in a dhh1 mutant, when formic acid was used as sole carbon source. Dhh1 interacts with the decapping activator Dcp1 and with the deadenylase complex. This led to the hypothesis that JEN1 expression is post-transcriptionally regulated by Dhh1 in formic acid. Analyses of JEN1 mRNAs decay in wild-type and dhh1 mutant strains confirmed this hypothesis. In these conditions, the stabilized JEN1 mRNA was associated to polysomes but no Jen1 protein could be detected, either by measurable lactate carrier activity, Jen1-GFP fluorescence detection or western blots. These results revealed the complexity of the expression regulation of JEN1 in S. cerevisiae and evidenced the importance of DHH1 in this process. Additionally, microarray analyses of dhh1 mutant indicated that Dhh1 plays a large role in metabolic adaptation, suggesting that carbon source changes triggers a complex interplay between transcriptional and post-transcriptional effects.
Asunto(s)
ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Regulación Fúngica de la Expresión Génica , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adaptación Fisiológica , Formiatos/metabolismo , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Mutación , Polirribosomas/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Simportadores/genética , Simportadores/metabolismoRESUMEN
Although the innate immune response is triggered by the formation of a stable assembly of pathogen-recognition receptors (PRRs) onto the pathogens, the driving force that enables this PRR-PRR interaction is unknown. Here, we show that serine proteases, which are activated during infection, participate in associating with the PRRs. Inhibition of serine proteases gravely impairs the PRR assembly. Using yeast two-hybrid and pull-down methods, we found that two serine proteases in the horseshoe crab Carcinoscorpius rotundicauda are able to bind to the following three core members of PRRs: galactose-binding protein, Carcinolectin-5 and C-reactive protein. These two serine proteases are (1) Factor C, which activates the coagulation pathway, and (2) C2/Bf, a protein from the complement pathway. By systematic molecular dissection, we show that these serine proteases interact with the core "pathogen-recognition complex" via their complement control protein modules.
Asunto(s)
Proteína C-Reactiva/metabolismo , Proteínas del Sistema Complemento/metabolismo , Precursores Enzimáticos/inmunología , Galectinas/metabolismo , Cangrejos Herradura/enzimología , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos , Activación de Complemento , Hemolinfa/metabolismo , Hemolinfa/microbiología , Cangrejos Herradura/inmunología , Inmunidad Innata , Técnicas In Vitro , Datos de Secuencia Molecular , Unión Proteica , Mapeo de Interacción de Proteínas , Pseudomonas aeruginosa/metabolismo , Serina Endopeptidasas/inmunología , Técnicas del Sistema de Dos HíbridosRESUMEN
Transcriptional activators work by recruiting transcription factors that are required for the process of transcription to their target genes. We have used the Split-Ubiquitin system to identify eight transcription factors that interacted with both the transcriptional activators Gal4p and Gcn4p in living cells. The over-expression of one of the activator-interacting proteins, Gal11p, partially suppressed GAL4 and GCN4 deletions. We have isolated two point mutants in Gal11p, F848L and F869S that were defective for the dosage compensation. We have identified 35 transcription factors that interacted with Gal11p in living cells, and the only protein-protein interaction affected by the Gal11p mutations was the one between Gal11p and Taf14p. We have further shown that the suppression of a GAL4 deletion by high levels of Gal11p required Taf14p, and that over-expression of Gal11p recruited Taf14p to the GAL1 promoter together with Tbp1p, Swi2p and Srb7p. Gal11p interacted with Mig1p, indicating that Mig1/2p could have recruited Gal11p to the GAL1 promoter in the absence of Gal4p. Our results suggest that transcriptional activators work by raising the local concentration of the limiting factor Gal11p, and that Gal11p works by recruiting Mediator and Taf14p-containing transcription factors like TFIID and SWI/SNF and by competing general repressors like Ssn6p-Tup1p off the target promoters.
Asunto(s)
Dosificación de Gen , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transactivadores/genética , Factor de Transcripción TFIID/genética , Transcripción Genética , Adenosina Trifosfatasas , Northern Blotting , Cromatina/metabolismo , ADN de Hongos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Galactoquinasa/genética , Galactoquinasa/metabolismo , Complejo Mediador , Plásmidos , Regiones Promotoras Genéticas/genética , Unión Proteica , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Transactivadores/metabolismo , Factor de Transcripción TFIID/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Although human C-reactive protein (CRP) becomes upregulated during septicemia, its role remains unclear, since purified CRP showed no binding to many common pathogens. Contrary to previous findings, we show that purified human CRP (hCRP) binds to Salmonella enterica, and that binding is enhanced in the presence of plasma factors. In the horseshoe crab, Carcinoscorpius rotundicauda, CRP is a major hemolymph protein. Incubation of hemolymph with a range of bacteria resulted in CRP binding to all the bacteria tested. Lipopolysaccharide-affinity chromatography of the hemolymph co-purified CRP, galactose-binding protein (GBP) and carcinolectin-5 (CL5). Yeast two-hybrid and pull-down assays suggested that these pattern recognition receptors (PRRs) form pathogen recognition complexes. We show the conservation of PRR crosstalk in humans, whereby hCRP interacts with ficolin (CL5 homologue). This interaction stabilizes CRP binding to bacteria and activates the lectin-mediated complement pathway. We propose that CRP does not act alone but collaborates with other plasma PRRs to form stable pathogen recognition complexes when targeting a wide range of bacteria for destruction.
Asunto(s)
Bacterias/inmunología , Proteína C-Reactiva/metabolismo , Cangrejos Herradura/inmunología , Inmunidad/inmunología , Lectinas/sangre , Secuencia de Aminoácidos , Animales , Bacterias/efectos de los fármacos , Proteínas Sanguíneas/metabolismo , Proteína C-Reactiva/química , Proteínas de Unión al Calcio/química , Activación de Complemento/efectos de los fármacos , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Lectina de Unión a Manosa de la Vía del Complemento/inmunología , Hemolinfa/química , Hemolinfa/efectos de los fármacos , Cangrejos Herradura/efectos de los fármacos , Humanos , Inmunidad/efectos de los fármacos , Lectinas/metabolismo , Lipopolisacáridos/farmacología , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/química , Proteínas de Unión Periplasmáticas/química , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Receptor Cross-Talk/efectos de los fármacos , Receptores de Reconocimiento de Patrones/metabolismo , Salmonella enterica/efectos de los fármacos , Salmonella enterica/inmunología , FicolinasRESUMEN
Rotaviruses are the main cause of infantile viral gastroenteritis worldwide leading to approximately 500,000 deaths each year mostly in the developing world. For unknown reasons, live attenuated viruses used in classical vaccine strategies were shown to be responsible for intussusception (a bowel obstruction). New strategies allowing production of safe recombinant non-replicating rotavirus candidate vaccine are thus clearly needed. In this study we utilized transgenic rabbit milk as a source of rotavirus antigens. Individual transgenic rabbit lines were able to produce several hundreds of micrograms per ml of secreted recombinant VP2 and VP6 proteins in their milk. Viral proteins expressed in our model were immunogenic and were shown to induce a significant reduction in viral antigen shedding after challenge with virulent rotavirus in the adult mouse model. To our knowledge, this is the first report of transgenic mammal bioreactors allowing the rapid co-production of two recombinant viral proteins in milk to be used as a vaccine.