RESUMEN
Here, we reveal an unanticipated role of the blood-brain barrier (BBB) in regulating complex social behavior in ants. Using scRNA-seq, we find localization in the BBB of a key hormone-degrading enzyme called juvenile hormone esterase (Jhe), and we show that this localization governs the level of juvenile hormone (JH3) entering the brain. Manipulation of the Jhe level reprograms the brain transcriptome between ant castes. Although ant Jhe is retained and functions intracellularly within the BBB, we show that Drosophila Jhe is naturally extracellular. Heterologous expression of ant Jhe into the Drosophila BBB alters behavior in fly to mimic what is seen in ants. Most strikingly, manipulation of Jhe levels in ants reprograms complex behavior between worker castes. Our study thus uncovers a remarkable, potentially conserved role of the BBB serving as a molecular gatekeeper for a neurohormonal pathway that regulates social behavior.
Asunto(s)
Hormigas , Animales , Hormigas/fisiología , Barrera Hematoencefálica , Encéfalo/metabolismo , Drosophila , Conducta Social , Conducta AnimalRESUMEN
Behavioral plasticity is key to animal survival. Harpegnathos saltator ants can switch between worker and queen-like status (gamergate) depending on the outcome of social conflicts, providing an opportunity to study how distinct behavioral states are achieved in adult brains. Using social and molecular manipulations in live ants and ant neuronal cultures, we show that ecdysone and juvenile hormone drive molecular and functional differences in the brains of workers and gamergates and direct the transcriptional repressor Kr-h1 to different target genes. Depletion of Kr-h1 in the brain caused de-repression of "socially inappropriate" genes: gamergate genes were upregulated in workers, whereas worker genes were upregulated in gamergates. At the phenotypic level, loss of Kr-h1 resulted in the emergence of worker-specific behaviors in gamergates and gamergate-specific traits in workers. We conclude that Kr-h1 is a transcription factor that maintains distinct brain states established in response to socially regulated hormones.
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Hormigas/genética , Ecdisterona/farmacología , Jerarquia Social , Proteínas de Insectos/metabolismo , Neuronas/metabolismo , Sesquiterpenos/farmacología , Conducta Social , Transcriptoma/genética , Animales , Hormigas/efectos de los fármacos , Hormigas/fisiología , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Genoma , Neuronas/efectos de los fármacos , Fenotipo , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Transcriptoma/efectos de los fármacosRESUMEN
Chromatin domains and their associated structures must be faithfully inherited through cellular division to maintain cellular identity. However, accessing the localized strategies preserving chromatin domain inheritance, specifically the transfer of parental, pre-existing nucleosomes with their associated post-translational modifications (PTMs) during DNA replication, is challenging in living cells. We devised an inducible, proximity-dependent labeling system to irreversibly mark replication-dependent H3.1 and H3.2 histone-containing nucleosomes at desired loci in mouse embryonic stem cells so that their fate after DNA replication could be followed. Strikingly, repressed chromatin domains are preserved through local re-deposition of parental nucleosomes. In contrast, nucleosomes decorating active chromatin domains do not exhibit such preservation. Notably, altering cell fate leads to an adjustment of the positional inheritance of parental nucleosomes that reflects the corresponding changes in chromatin structure. These findings point to important mechanisms that contribute to parental nucleosome segregation to preserve cellular identity.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , Epigénesis Genética , Nucleosomas/genética , Animales , Diferenciación Celular/genética , División Celular/genética , Linaje de la Célula/genética , Replicación del ADN/genética , Histonas/genética , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional/genéticaRESUMEN
CBP/p300 are transcription co-activators whose binding is a signature of enhancers, cis-regulatory elements that control patterns of gene expression in multicellular organisms. Active enhancers produce bi-directional enhancer RNAs (eRNAs) and display CBP/p300-dependent histone acetylation. Here, we demonstrate that CBP binds directly to RNAs in vivo and in vitro. RNAs bound to CBP in vivo include a large number of eRNAs. Using steady-state histone acetyltransferase (HAT) assays, we show that an RNA binding region in the HAT domain of CBP-a regulatory motif unique to CBP/p300-allows RNA to stimulate CBP's HAT activity. At enhancers where CBP interacts with eRNAs, stimulation manifests in RNA-dependent changes in the histone acetylation mediated by CBP, such as H3K27ac, and by corresponding changes in gene expression. By interacting directly with CBP, eRNAs contribute to the unique chromatin structure at active enhancers, which, in turn, is required for regulation of target genes.
Asunto(s)
Histona Acetiltransferasas/metabolismo , ARN no Traducido/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Línea Celular , Elementos de Facilitación Genéticos , Fibroblastos/metabolismo , Histonas/metabolismo , RatonesRESUMEN
Social insects are emerging models to study how gene regulation affects behavior because their colonies comprise individuals with the same genomes but greatly different behavioral repertoires. To investigate the molecular mechanisms that activate distinct behaviors in different castes, we exploit a natural behavioral plasticity in Harpegnathos saltator, where adult workers can transition to a reproductive, queen-like state called gamergate. Analysis of brain transcriptomes during the transition reveals that corazonin, a neuropeptide homologous to the vertebrate gonadotropin-releasing hormone, is downregulated as workers become gamergates. Corazonin is also preferentially expressed in workers and/or foragers from other social insect species. Injection of corazonin in transitioning Harpegnathos individuals suppresses expression of vitellogenin in the brain and stimulates worker-like hunting behaviors, while inhibiting gamergate behaviors, such as dueling and egg deposition. We propose that corazonin is a central regulator of caste identity and behavior in social insects.
Asunto(s)
Hormigas/metabolismo , Proteínas de Insectos/metabolismo , Neuropéptidos/metabolismo , Animales , Hormigas/genética , Hormigas/crecimiento & desarrollo , Conducta Animal , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Conducta SocialRESUMEN
Ants exhibit cooperative behaviors and advanced forms of sociality that depend on pheromone-mediated communication. Odorant receptor neurons (ORNs) express specific odorant receptors (ORs) encoded by a dramatically expanded gene family in ants. In most eusocial insects, only the queen can transmit genetic information, restricting genetic studies. In contrast, workers in Harpegnathos saltator ants can be converted into gamergates (pseudoqueens) that can found entire colonies. This feature facilitated CRISPR-Cas9 generation of germline mutations in orco, the gene that encodes the obligate co-receptor of all ORs. orco mutations should significantly impact olfaction. We demonstrate striking functions of Orco in odorant perception, reproductive physiology, and social behavior plasticity. Surprisingly, unlike in other insects, loss of OR functionality also dramatically impairs development of the antennal lobe to which ORNs project. Therefore, the development of genetics in Harpegnathos establishes this ant species as a model organism to study the complexity of eusociality.
Asunto(s)
Hormigas/crecimiento & desarrollo , Hormigas/genética , Proteínas de Insectos/genética , Receptores Odorantes/genética , Conducta Social , Secuencia de Aminoácidos , Animales , Hormigas/anatomía & histología , Hormigas/fisiología , Antenas de Artrópodos/anatomía & histología , Antenas de Artrópodos/metabolismo , Secuencia de Bases , Conducta Animal , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Femenino , Técnicas de Inactivación de Genes , Proteínas de Insectos/química , Masculino , Mutación , Feromonas/metabolismo , Receptores Odorantes/químicaRESUMEN
Despite the numerous sequencing methods available, the diversity in RNA size and chemical modification makes it difficult to capture all RNAs in a cell. We developed a method that combines quasi-random priming with template switching to construct sequencing libraries from RNA molecules of any length and with any type of 3' modifications, allowing for the sequencing of virtually all RNA species. Our ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to identify and quantify all classes of coding and non-coding RNAs. With LIDAR, we comprehensively characterized the transcriptomes of mouse embryonic stem cells, neural progenitor cells, mouse tissues, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared with traditional ligation-dependent sequencing methods and uncovered tDRs with blocked 3' ends that had previously escaped detection. Therefore, LIDAR can capture all RNAs in a sample and uncover RNA species with potential regulatory functions.
RESUMEN
Transcription factors (TFs) orchestrate the gene expression programs that define each cell's identity. The canonical TF accomplishes this with two domains, one that binds specific DNA sequences and the other that binds protein coactivators or corepressors. We find that at least half of TFs also bind RNA, doing so through a previously unrecognized domain with sequence and functional features analogous to the arginine-rich motif of the HIV transcriptional activator Tat. RNA binding contributes to TF function by promoting the dynamic association between DNA, RNA, and TF on chromatin. TF-RNA interactions are a conserved feature important for vertebrate development and disrupted in disease. We propose that the ability to bind DNA, RNA, and protein is a general property of many TFs and is fundamental to their gene regulatory function.
Asunto(s)
ARN , Factores de Transcripción , Factores de Transcripción/metabolismo , ARN/metabolismo , Sitios de Unión , Unión Proteica , ADN/genéticaRESUMEN
Eusocial insect reproductive females show strikingly longer life spans than nonreproductive female workers despite high genetic similarity. In the ant Harpegnathos saltator (Hsal), workers can transition to reproductive "gamergates," acquiring a fivefold prolonged life span by mechanisms that are poorly understood. We found that gamergates have elevated expression of heat shock response (HSR) genes in the absence of heat stress and enhanced survival with heat stress. This HSR gene elevation is driven in part by gamergate-specific up-regulation of the gene encoding a truncated form of a heat shock factor most similar to mammalian HSF2 (hsalHSF2). In workers, hsalHSF2 was bound to DNA only upon heat stress. In gamergates, hsalHSF2 bound to DNA even in the absence of heat stress and was localized to gamergate-biased HSR genes. Expression of hsalHSF2 in Drosophila melanogaster led to enhanced heat shock survival and extended life span in the absence of heat stress. Molecular characterization illuminated multiple parallels between long-lived flies and gamergates, underscoring the centrality of hsalHSF2 to extended ant life span. Hence, ant caste-specific heat stress resilience and extended longevity can be transferred to flies via hsalHSF2. These findings reinforce the critical role of proteostasis in health and aging and reveal novel mechanisms underlying facultative life span extension in ants.
Asunto(s)
Hormigas , Longevidad , Animales , Femenino , Longevidad/genética , Hormigas/genética , Drosophila melanogaster/genética , Envejecimiento , Respuesta al Choque Térmico/genética , MamíferosRESUMEN
The Polycomb repressive complex 2 (PRC2) is an essential epigenetic regulator that deposits repressive H3K27me3. PRC2 subunits form two holocomplexes-PRC2.1 and PRC2.2-but the roles of these two PRC2 assemblies during differentiation are unclear. We employed auxin-inducible degradation to deplete PRC2.1 subunit MTF2 or PRC2.2 subunit JARID2 during differentiation of embryonic stem cells (ESCs) to neural progenitors (NPCs). Depletion of either MTF2 or JARID2 resulted in incomplete differentiation due to defects in gene regulation. Distinct sets of Polycomb target genes were derepressed in the absence of MTF2 or JARID2. MTF2-sensitive genes were marked by H3K27me3 in ESCs and remained silent during differentiation, whereas JARID2-sensitive genes were preferentially active in ESCs and became newly repressed in NPCs. Thus, MTF2 and JARID2 contribute non-redundantly to Polycomb silencing, suggesting that PRC2.1 and PRC2.2 have distinct functions in maintaining and establishing, respectively, Polycomb repression during differentiation.
Asunto(s)
Complejo Represivo Polycomb 2/metabolismo , Animales , Diferenciación Celular/fisiología , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/metabolismo , Complejo Represivo Polycomb 2/fisiología , Proteínas del Grupo Polycomb/metabolismo , Proteínas del Grupo Polycomb/fisiología , Unión Proteica/genéticaRESUMEN
Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.
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5-Metilcitosina/metabolismo , Reprogramación Celular , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/metabolismo , Elementos de Facilitación Genéticos , Epigénesis Genética , Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Proteínas de Unión al ADN/genética , Dioxigenasas , Embrión de Mamíferos/citología , Fibroblastos/citología , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Mutación , Células 3T3 NIH , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Up to 40% of patients with DLBCL display refractory disease or relapse after standard chemotherapy treatment (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R-CHOP]), leading to significant morbidity and mortality. The molecular mechanisms of chemoresistance in DLBCL remain incompletely understood. Using a cullin-really interesting new gene (RING) ligase-based CRISPR-Cas9 library, we identify that inactivation of the E3 ubiquitin ligase KLHL6 promotes DLBCL chemoresistance. Furthermore, proteomic approaches helped identify KLHL6 as a novel master regulator of plasma membrane-associated NOTCH2 via proteasome-dependent degradation. In CHOP-resistant DLBCL tumors, mutations of NOTCH2 result in a protein that escapes the mechanism of ubiquitin-dependent proteolysis, leading to protein stabilization and activation of the oncogenic RAS signaling pathway. Targeting CHOP-resistant DLBCL tumors with the phase 3 clinical trial molecules nirogacestat, a selective γ-secretase inhibitor, and ipatasertib, a pan-AKT inhibitor, synergistically promotes DLBCL destruction. These findings establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in KLHL6- or NOTCH2-mutated DLBCL.
Asunto(s)
Resistencia a Antineoplásicos , Linfoma de Células B Grandes Difuso , Humanos , Resistencia a Antineoplásicos/genética , Ubiquitina , Proteómica , Recurrencia Local de Neoplasia/tratamiento farmacológico , Rituximab/uso terapéutico , Vincristina , Ciclofosfamida , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Prednisona , Mutación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Receptor Notch2/genéticaRESUMEN
Interactions between noncoding RNAs and chromatin proteins play important roles in gene regulation, but the molecular details of most of these interactions are unknown. Using protein-RNA photocrosslinking and mass spectrometry on embryonic stem cell nuclei, we identified and mapped, at peptide resolution, the RNA-binding regions in â¼800 known and previously unknown RNA-binding proteins, many of which are transcriptional regulators and chromatin modifiers. In addition to known RNA-binding motifs, we detected several protein domains previously unknown to function in RNA recognition, as well as non-annotated and/or disordered regions, suggesting that many functional protein-RNA contacts remain unexplored. We identified RNA-binding regions in several chromatin regulators, including TET2, and validated their ability to bind RNA. Thus, proteomic identification of RNA-binding regions (RBR-ID) is a powerful tool to map protein-RNA interactions and will allow rational design of mutants to dissect their function at a mechanistic level.
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Cromatina/química , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Nucleares/química , Proteoma/química , ARN no Traducido/química , Proteínas de Unión al ARN/química , Animales , Sitios de Unión , Cromatina/metabolismo , Cromatina/efectos de la radiación , Expresión Génica , Células HEK293 , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de la radiación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Mapeo Peptídico/métodos , Procesos Fotoquímicos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteoma/genética , Proteoma/metabolismo , Proteómica/métodos , ARN no Traducido/genética , ARN no Traducido/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Rayos UltravioletaRESUMEN
Germline pathogenic variants in chromatin-modifying enzymes are a common cause of pediatric developmental disorders. These enzymes catalyze reactions that regulate epigenetic inheritance via histone post-translational modifications and DNA methylation. Cytosine methylation (5-methylcytosine [5mC]) of DNA is the quintessential epigenetic mark, yet no human Mendelian disorder of DNA demethylation has yet been delineated. Here, we describe in detail a Mendelian disorder caused by the disruption of DNA demethylation. TET3 is a methylcytosine dioxygenase that initiates DNA demethylation during early zygote formation, embryogenesis, and neuronal differentiation and is intolerant to haploinsufficiency in mice and humans. We identify and characterize 11 cases of human TET3 deficiency in eight families with the common phenotypic features of intellectual disability and/or global developmental delay; hypotonia; autistic traits; movement disorders; growth abnormalities; and facial dysmorphism. Mono-allelic frameshift and nonsense variants in TET3 occur throughout the coding region. Mono-allelic and bi-allelic missense variants localize to conserved residues; all but one such variant occur within the catalytic domain, and most display hypomorphic function in an assay of catalytic activity. TET3 deficiency and other Mendelian disorders of the epigenetic machinery show substantial phenotypic overlap, including features of intellectual disability and abnormal growth, underscoring shared disease mechanisms.
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Desmetilación del ADN , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Dioxigenasas/deficiencia , Adulto , Secuencia de Aminoácidos , Trastorno Autístico/genética , Trastorno Autístico/patología , Niño , Preescolar , Dioxigenasas/química , Dioxigenasas/genética , Desarrollo Embrionario , Femenino , Regulación del Desarrollo de la Expresión Génica , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/patología , Humanos , Lactante , Masculino , Persona de Mediana Edad , Trastornos del Movimiento/genética , Trastornos del Movimiento/patología , Linaje , Conformación Proteica , Homología de Secuencia , Adulto JovenRESUMEN
Post-transcriptional modifications of RNA strongly influence the RNA structure and function. Recent advances in RNA sequencing and mass spectrometry (MS) methods have identified over 140 of these modifications on a wide variety of RNA species. Most next-generation sequencing approaches can only map one RNA modification at a time, and while MS can assign multiple modifications simultaneously in an unbiased manner, MS cannot accurately catalog and assign RNA modifications in complex biological samples due to limitations in the fragment length and coverage depth. Thus, a facile method to identify novel RNA modifications while simultaneously locating them in the context of their RNA sequences is still lacking. We combined two orthogonal modes of RNA ion separation before MS identification: high-field asymmetric ion mobility separation (FAIMS) and electrochemically modulated liquid chromatography (EMLC). FAIMS RNA MS increases both coverage and throughput, while EMLC LC-MS orthogonally separates RNA molecules of different lengths and charges. The combination of the two methods offers a broadly applicable platform to improve the length and depth of MS-based RNA sequencing while providing contextual access to the analysis of RNA modifications.
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Espectrometría de Movilidad Iónica , ARN , Secuencia de Bases , Espectrometría de Masas/métodos , Cromatografía Liquida , Espectrometría de Movilidad Iónica/métodosRESUMEN
Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, and degradation, as well as human disease states, such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to first efficiently derivatize the ribonucleosides and quantitatively analyze them by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate that this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlight the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.
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Ribonucleósidos , Animales , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida/métodos , Ratones , ARN/química , Procesamiento Postranscripcional del ARN , Ribonucleósidos/análisis , Ribonucleósidos/química , Ribonucleósidos/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Over the past decade there has been a greater understanding of genomic complexity in eukaryotes ushered in by the immense technological advances in high-throughput sequencing of DNA and its corresponding RNA transcripts. This has resulted in the realization that beyond protein-coding genes, there are a large number of transcripts that do not encode for proteins and, therefore, may perform their function through RNA sequences and/or through secondary and tertiary structural determinants. This review is focused on the latest findings on a class of noncoding RNAs that are relatively large (>200 nucleotides), display nuclear localization, and use different strategies to regulate transcription. These are exciting times for discovering the biological scope and the mechanism of action for these RNA molecules, which have roles in dosage compensation, imprinting, enhancer function, and transcriptional regulation, with a great impact on development and disease.
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Cromatina/genética , Elementos de Facilitación Genéticos , ARN Largo no Codificante/genética , Transcripción Genética , Animales , Compensación de Dosificación (Genética) , Drosophila , Silenciador del Gen , Secuenciación de Nucleótidos de Alto Rendimiento , Mamíferos , ARN Polimerasa II/genéticaRESUMEN
JARID2 is an accessory component of Polycomb repressive complex-2 (PRC2) required for the differentiation of embryonic stem cells (ESCs). A role for JARID2 in the recruitment of PRC2 to target genes silenced during differentiation has been put forward, but the molecular details remain unclear. We identified a 30-amino-acid region of JARID2 that mediates interactions with long noncoding RNAs (lncRNAs) and found that the presence of lncRNAs stimulated JARID2-EZH2 interactions in vitro and JARID2-mediated recruitment of PRC2 to chromatin in vivo. Native and crosslinked RNA immunoprecipitations of JARID2 revealed that Meg3 and other lncRNAs from the imprinted Dlk1-Dio3 locus, an important regulator of development, interacted with PRC2 via JARID2. Lack of MEG3 expression in human induced pluripotent cells altered the chromatin distribution of JARID2, PRC2, and H3K27me3. Our findings show that lncRNAs facilitate JARID2-PRC2 interactions on chromatin and suggest a mechanism by which lncRNAs contribute to PRC2 recruitment.
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Cromatina/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Complejo Represivo Polycomb 2/fisiología , ARN no Traducido/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Proteína Potenciadora del Homólogo Zeste 2 , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas , Ratones , Complejo Represivo Polycomb 2/química , ARN Largo no Codificante/metabolismoRESUMEN
BACKGROUND: Functional genomic analyses rely on high-quality genome assemblies and annotations. Highly contiguous genome assemblies have become available for a variety of species, but accurate and complete annotation of gene models, inclusive of alternative splice isoforms and transcription start and termination sites, remains difficult with traditional approaches. RESULTS: Here, we utilized full-length isoform sequencing (Iso-Seq), a long-read RNA sequencing technology, to obtain a comprehensive annotation of the transcriptome of the ant Harpegnathos saltator. The improved genome annotations include additional splice isoforms and extended 3' untranslated regions for more than 4000 genes. Reanalysis of RNA-seq experiments using these annotations revealed several genes with caste-specific differential expression and tissue- or caste-specific splicing patterns that were missed in previous analyses. The extended 3' untranslated regions afforded great improvements in the analysis of existing single-cell RNA-seq data, resulting in the recovery of the transcriptomes of 18% more cells. The deeper single-cell transcriptomes obtained with these new annotations allowed us to identify additional markers for several cell types in the ant brain, as well as genes differentially expressed across castes in specific cell types. CONCLUSIONS: Our results demonstrate that Iso-Seq is an efficient and effective approach to improve genome annotations and maximize the amount of information that can be obtained from existing and future genomic datasets in Harpegnathos and other organisms.
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Hormigas , Regiones no Traducidas 3' , Animales , Hormigas/genética , Encéfalo , Anotación de Secuencia Molecular , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual , TranscriptomaRESUMEN
Polycomb-repressive complex 2 (PRC2) facilitates the maintenance and inheritance of chromatin domains repressive to transcription through catalysis of methylation of histone H3 at Lys27 (H3K27me2/3). However, through its EZH2 subunit, PRC2 also binds to nascent transcripts from active genes that are devoid of H3K27me2/3 in embryonic stem cells. Here, biochemical analyses indicated that RNA interaction inhibits SET domain-containing proteins, such as PRC2, nonspecifically in vitro. However, CRISPR-mediated truncation of a PRC2-interacting nascent RNA rescued PRC2-mediated deposition of H3K27me2/3. That PRC2 activity is inhibited by interactions with nascent transcripts supports a model in which PRC2 can only mark for repression those genes silenced by transcriptional repressors.