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BACKGROUND: Organisms from many distinct evolutionary lineages acquired the capacity to enter a dormant state in response to environmental conditions incompatible with maintaining normal life activities. Most studied organisms exhibit seasonal or annual episodes of dormancy, but numerous less studied organisms enter long-term dormancy, lasting decades or even centuries. Intriguingly, many planktonic animals produce encased embryos known as resting eggs or cysts that, like plant seeds, may remain dormant for decades. Herein, we studied a rotifer Brachionus plicatilis as a model planktonic species that forms encased dormant embryos via sexual reproduction and non-dormant embryos via asexual reproduction and raised the following questions: Which genes are expressed at which time points during embryogenesis? How do temporal transcript abundance profiles differ between the two types of embryos? When does the cell cycle arrest? How do dormant embryos manage energy? RESULTS: As the molecular developmental kinetics of encased embryos remain unknown, we employed single embryo RNA sequencing (CEL-seq) of samples collected during dormant and non-dormant embryogenesis. We identified comprehensive and temporal transcript abundance patterns of genes and their associated enriched functional pathways. Striking differences were uncovered between dormant and non-dormant embryos. In early development, the cell cycle-associated pathways were enriched in both embryo types but terminated with fewer nuclei in dormant embryos. As development progressed, the gene transcript abundance profiles became increasingly divergent between dormant and non-dormant embryos. Organogenesis was suspended in dormant embryos, concomitant with low transcript abundance of homeobox genes, and was replaced with an ATP-poor preparatory phase characterized by very high transcript abundance of genes encoding for hallmark dormancy proteins (e.g., LEA proteins, sHSP, and anti-ROS proteins, also found in plant seeds) and proteins involved in dormancy exit. Surprisingly, this period appeared analogous to the late maturation phase of plant seeds. CONCLUSIONS: The study highlights novel divergent temporal transcript abundance patterns between dormant and non-dormant embryos. Remarkably, several convergent functional solutions appear during the development of resting eggs and plant seeds, suggesting a similar preparatory phase for long-term dormancy. This study accentuated the broad novel molecular features of long-term dormancy in encased animal embryos that behave like "animal seeds".
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Rotíferos , Animales , Rotíferos/genética , Perfilación de la Expresión Génica , Transcriptoma , Proteínas/metabolismo , Semillas , Latencia en las Plantas , Germinación/genéticaRESUMEN
Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development--known as the phylotypic period--is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent 'mid-developmental transition' that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.
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Tipificación del Cuerpo , Desarrollo Embrionario , Filogenia , Animales , Tipificación del Cuerpo/genética , Secuencia Conservada/genética , Desarrollo Embrionario/genética , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Genes del Desarrollo/genética , Modelos Biológicos , Fenotipo , Especificidad de la Especie , Transcriptoma/genéticaRESUMEN
Alternative polyadenylation (APA) produces isoforms with distinct 3'-ends, yet their functional differences remain largely unknown. Here, we introduce the APA-seq method to detect the expression levels of APA isoforms from 3'-end RNA-Seq data by exploiting both paired-end reads for gene isoform identification and quantification. We detected the expression levels of APA isoforms in individual Caenorhabditis elegans embryos at different stages throughout embryogenesis. Examining the correlation between the temporal profiles of isoforms led us to distinguish two classes of genes: those with highly correlated isoforms (HCI) and those with lowly correlated isoforms (LCI) across time. We hypothesized that variants with similar expression profiles may be the product of biological noise, while the LCI variants may be under tighter selection and consequently their distinct 3' UTR isoforms are more likely to have functional consequences. Supporting this notion, we found that LCI genes have significantly more miRNA binding sites, more correlated expression profiles with those of their targeting miRNAs and a relative lack of correspondence between their transcription and protein abundances. Collectively, our results suggest that a lack of coherence among the regulation of 3' UTR isoforms is a proxy for selective pressures acting upon APA usage and consequently for their functional relevance.
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Regiones no Traducidas 3'/genética , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Regulación del Desarrollo de la Expresión Génica , Poli A/análisis , Poliadenilación , Animales , Drosophila melanogaster , Desarrollo Embrionario/genética , MicroARNs/genética , MicroARNs/metabolismo , Conformación de Ácido Nucleico , Xenopus laevisRESUMEN
The concept of germ layers has been one of the foremost organizing principles in developmental biology, classification, systematics and evolution for 150 years (refs 1 - 3). Of the three germ layers, the mesoderm is found in bilaterian animals but is absent in species in the phyla Cnidaria and Ctenophora, which has been taken as evidence that the mesoderm was the final germ layer to evolve. The origin of the ectoderm and endoderm germ layers, however, remains unclear, with models supporting the antecedence of each as well as a simultaneous origin. Here we determine the temporal and spatial components of gene expression spanning embryonic development for all Caenorhabditis elegans genes and use it to determine the evolutionary ages of the germ layers. The gene expression program of the mesoderm is induced after those of the ectoderm and endoderm, thus making it the last germ layer both to evolve and to develop. Strikingly, the C. elegans endoderm and ectoderm expression programs do not co-induce; rather the endoderm activates earlier, and this is also observed in the expression of endoderm orthologues during the embryology of the frog Xenopus tropicalis, the sea anemone Nematostella vectensis and the sponge Amphimedon queenslandica. Querying the phylogenetic ages of specifically expressed genes reveals that the endoderm comprises older genes. Taken together, we propose that the endoderm program dates back to the origin of multicellularity, whereas the ectoderm originated as a secondary germ layer freed from ancestral feeding functions.
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Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Endodermo/metabolismo , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica/genética , Análisis Espacio-Temporal , Transcriptoma/genética , Animales , Caenorhabditis elegans/citología , Linaje de la Célula , Ingestión de Alimentos , Ectodermo/citología , Ectodermo/embriología , Ectodermo/metabolismo , Endodermo/citología , Endodermo/embriología , Perfilación de la Expresión Génica , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Modelos Biológicos , Poríferos/citología , Poríferos/embriología , Poríferos/genética , Anémonas de Mar/citología , Anémonas de Mar/embriología , Anémonas de Mar/genética , Factores de Tiempo , Xenopus/embriología , Xenopus/genéticaRESUMEN
The evolution of development has been studied through the lens of gene regulation by examining either closely related species or extremely distant animals of different phyla. In nematodes, detailed cell- and stage-specific expression analyses are focused on the model Caenorhabditis elegans, in part leading to the view that the developmental expression of gene cascades in this species is archetypic for the phylum. Here, we compared two species of an intermediate evolutionary distance: the nematodes C. elegans (clade V) and Acrobeloides nanus (clade IV). To examine A. nanus molecularly, we sequenced its genome and identified the expression profiles of all genes throughout embryogenesis. In comparison with C. elegans, A. nanus exhibits a much slower embryonic development and has a capacity for regulative compensation of missing early cells. We detected conserved stages between these species at the transcriptome level, as well as a prominent middevelopmental transition, at which point the two species converge in terms of their gene expression. Interestingly, we found that genes originating at the dawn of the Ecdysozoa supergroup show the least expression divergence between these two species. This led us to detect a correlation between the time of expression of a gene and its phylogenetic age: evolutionarily ancient and young genes are enriched for expression in early and late embryogenesis, respectively, whereas Ecdysozoa-specific genes are enriched for expression during the middevelopmental transition. Our results characterize the developmental constraints operating on each individual embryo in terms of developmental stages and genetic evolutionary history.
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Evolución Molecular , Regulación del Desarrollo de la Expresión Génica/fisiología , Filogenia , Rabdítidos/embriología , Transcriptoma/fisiología , Animales , Rabdítidos/clasificación , Rabdítidos/genéticaRESUMEN
BACKGROUND: The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a current pandemic of unprecedented scale. Although diagnostic tests are fundamental to the ability to detect and respond, overwhelmed healthcare systems are already experiencing shortages of reagents associated with this test, calling for a lean immediately applicable protocol. METHODS: RNA extracts of positive samples were tested for the presence of SARS-CoV-2 using reverse transcription quantitative polymerase chain reaction, alone or in pools of different sizes (2-, 4-, 8-, 16-, 32-, and 64-sample pools) with negative samples. Transport media of additional 3 positive samples were also tested when mixed with transport media of negative samples in pools of 8. RESULTS: A single positive sample can be detected in pools of up to 32 samples, using the standard kits and protocols, with an estimated false negative rate of 10%. Detection of positive samples diluted in even up to 64 samples may also be attainable, although this may require additional amplification cycles. Single positive samples can be detected when pooling either after or prior to RNA extraction. CONCLUSIONS: As it uses the standard protocols, reagents, and equipment, this pooling method can be applied immediately in current clinical testing laboratories. We hope that such implementation of a pool test for coronavirus disease 2019 would allow expanding current screening capacities, thereby enabling the expansion of detection in the community, as well as in close organic groups, such as hospital departments, army units, or factory shifts.
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COVID-19/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , COVID-19/virología , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2/genética , SARS-CoV-2/patogenicidadRESUMEN
Productive rearrangement of the immunoglobulin heavy-chain locus triggers a major developmental checkpoint that promotes limited clonal expansion of pre-B cells, thereby culminating in cell-cycle arrest and rearrangement of light-chain loci. By using Irf4-/-Irf8-/- pre-B cells, we demonstrated that two pathways converge to synergistically drive light-chain rearrangement, but not simply as a consequence of cell-cycle exit. One pathway was directly dependent on transcription factor IRF-4, whose expression was elevated by pre-B cell receptor signaling. IRF-4 targeted the immunoglobulin 3'Ekappa and Elambda enhancers and positioned a kappa allele away from pericentromeric heterochromatin. The other pathway was triggered by attenuation of IL-7 signaling and activated the iEkappa enhancer via binding of the transcription factor E2A. IRF-4 also regulated expression of chemokine receptor Cxcr4 and promoted migration of pre-B cells in response to the chemokine ligand CXCL12. We propose that IRF-4 coordinates the two pathways regulating light-chain recombination by positioning pre-B cells away from IL-7-expressing stromal cells.
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Linfocitos B/inmunología , Diferenciación Celular/inmunología , Reordenamiento Génico de Cadena Ligera de Linfocito B/inmunología , Factores Reguladores del Interferón/inmunología , Interleucina-7/inmunología , Transducción de Señal/inmunología , Animales , Linfocitos B/citología , Western Blotting , Movimiento Celular/inmunología , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Inmunoprecipitación , Hibridación Fluorescente in Situ , Factores Reguladores del Interferón/metabolismo , Interleucina-7/metabolismo , Ratones , Ratones Mutantes , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/citología , Células Madre/inmunologíaRESUMEN
Core promoters mediate transcription initiation by the integration of diverse regulatory signals encoded in the proximal promoter and enhancers. It has been suggested that genes under simple regulation may have low-complexity permissive promoters. For these genes, the core promoter may serve as the principal regulatory element; however, the mechanism by which this occurs is unclear. We report here a periodic poly-thymine motif, which we term T-blocks, enriched in occurrences within core promoter forward strands in Caenorhabditis elegans. An increasing number of T-blocks on either strand is associated with increasing nucleosome eviction. Strikingly, only forward strand T-blocks are correlated with expression levels, whereby genes with ≥6 T-blocks have fivefold higher expression levels than genes with ≤3 T-blocks. We further demonstrate that differences in T-block numbers between strains predictably affect expression levels of orthologs. Highly expressed genes and genes in operons tend to have a large number of T-blocks, as well as the previously characterized SL1 motif involved in trans-splicing. The presence of T-blocks thus correlates with low nucleosome occupancy and the precision of a trans-splicing motif, suggesting its role at both the DNA and RNA levels. Collectively, our results suggest that core promoters may tune gene expression levels through the occurrences of T-blocks, independently of the spatio-temporal regulation mediated by the proximal promoter.
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Secuencia de Bases , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Nucleosomas/metabolismo , Nucleosomas/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Trans-EmpalmeRESUMEN
Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. In Drosophila, classic work has shown that segmentation in the embryo is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium - such as Caenorhabditis elegans - where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies in C. elegans have analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28- to 102-cells stages) missed by these approaches, here we determine the transcriptomes of the 1- to 102-cell stage to identify 119 embryonic cell states during cell fate specification, including 'equivalence-group' cell identities. We find that gene expression programs are modular according to the sub-cell lineages, each establishing a set of stripes by combinations of transcription factor gene expression across the anterior-posterior axis. In particular, expression of the homeodomain genes establishes a comprehensive lineage-specific positioning system throughout the embryo beginning at the 28-cell stage. Moreover, we find that genes that segment the entire embryo in Drosophila have orthologs in C. elegans that exhibit sub-lineage-specific expression. These results suggest that the C. elegans embryo is patterned by a juxtaposition of distinct lineage-specific gene regulatory programs each with a unique encoding of cell location and fate. This use of homologous gene regulatory patterning codes suggests a deep homology of cell fate specification programs across diverse modes of development.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Regulación del Desarrollo de la Expresión Génica , Diferenciación Celular/genética , Linaje de la Célula/genética , Drosophila/genética , Tipificación del Cuerpo/genética , Embrión no Mamífero/metabolismoRESUMEN
Congenital cytomegalovirus (cCMV) is the most common intrauterine infection, leading to infant neurodevelopmental disabilities. An improved knowledge of correlates of protection against cCMV is needed to guide prevention strategies. Here, we employ an ex vivo model of human CMV (HCMV) infection in decidual tissues of women with and without preconception immunity against CMV, recapitulating nonprimary vs. primary infection at the authentic maternofetal transmission site. We show that decidual tissues of women with preconception immunity against CMV exhibit intrinsic resistance to HCMV, mounting a rapid activation of tissue-resident memory CD8+ and CD4+ T cells upon HCMV reinfection. We further reveal the role of HCMV-specific decidual-tissue-resident CD8+ T cells in local protection against nonprimary HCMV infection. The findings could inform the development of a vaccine against cCMV and provide insights for further studies of the integrity of immune defense against HCMV and other pathogens at the human maternal-fetal interface.
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Infecciones por Citomegalovirus , Citomegalovirus , Lactante , Humanos , Femenino , Linfocitos T CD8-positivos , Células T de Memoria , FetoRESUMEN
The phenotype of an organism is determined by its genotype and environment. An interaction between these two arises from the differential effect of the environment on gene expression in distinct genotypes; however, the genomic properties identifying these are not well understood. Here we analyze the transcriptomes of five C. elegans strains (genotype) cultivated in five growth conditions (environment), and find that highly regulated genes, as distinguished by intergenic lengths, motif concentration, and expression levels, are particularly biased toward genotype-environment interactions. Sequencing these strains, we find that genes with expression variation across genotypes are enriched for promoter single-nucleotide polymorphisms (SNPs), as expected. However, genes with genotype-environment interactions do not significantly differ from background in terms of their promoter SNPs. Collectively, these results indicate that the highly regulated nature of particular genes predispose them for exhibiting genotype-environment interaction as a consequence of changes to upstream regulators. This observation may provide a deeper understanding into the origin of the extraordinary gene expression diversity present in even closely related species.
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Caenorhabditis elegans/genética , Interacción Gen-Ambiente , Regiones Promotoras Genéticas , Animales , Regulación de la Expresión Génica , Genómica/métodos , Genotipo , Polimorfismo de Nucleótido Simple , TranscriptomaRESUMEN
DNA methylation inhibits gene expression in animal cells, probably by affecting chromatin structure. Biochemical studies suggest that this process may be mediated by methyl-specific binding proteins that recruit enzymatic machinery capable of locally altering histone modification. To test whether DNA methylation actually has a role in the assembly of chromatin during normal development, we used cell transfection and a transgene construct genetically programmed to be either methylated or unmethylated in all cell types of the mouse. Chromatin immunoprecipitation (ChIP) analysis shows that the presence of DNA methylation brings about the deacetylation of histone H4 and methylation of Lys9 of histone H3 (H3 Lys9) and prevents methylation of Lys4 of histone H3 (H3 Lys4), thus generating a structure identical to that of methylated sequences in the genome. These results indicate that the methylation pattern established in early embryogenesis is profoundly important in setting up the structural profile of the genome.
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Cromatina/genética , Cromatina/metabolismo , Metilación de ADN , Acetilación , Animales , Islas de CpG , Regulación del Desarrollo de la Expresión Génica , Globinas/genética , Histonas/química , Histonas/metabolismo , Humanos , Lisina/química , Metilación , Ratones , Ratones Transgénicos , Modelos Genéticos , TransfecciónRESUMEN
Embryonic stem cell (ESC) self-renewal and cell fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carry out a proteome-wide screen and identify 810 proteins that bind RNA in hESCs. We reveal that RBPs are preferentially expressed in hESCs and dynamically regulated during early stem cell differentiation. Notably, many RBPs are affected by knockdown of OCT4, a master regulator of pluripotency, several dozen of which are directly targeted by this factor. Using cross-linking and immunoprecipitation (CLIP-seq), we find that the pluripotency-associated STAT3 and OCT4 transcription factors interact with RNA in hESCs and confirm the binding of STAT3 to the conserved NORAD long-noncoding RNA. Our findings indicate that RBPs have a more widespread role in human pluripotency than previously appreciated.
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Células Madre Embrionarias Humanas/metabolismo , Proteínas de Unión al ARN/metabolismo , Diferenciación Celular/genética , Línea Celular , ADN/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Unión Proteica , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
Single-cell RNA sequencing has revolutionized the way we look at cell populations. Of the methods available, CEL-Seq was the first to use linear RNA amplification. With early barcoding and 3' sequencing, it is sensitive, cost-effective and easy to perform. Here we describe a protocol for performing CEL-Seq2 on sorted cells, which can be performed without any special equipment.
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Perfilación de la Expresión Génica/métodos , ARN/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Secuencia de Bases , ADN Complementario/genética , Citometría de Flujo , Perfilación de la Expresión Génica/economía , Biblioteca de Genes , Humanos , Técnicas de Amplificación de Ácido Nucleico/economía , Técnicas de Amplificación de Ácido Nucleico/métodos , Análisis de Secuencia de ARN/economía , Análisis de la Célula Individual/economíaRESUMEN
Developmental programs are executed by tightly controlled gene regulatory pathways. Here, we combined the unique sample retrieval capacity afforded by laser capture microscopy with analysis of mRNA abundance by CEL-Seq (cell expression by linear amplification and sequencing) to generate a spatiotemporal gene expression map of the Caenorhabditis elegans syncytial germline from adult hermaphrodites and males. We found that over 6000 genes exhibit spatiotemporally dynamic expression patterns throughout the hermaphrodite germline, with two dominant groups of genes exhibiting reciprocal shifts in expression at late pachytene during meiotic prophase I. We found a strong correlation between restricted spatiotemporal expression and known developmental and cellular processes, indicating that these gene expression changes may be an important driver of germ cell progression. Analysis of the male gonad revealed a shift in gene expression at early pachytene and upregulation of subsets of genes following the meiotic divisions, specifically in early and late spermatids, mostly transcribed from the X chromosome. We observed that while the X chromosome is silenced throughout the first half of the gonad, some genes escape this control and are highly expressed throughout the germline. Although we found a strong correlation between the expression of genes corresponding to CSR-1-interacting 22G-RNAs during germ cell progression, we also found that a large fraction of genes may bypass the need for CSR-1-mediated germline licensing. Taken together, these findings suggest the existence of mechanisms that enable a shift in gene expression during prophase I to promote germ cell progression.
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Gametogénesis , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Células Germinativas/citología , Meiosis , Cromosoma X/genética , Inactivación del Cromosoma XRESUMEN
The interaction between a pathogen and a host is a highly dynamic process in which both agents activate complex programs. Here, we introduce a single-cell RNA-sequencing method, scDual-Seq, that simultaneously captures both host and pathogen transcriptomes. We use it to study the process of infection of individual mouse macrophages with the intracellular pathogen Salmonella typhimurium. Among the infected macrophages, we find three subpopulations and we show evidence for a linear progression through these subpopulations, supporting a model in which these three states correspond to consecutive stages of infection.
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Perfilación de la Expresión Génica/métodos , Macrófagos/metabolismo , Macrófagos/microbiología , Salmonella typhimurium/genética , Análisis de Secuencia de ARN/métodos , Animales , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Análisis de la Célula IndividualRESUMEN
Single-cell transcriptomics requires a method that is sensitive, accurate, and reproducible. Here, we present CEL-Seq2, a modified version of our CEL-Seq method, with threefold higher sensitivity, lower costs, and less hands-on time. We implemented CEL-Seq2 on Fluidigm's C1 system, providing its first single-cell, on-chip barcoding method, and we detected gene expression changes accompanying the progression through the cell cycle in mouse fibroblast cells. We also compare with Smart-Seq to demonstrate CEL-Seq2's increased sensitivity relative to other available methods. Collectively, the improvements make CEL-Seq2 uniquely suited to single-cell RNA-Seq analysis in terms of economics, resolution, and ease of use.
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Algoritmos , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Ciclo Celular , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Ratones , Sensibilidad y EspecificidadRESUMEN
New methods employ RNA-seq to study single cells within complex tissues by in situ sequencing or mRNA capture from single photoactivated cells.
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Encéfalo/metabolismo , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Hipocampo/metabolismo , Neuronas/metabolismo , Análisis de Secuencia de ARN/métodos , Transcriptoma , Animales , HumanosRESUMEN
In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (large) and Rpd3S (small) that exert opposing effects on the transcription of meiosis-specific genes. By introducing mutations that disrupt the integrity and function of either Rpd3L or Rpd3S, we show here that Rpd3 function is determined by its association with either of these complexes. Specifically, the catalytic activity of Rpd3S activates the transcription of the two major positive regulators of meiosis, IME1 and IME2, under all growth conditions and activates the transcription of NDT80 only during vegetative growth. In contrast, the effects of Rpd3L depends on nutrients; it represses or activates transcription in the presence or absence of a nitrogen source, respectively. Further, we show that transcriptional activation does not correlate with histone H4 deacetylation, suggesting an effect on a nonhistone protein. Comparison of rpd3-null and catalytic-site point mutants revealed an inhibitory activity that is independent of either the catalytic activity of Rpd3 or the integrity of Rpd3L and Rpd3S.