RESUMEN
Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.
Asunto(s)
Proteínas Argonautas/metabolismo , Proteínas del Helminto/metabolismo , Planarias/fisiología , Tetraspaninas/metabolismo , Animales , Proteínas Argonautas/antagonistas & inhibidores , Proteínas Argonautas/genética , Ciclo Celular/efectos de la radiación , Regulación de la Expresión Génica , Proteínas del Helminto/antagonistas & inhibidores , Proteínas del Helminto/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/trasplante , Análisis de Componente Principal , Interferencia de ARN , ARN Bicatenario/metabolismo , ARN de Helminto/química , ARN de Helminto/aislamiento & purificación , ARN de Helminto/metabolismo , Regeneración/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Tetraspaninas/genética , Irradiación Corporal TotalRESUMEN
The optimal relational experiences of infants and young children demand a cross-sector workforce informed by Infant and Early Childhood Mental Health (IECMH) principles and practices. A recent review by the Alliance for the Advancement of Infant Mental Health, Inc identified seven themes that help define "What makes an IECMH association strong?": (1) Identity, (2) Cross-Systems Collaborations, (3) Sound Organizational Structure, (4) Competency-Informed Training, (5) Reflective Supervision Capacity, (6) Policy, and (7) Higher Education. The present paper documents the story of the Association of Infant Mental Health in Tennessee (AIMHiTN) and the role of the Endorsement for Culturally Sensitive, Relationship-Focused Practice Promoting IECMH in that growth across those seven themes with the additional themes of (8) Funding and, (9) Diversity, Equity, Inclusion, and Belonging. First, foundational literature is reviewed to summarize IECMH-informed workforce development. Next, AIMHiTN's story of workforce development is mapped onto the nine themes and challenges and lessons learned are summarized. The article aims to serve as a roadmap for other states, provinces, territories, or nations hoping to develop their own Association for Infant Mental Health (AIMH) as well as a guide for those with existing AIMHs for promoting continued growth and sustainability.
Las óptimas experiencias de relaciones de los infantes y niños pequeñitos requieren de un sector heterogéneo de la fuerza laboral que esté informado acerca de los principios y prácticas de la salud mental en los infantes y en la temprana niñez (IECMH). Una reciente revisión por parte de la Alianza para el Avance de la Salud Mental Infantil, Inc.® identificó siete temas que ayudan a definir "¿qué hace fuerte a una asociación de IECMH?:" 1. Identidad, 2. Colaboraciones entre sistemas heterogéneos, 3. Buena estructura organizacional, 4. Entrenamiento con base en la competencia, 5. Capacidad para la supervisión con reflexión, 6. Políticas, y 7. Educación postsecundaria. El presente estudio documenta la historia de la Asociación de Salud Mental en Tennessee (AIMHiTN) y el papel que la Aprobación a Promover la Salud Mental Infantil y en la Temprana Niñez® para la Práctica de Sensibilidad Cultural Enfocada en la Relación juega en ese crecimiento a través de esos siete temas más los temas adicionales de 8. Fondos económicos y, 9. Diversidad, equidad, inclusión y sentido de pertenencia. Primero, la literatura básica se revisa para resumir el desarrollo de la fuerza laboral informada sobre IECMH. Después, la historia de AIMHiTN en cuanto al desarrollo de la fuerza laboral se despliega en los nueve temas y se resumen los retos y lecciones aprendidas. El artículo se propone servir como una especie de mapa de caminos a seguir para otros estados, provincias, territorios, o naciones que desean desarrollar su propia asociación de salud mental infantil (AIMH) así como también ser una guía para aquellos en donde ya existe una AIMH con el fin de promover el continuo crecimiento y sostenimiento.
Les expériences relationnelles optimales des bébés et des jeunes enfants exigent des effectifs intersectoriels ayant connaissance des principes et des pratiques de la santé mentale du nourrisson et de la petite enfance (IECMH). Une revue récente de l'Alliance for the Advancement of Infant Mental Health, Inc® a identifié sept thèmes qui définissent « Les forces de l'Association IECMH ¼ : 1. L'identité, 2. Les collaborations intersectorielles, 3. Sa structure organisationnelle saine, 4. Sa formation centrée sur la connaissance des compétences, 5. Sa capacité de supervision réfléchie, et 7. L'enseignement supérieur. Cet article documente l'histoire de l'Association de Santé Mentale du Nourrisson de l'état du Tennessee aux Etats-Unis (AIMHiTN) et le rôle de l'Adoption d'une Pratique Culturellement Adaptée et Focalisée sur la Relation, promouvant la Santé Mentale du Nourrisson et de la Petite Enfance dans ce développement au travers de ces sept thèmes avec deux thèmes supplémentaires : 8. Le financement et, 9. La diversité, l'équité, l'inclusion et l'appartenance. Les recherches fondatrices sont d'abord passées en revue afin de résumer le développement des effectifs suivi par l'IECHM. Ensuite l'histoire du développement des effectifs de l'AIMHiTN est mappé sur les neuf thèmes, les défis et les leçons apprises étant résumés. Cet article a pour but de servir de feuille de route aux autres états, provinces, territoires ou nations espérant développer leur propre association de santé mentale du nourrisson (AIMH) ainsi que de servir de guide à ceux ou celles ayant déjà des AIMH pour la promotion d'une croissance soutenue et d'une durabilité.
Asunto(s)
Fuerza Laboral en Salud , Salud Mental , Niño , Preescolar , Personal de Salud , Humanos , Lactante , Salud del Lactante , TennesseeRESUMEN
The Spt-Ada-Gcn5-acetyltransferase (SAGA) complex was discovered from Saccharomyces cerevisiae and has been well characterized as an important transcriptional coactivator that interacts both with sequence-specific transcription factors and the TATA-binding protein TBP. SAGA contains a histone acetyltransferase and a ubiquitin protease. In metazoans, SAGA is essential for development, yet little is known about the function of SAGA in differentiating tissue. We analyzed the composition, interacting proteins, and genomic distribution of SAGA in muscle and neuronal tissue of late stage Drosophila melanogaster embryos. The subunit composition of SAGA was the same in each tissue; however, SAGA was associated with considerably more transcription factors in muscle compared with neurons. Consistent with this finding, SAGA was found to occupy more genes specifically in muscle than in neurons. Strikingly, SAGA occupancy was not limited to enhancers and promoters but primarily colocalized with RNA polymerase II within transcribed sequences. SAGA binding peaks at the site of RNA polymerase pausing at the 5' end of transcribed sequences. In addition, many tissue-specific SAGA-bound genes required its ubiquitin protease activity for full expression. These data indicate that in metazoans SAGA plays a prominent post-transcription initiation role in tissue-specific gene expression.
Asunto(s)
Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Músculos/metabolismo , Neuronas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Activación Transcripcional , Acetilación , Animales , ADN Polimerasa II/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Músculos/enzimología , Neuronas/enzimología , Sistemas de Lectura Abierta , Péptido Hidrolasas/metabolismo , Regiones Promotoras Genéticas , Transporte de Proteínas , Factores de Transcripción/metabolismo , Ubiquitina/metabolismoRESUMEN
The clustered Hox genes, which are highly conserved across metazoans, encode homeodomain-containing transcription factors that provide a blueprint for segmental identity along the body axis. Recent studies have underscored that in addition to encoding Hox genes, the homeotic clusters contain key noncoding RNA genes that play a central role in development. In this study, we have taken advantage of genome-wide approaches to provide a detailed analysis of retinoic acid (RA)-induced transcriptional and epigenetic changes within the homeotic clusters of mouse embryonic stem cells. Although there is a general colinear response, our analyses suggest a lack of strict colinearity for several genes in the HoxA and HoxB clusters. We have identified transcribed novel noncoding RNAs (ncRNAs) and their cis-regulatory elements that function in response to RA and demonstrated that the expression of these ncRNAs from both strands represent some of the most rapidly induced transcripts in ES cells. Finally, we have provided dynamic analyses of chromatin modifications for the coding and noncoding genes expressed upon activation and suggest that active transcription can occur in the presence of chromatin modifications and machineries associated with repressed transcription state over the clusters. Overall, our data provide a resource for a better understanding of the dynamic nature of the coding and noncoding transcripts and their associated chromatin marks in the regulation of homeotic gene transcription during development.
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Epigénesis Genética/efectos de los fármacos , Proteínas de Homeodominio/genética , ARN no Traducido/genética , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacología , Animales , Línea Celular , Cromatina/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Elementos Reguladores de la Transcripción/efectos de los fármacosRESUMEN
The highly conserved yeast R2TP complex, consisting of Rvb1, Rvb2, Pih1, and Tah1, participates in diverse cellular processes ranging from assembly of protein complexes to apoptosis. Rvb1 and Rvb2 are closely related proteins belonging to the AAA+ superfamily and are essential for cell survival. Although Rvbs have been shown to be associated with various protein complexes including the Ino80 and Swr1chromatin remodeling complexes, we performed a systematic quantitative proteomic analysis of their associated proteins and identified two additional complexes that associate with Rvb1 and Rvb2: the chaperonin-containing T-complex and the 19S regulatory particle of the proteasome complex. We also analyzed Rvb1 and Rvb2 purified from yeast strains devoid of PIH1 and TAH1. These analyses revealed that both Rvb1 and Rvb2 still associated with Hsp90 and were highly enriched with RNA polymerase II complex components. Our analyses also revealed that both Rvb1 and Rvb2 were recruited to the Ino80 and Swr1 chromatin remodeling complexes even in the absence of Pih1 and Tah1 proteins. Using further biochemical analysis, we showed that Rvb1 and Rvb2 directly interacted with Hsp90 as well as with the RNA polymerase II complex. RNA-Seq analysis of the deletion strains compared with the wild-type strains revealed an up-regulation of ribosome biogenesis and ribonucleoprotein complex biogenesis genes, down-regulation of response to abiotic stimulus genes, and down-regulation of response to temperature stimulus genes. A Gene Ontology analysis of the 80 proteins whose protein associations were altered in the PIH1 or TAH1 deletion strains found ribonucleoprotein complex proteins to be the most enriched category. This suggests an important function of the R2TP complex in ribonucleoprotein complex biogenesis at both the proteomic and genomic levels. Finally, these results demonstrate that deletion network analyses can provide novel insights into cellular systems.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , ADN Helicasas/metabolismo , Eliminación de Gen , Redes Reguladoras de Genes , Proteómica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ARN/métodos , Factores de Transcripción/metabolismo , Ensamble y Desensamble de Cromatina , Ontología de Genes , Genoma Fúngico , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The NF-κB family of transcription factors is pivotal in controlling cellular responses to environmental stresses; abnormal nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling features in many autoimmune diseases and cancers. Several components of the NF-κB signaling pathway have been reported to interact with the protein TNIP2 (also known as ABIN2), and TNIP2 can both positively and negatively regulate NF-κB- dependent transcription of target genes. However, the function of TNIP2 remains elusive and the cellular machinery associating with TNIP2 has not been systematically defined. Here we first used a broad MudPIT/Halo Affinity Purification Mass Spectrometry (AP-MS) approach to map the network of proteins associated with the NF-κB transcription factors, and establish TNIP2 as an NF-κB network hub protein. We then combined AP-MS with biochemical approaches in a more focused study of truncated and mutated forms of TNIP2 to map protein associations with distinct regions of TNIP2. NF-κB interacted with the N-terminal region of TNIP2. A central region of TNIP2 interacted with the endosomal sorting complex ESCRT-I via its TSG101 subunit, a protein essential for HIV-1 budding, and a single point mutant in TNIP2 disrupted this interaction. The major gene ontology category for TNIP2 associated proteins was mRNA metabolism, and several of these associations, like KHDRBS1, were lost upon depletion of RNA. Given the major association of TNIP2 with mRNA metabolism proteins, we analyzed the RNA content of affinity purified TNIP2 using RNA-Seq. Surprisingly, a specific limited number of mRNAs was associated with TNIP2. These RNAs were enriched for transcription factor binding, transcription factor cofactor activity, and transcription regulator activity. They included mRNAs of genes in the Sin3A complex, the Mediator complex, JUN, HOXC6, and GATA2. Taken together, our findings suggest an expanded role for TNIP2, establishing a link between TNIP2, cellular transport machinery, and RNA transcript processing.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , FN-kappa B/metabolismo , Mapeo de Interacción de Proteínas/métodos , Análisis de Secuencia de ARN/métodos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Células HEK293 , Células HeLa , Humanos , Espectrometría de Masas/métodos , Mutación , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The Scc2-Scc4 complex is essential for loading the cohesin complex onto DNA. Cohesin has important roles in chromosome segregation, DSB repair, and chromosome condensation. Here we report that Scc2 is important for gene expression in budding yeast. Scc2 and the transcriptional regulator Paf1 collaborate to promote the production of Box H/ACA snoRNAs which guide pseudouridylation of RNAs including ribosomal RNA. Mutation of SCC2 was associated with defects in the production of ribosomal RNA, ribosome assembly, and splicing. While the scc2 mutant does not have a general defect in protein synthesis, it shows increased frameshifting and reduced cap-independent translation. These findings suggest Scc2 normally promotes a gene expression program that supports translational fidelity. We hypothesize that translational dysfunction may contribute to the human disorder Cornelia de Lange syndrome, which is caused by mutations in NIPBL, the human ortholog of SCC2.
Asunto(s)
Proteínas Cromosómicas no Histona/biosíntesis , Síndrome de Cornelia de Lange/genética , Biosíntesis de Proteínas , Proteínas/genética , ARN Largo no Codificante/biosíntesis , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Síndrome de Cornelia de Lange/patología , Regulación Fúngica de la Expresión Génica , Humanos , Empalme del ARN/genética , ARN Largo no Codificante/genética , ARN Ribosómico/biosíntesis , ARN Ribosómico/genética , ARN Nucleolar Pequeño/biosíntesis , ARN Nucleolar Pequeño/genética , Ribosomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , CohesinasRESUMEN
Histone deacetylases (HDACs) are targets for cancer therapy. Suberoylanilide hydroxamic acid (SAHA) is an HDAC inhibitor approved by the U.S. Food and Drug Administration for the treatment of cutaneous T-cell lymphoma. To obtain a better mechanistic understanding of the Sin3/HDAC complex in cancer, we extended its protein-protein interaction network and identified a mutually exclusive pair within the complex. We then assessed the effects of SAHA on the disruption of the complex network through six homologous baits. SAHA perturbs multiple protein interactions and therefore compromises the composition of large parts of the Sin3/HDAC network. A comparison of the effect of SAHA treatment on gene expression in breast cancer cells to a knockdown of the ING2 subunit indicated that a portion of the anticancer effects of SAHA may be attributed to the disruption of ING2's association with the complex. Our dynamic protein interaction network resource provides novel insights into the molecular mechanism of SAHA action and demonstrates the potential for drugs to rewire networks.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Proteínas de Homeodominio/genética , Ácidos Hidroxámicos/farmacología , Mapas de Interacción de Proteínas , Receptores Citoplasmáticos y Nucleares/genética , Complejo Correpresor Histona Desacetilasa y Sin3/metabolismo , Proteínas Supresoras de Tumor/genética , Línea Celular Tumoral , Femenino , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Unión Proteica , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , VorinostatRESUMEN
Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of cohesin in gene expression is not well understood. We used budding yeast strains bearing mutations analogous to the human cohesinopathy disease alleles under control of their native promoter to study gene expression. These mutations do not significantly affect chromosome segregation. Transcriptional profiling reveals that many targets of the transcriptional activator Gcn4 are induced in the eco1-W216G mutant background. The upregulation of Gcn4 was observed in many cohesin mutants, and this observation suggested protein translation was reduced. We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and (35)S-methionine incorporation, all of which indicate a deficit in protein translation. Metabolic labeling shows that the eco1-W216G and smc1-Q843Δ mutants produce less ribosomal RNA, which is expected to constrain ribosome biogenesis. Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged. Similar defects in rRNA production and protein translation are observed in a human Roberts syndrome cell line. In addition, cohesion is defective specifically at the rDNA locus in the eco1-W216G mutant, as has been previously reported for Roberts syndrome. Collectively, our data suggest that cohesin proteins normally facilitate production of ribosomal RNA and protein translation, and this is one way they can influence gene expression. Reduced translational capacity could contribute to the human cohesinopathies.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Anomalías Craneofaciales , Ectromelia , Hipertelorismo , Biosíntesis de Proteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Acetiltransferasas/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/metabolismo , Ectromelia/genética , Ectromelia/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Hipertelorismo/genética , Hipertelorismo/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Polirribosomas/genética , ARN Ribosómico/biosíntesis , ARN Ribosómico/genética , Ribosomas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , CohesinasRESUMEN
A significant challenge in biology is to functionally annotate novel and uncharacterized proteins. Several approaches are available for deducing the function of proteins in silico based upon sequence homology and physical or genetic interaction, yet this approach is limited to proteins with well-characterized domains, paralogs and/or orthologs in other species, as well as on the availability of suitable large-scale data sets. Here, we present a quantitative proteomics approach extending the protein network of core histones H2A, H2B, H3, and H4 in Saccharomyces cerevisiae, among which a novel associated protein, the previously uncharacterized Ydl156w, was identified. In order to predict the role of Ydl156w, we designed and applied integrative bioinformatics, quantitative proteomics and biochemistry approaches aiming to infer its function. Reciprocal analysis of Ydl156w protein interactions demonstrated a strong association with all four histones and also to proteins strongly associated with histones including Rim1, Rfa2 and 3, Yku70, and Yku80. Through a subsequent combination of the focused quantitative proteomics experiments with available large-scale genetic interaction data and Gene Ontology functional associations, we provided sufficient evidence to associate Ydl156w with multiple processes including chromatin remodeling, transcription and DNA repair/replication. To gain deeper insights into the role of Ydl156w in histone biology we investigated the effect of the genetic deletion of ydl156w on H4 associated proteins, which lead to a dramatic decrease in the association of H4 with RNA polymerase III proteins. The implication of a role for Ydl156w in RNA Polymerase III mediated transcription was consequently verified by RNA-Seq experiments. Finally, using these approaches we generated a refined network of Ydl156w-associated proteins.
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Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Proteómica/métodos , ARN Polimerasa III/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al ADN/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
Piwi-interacting RNAs (piRNAs) are important for repressing transposable elements (TEs) and modulating gene expression in germ cells, thereby maintaining genome stability and germ cell function. Although they are also important for maintaining germline stem cells (GSCs) in the Drosophila ovary by repressing TEs and preventing DNA damage, piRNA expression has not been investigated in GSCs or their early progeny. Here, we show that the canonical piRNA clusters are more active in GSCs and their early progeny than late germ cells and also identify more than 3,000 new piRNA clusters from deep sequencing data. The increase in piRNAs in GSCs and early progeny can be attributed to both canonical and newly identified piRNA clusters. As expected, piRNA clusters in GSCs, but not those in somatic support cells (SCs), exhibit ping-pong signatures. Surprisingly, GSCs and early progeny express more TE transcripts than late germ cells, suggesting that the increase in piRNA levels may be related to the higher levels of TE transcripts in GSCs and early progeny. GSCs also have higher piRNA levels and lower TE levels than SCs. Furthermore, the 3' UTRs of 171 mRNA transcripts may produce sense, antisense, or dual-stranded piRNAs. Finally, we show that alternative promoter usage and splicing are frequently used to modulate gene function in GSCs and SCs. Overall, this study has provided important insight into piRNA production and TE repression in GSCs and SCs. The rich information provided by this study will be a beneficial resource to the fields of piRNA biology and germ cell development.
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Elementos Transponibles de ADN , Drosophila/genética , Perfilación de la Expresión Génica/métodos , ARN Interferente Pequeño/genética , Regiones no Traducidas 3' , Animales , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Ovario/química , Ovario/citología , ARN Mensajero/genética , ARN de Transferencia de Leucina , Células Madre/química , Células Madre/citologíaRESUMEN
Loss of sensory hair cells leads to deafness and balance deficiencies. In contrast to mammalian hair cells, zebrafish ear and lateral line hair cells regenerate from poorly characterized support cells. Equally ill-defined is the gene regulatory network underlying the progression of support cells to differentiated hair cells. scRNA-Seq of lateral line organs uncovered five different support cell types, including quiescent and activated stem cells. Ordering of support cells along a developmental trajectory identified self-renewing cells and genes required for hair cell differentiation. scRNA-Seq analyses of fgf3 mutants, in which hair cell regeneration is increased, demonstrates that Fgf and Notch signaling inhibit proliferation of support cells in parallel by inhibiting Wnt signaling. Our scRNA-Seq analyses set the foundation for mechanistic studies of sensory organ regeneration and is crucial for identifying factors to trigger hair cell production in mammals. The data is searchable and publicly accessible via a web-based interface.
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Proliferación Celular , Factores de Crecimiento de Fibroblastos/metabolismo , Células Ciliadas Auditivas/citología , ARN Citoplasmático Pequeño/genética , Receptores Notch/metabolismo , Transducción de Señal , Células Madre/metabolismo , Animales , Pez CebraRESUMEN
In the developing brain, heightened plasticity during the critical period enables the proper formation of neural circuits. Here, we identify the "navigator" neurons, a group of perinatally born olfactory sensory neurons, as playing an essential role in establishing the olfactory map during the critical period. The navigator axons project circuitously in the olfactory bulb and traverse multiple glomeruli before terminating in perspective glomeruli. These neurons undergo a phase of exuberant axon growth and exhibit a shortened lifespan. Single-cell transcriptome analyses reveal distinct molecular signatures for the navigators. Extending their lifespan prolongs the period of exuberant growth and perturbs axon convergence. Conversely, a genetic ablation experiment indicates that, despite postnatal neurogenesis, only the navigators are endowed with the ability to establish a convergent map. The presence and the proper removal of the navigator neurons are both required to establish tight axon convergence into the glomeruli.
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Axones/fisiología , Bulbo Olfatorio/crecimiento & desarrollo , Neuronas Receptoras Olfatorias/fisiología , Animales , Femenino , Células HEK293 , Humanos , Masculino , Ratones Transgénicos , Neurogénesis , Bulbo Olfatorio/metabolismo , Vías Olfatorias/crecimiento & desarrollo , Vías Olfatorias/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , TranscriptomaRESUMEN
Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m6A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m6A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m6A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knocking down one of YTHDF2's key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.
Asunto(s)
Adenosina/análogos & derivados , Autorrenovación de las Células , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Adenosina/metabolismo , Animales , Células Madre Hematopoyéticas/patología , Ratones , Ratones NoqueadosRESUMEN
Hox genes modulate the properties of hematopoietic stem cells (HSCs) and reacquired Hox expression in progenitors contributes to leukemogenesis. Here, our transcriptome and DNA methylome analyses revealed that Hoxb cluster and retinoid signaling genes are predominantly enriched in LT-HSCs, and this coordinate regulation of Hoxb expression is mediated by a retinoid-dependent cis-regulatory element, distal element RARE (DERARE). Deletion of the DERARE reduced Hoxb expression, resulting in changes to many downstream signaling pathways (e.g., non-canonical Wnt signaling) and loss of HSC self-renewal and reconstitution capacity. DNA methyltransferases mediate DNA methylation on the DERARE, leading to reduced Hoxb cluster expression. Acute myeloid leukemia patients with DNMT3A mutations exhibit DERARE hypomethylation, elevated HOXB expression, and adverse outcomes. CRISPR-Cas9-mediated specific DNA methylation at DERARE attenuated HOXB expression and alleviated leukemogenesis. Collectively, these findings demonstrate pivotal roles for retinoid signaling and the DERARE in maintaining HSCs and preventing leukemogenesis by coordinate regulation of Hoxb genes.
Asunto(s)
Epigénesis Genética/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Proteínas de Homeodominio/antagonistas & inhibidores , Retinoides/farmacología , Animales , Elementos de Facilitación Genéticos/efectos de los fármacos , Elementos de Facilitación Genéticos/genética , Epigénesis Genética/genética , Células HEK293 , Hematopoyesis/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Retinoides/químicaRESUMEN
In the initial published version of this article, there was an inadvertent omission from the Acknowledgements that this work was supported by Stowers Institute for Medical Research (SIMR-1004) and NIH National Cancer Institute grant to University of Kansas Cancer Center (P30 CA168524). This omission does not affect the description of the results or the conclusions of this work.
RESUMEN
Piwi family protein Aubergine (Aub) maintains genome integrity in late germ cells of the Drosophila ovary through Piwi-associated RNA-mediated repression of transposon activities. Although it is highly expressed in germline stem cells (GSCs) and early progeny, it remains unclear whether it plays any roles in early GSC lineage development. Here we report that Aub promotes GSC self-renewal and GSC progeny differentiation. RNA-iCLIP results show that Aub binds the mRNAs encoding self-renewal and differentiation factors in cultured GSCs. Aub controls GSC self-renewal by preventing DNA-damage-induced Chk2 activation and by translationally controlling the expression of self-renewal factors. It promotes GSC progeny differentiation by translationally controlling the expression of differentiation factors, including Bam. Therefore, this study reveals a function of Aub in GSCs and their progeny, which promotes translation of self-renewal and differentiation factors by directly binding to its target mRNAs and interacting with translational initiation factors.
Asunto(s)
División Celular Asimétrica/fisiología , Diferenciación Celular/fisiología , Autorrenovación de las Células/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Células Madre/citología , Animales , Quinasa de Punto de Control 2/metabolismo , Drosophila melanogaster/citología , Femenino , Ovario/citologíaRESUMEN
Aneuploidy and epigenetic alterations have long been associated with carcinogenesis, but it was unknown whether aneuploidy could disrupt the epigenetic states required for cellular differentiation. In this study, we found that ~3% of random aneuploid karyotypes in yeast disrupt the stable inheritance of silenced chromatin during cell proliferation. Karyotype analysis revealed that this phenotype was significantly correlated with gains of chromosomes III and X. Chromosome X disomy alone was sufficient to disrupt chromatin silencing and yeast mating-type identity as indicated by a lack of growth response to pheromone. The silencing defect was not limited to cryptic mating type loci and was associated with broad changes in histone modifications and chromatin localization of Sir2 histone deacetylase. The chromatin-silencing defect of disome X can be partially recapitulated by an extra copy of several genes on chromosome X. These results suggest that aneuploidy can directly cause epigenetic instability and disrupt cellular differentiation.
Asunto(s)
Aneuploidia , Cromatina/metabolismo , Epigénesis Genética , Regulación Fúngica de la Expresión Génica , Genes del Tipo Sexual de los Hongos , Saccharomycetales/crecimiento & desarrollo , Saccharomycetales/genética , Ciclo CelularRESUMEN
This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome analysis. Vendor-specific protocols used for RNA library construction often require at least 100 ng total RNA. However, under certain conditions, much less RNA is available for library construction. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several commercial RNA amplification kits are available for amplification prior to library construction for next-generation sequencing, but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-seq for picogram amounts of RNA. To address this, 4 types of amplification kits were tested with 3 different concentrations, from 5 ng to 50 pg, of a commercially available RNA. Kits were tested at multiple sites to assess reproducibility and ease of use. The human total reference RNA used was spiked with a control pool of RNA molecules in order to further evaluate quantitative recovery of input material. Additional control data sets were generated from libraries constructed following polyA selection or ribosomal depletion using established kits and protocols. cDNA was collected from the different sites, and libraries were synthesized at a single site using established protocols. Sequencing runs were carried out on the Illumina platform. Numerous metrics were compared among the kits and dilutions used. Overall, no single kit appeared to meet all the challenges of small input material. However, it is encouraging that excellent data can be recovered with even the 50 pg input total RNA.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/normas , Análisis de Secuencia de ARN/normas , Animales , Secuencia de Bases , ADN Complementario/genética , Humanos , Límite de Detección , Ratones , Poliadenilación , ARN/genética , Ratas , Estándares de ReferenciaRESUMEN
The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary.