RESUMEN
Genetics have nominated many schizophrenia risk genes and identified convergent signals between schizophrenia and neurodevelopmental disorders. However, functional interpretation of the nominated genes in the relevant brain cell types is often lacking. We executed interaction proteomics for six schizophrenia risk genes that have also been implicated in neurodevelopment in human induced cortical neurons. The resulting protein network is enriched for common variant risk of schizophrenia in Europeans and East Asians, is down-regulated in layer 5/6 cortical neurons of individuals affected by schizophrenia, and can complement fine-mapping and eQTL data to prioritize additional genes in GWAS loci. A sub-network centered on HCN1 is enriched for common variant risk and contains proteins (HCN4 and AKAP11) enriched for rare protein-truncating mutations in individuals with schizophrenia and bipolar disorder. Our findings showcase brain cell-type-specific interactomes as an organizing framework to facilitate interpretation of genetic and transcriptomic data in schizophrenia and its related disorders.
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Autism spectrum disorders (ASDs) have been linked to genes with enriched expression in the brain, but it is unclear how these genes converge into cell-type-specific networks. We built a protein-protein interaction network for 13 ASD-associated genes in human excitatory neurons derived from induced pluripotent stem cells (iPSCs). The network contains newly reported interactions and is enriched for genetic and transcriptional perturbations observed in individuals with ASDs. We leveraged the network data to show that the ASD-linked brain-specific isoform of ANK2 is important for its interactions with synaptic proteins and to characterize a PTEN-AKAP8L interaction that influences neuronal growth. The IGF2BP1-3 complex emerged as a convergent point in the network that may regulate a transcriptional circuit of ASD-associated genes. Our findings showcase cell-type-specific interactomes as a framework to complement genetic and transcriptomic data and illustrate how both individual and convergent interactions can lead to biological insights into ASDs.
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Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron degeneration accompanied by aberrant accumulation and loss of function of the RNA-binding protein TDP43. Thus far, it remains unresolved to what extent TDP43 loss of function directly contributes to motor system dysfunction. Here, we employed gene editing to find whether the mouse ortholog of the TDP43-regulated gene STMN2 has an important function in maintaining the motor system. Both mosaic founders and homozygous loss-of-function Stmn2 mice exhibited neuromuscular junction denervation and fragmentation, resulting in muscle atrophy and impaired motor behavior, accompanied by an imbalance in neuronal microtubule dynamics in the spinal cord. The introduction of human STMN2 through BAC transgenesis was sufficient to rescue the motor phenotypes observed in Stmn2 mutant mice. Collectively, our results demonstrate that disrupting the ortholog of a single TDP43-regulated RNA is sufficient to cause substantial motor dysfunction, indicating that disruption of TDP43 function is likely a contributor to ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Estatmina , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Modelos Animales de Enfermedad , Homocigoto , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Unión Neuromuscular/metabolismo , Estatmina/genética , Estatmina/metabolismoRESUMEN
Gene networks have yielded numerous neurobiological insights, yet an integrated view across brain regions is lacking. We leverage RNA sequencing in 864 samples representing 12 brain regions to robustly identify 12 brain-wide, 50 cross-regional and 114 region-specific coexpression modules. Nearly 40% of genes fall into brain-wide modules, while 25% comprise region-specific modules reflecting regional biology, such as oxytocin signaling in the hypothalamus, or addiction pathways in the nucleus accumbens. Schizophrenia and autism genetic risk are enriched in brain-wide and multiregional modules, indicative of broad impact; these modules implicate neuronal proliferation and activity-dependent processes, including endocytosis and splicing, in disease pathophysiology. We find that cell-type-specific long noncoding RNA and gene isoforms contribute substantially to regional synaptic diversity and that constrained, mutation-intolerant genes are primarily enriched in neurons. We leverage these data using an omnigenic-inspired network framework to characterize how coexpression and gene regulatory networks reflect neuropsychiatric disease risk, supporting polygenic models.
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Encéfalo/fisiopatología , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/fisiología , Predisposición Genética a la Enfermedad/genética , Trastornos Mentales/genética , Humanos , Trastornos Mentales/fisiopatología , TranscriptomaRESUMEN
RNA N 6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed cotranscriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here, we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells. We find that around 10% of m6A peaks are located in alternative introns/exons, often close to 5' splice sites. m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5' splice sites that are proximal to m6A peaks. For terminal or variable-length exons, m6A peaks are generally located on or immediately downstream from a 5' splice site that is suppressed in the presence of m6A and upstream of a 5' splice site that is promoted in the presence of m6A. Genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Collectively, our findings demonstrate crosstalk between the m6A machinery and the regulation of RNA splicing.
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Exones , Intrones , Empalme del ARN , Transcriptoma , Empalme Alternativo , Animales , Exones/genética , Intrones/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Sitios de Empalme de ARNRESUMEN
Combining genetic and cell-type-specific proteomic datasets can generate biological insights and therapeutic hypotheses, but a technical and statistical framework for such analyses is lacking. Here, we present an open-source computational tool called Genoppi (lagelab.org/genoppi) that enables robust, standardized, and intuitive integration of quantitative proteomic results with genetic data. We use Genoppi to analyze 16 cell-type-specific protein interaction datasets of four proteins (BCL2, TDP-43, MDM2, PTEN) involved in cancer and neurological disease. Through systematic quality control of the data and integration with published protein interactions, we show a general pattern of both cell-type-independent and cell-type-specific interactions across three cancer cell types and one human iPSC-derived neuronal cell type. Furthermore, through the integration of proteomic and genetic datasets in Genoppi, our results suggest that the neuron-specific interactions of these proteins are mediating their genetic involvement in neurodegenerative diseases. Importantly, our analyses suggest that human iPSC-derived neurons are a relevant model system for studying the involvement of BCL2 and TDP-43 in amyotrophic lateral sclerosis.
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Biología Computacional/métodos , Estudio de Asociación del Genoma Completo/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neuronas/metabolismo , Programas Informáticos , Línea Celular Tumoral , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genómica , Humanos , Mutación , Polimorfismo de Nucleótido Simple , Unión Proteica , Proteómica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Espectrometría de Masas en TándemRESUMEN
Transactive response DNA-binding protein 43 kDa (TDP-43), a multifunctional nucleic acid-binding protein, is a primary component of insoluble aggregates associated with several devastating nervous system disorders; mutations in TARDBP, its encoding gene, are a cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we review established and emerging roles of TDP-43 and consider how its dysfunction impinges on RNA homeostasis in the nervous system, thereby contributing to neural degeneration. Notably, improper splicing of the axonal growth-associated factor STMN2 has recently been connected to TDP-43 dysfunction, providing a mechanistic link between TDP-43 proteinopathies and neuropathy. This review highlights how a deep understanding of the function of TDP-43 in the brain might be leveraged to develop new targeted therapies for several neurological disorders.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Esclerosis Amiotrófica Lateral/genética , Axones , Proteínas de Unión al ADN/genética , Humanos , MutaciónRESUMEN
Autism spectrum disorder (ASD) comprises a group of neurodevelopmental disorders characterized by impaired social interactions as well as the presentation of restrictive and repetitive behaviors. ASD is highly heritable but genetically heterogenous with both common and rare genetic variants collaborating to predispose individuals to the disorder. In this review, we synthesize recent efforts to develop human induced pluripotent stem cell (iPSC)-derived models of ASD-related phenotypes. We firstly address concerns regarding the relevance and validity of available neuronal iPSC-derived models. We then critically evaluate the robustness of various differentiation and cell culture protocols used for producing cell types of relevance to ASD. By exploring iPSC models of ASD reported thus far, we examine to what extent cellular and neuronal phenotypes with potential relevance to ASD can be linked to genetic variants found to underlie it. Lastly, we outline promising strategies by which iPSC technology can both enhance the power of genetic studies to identify ASD risk factors and nominate pathways that are disrupted across groups of ASD patients that might serve as common points for therapeutic intervention.
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Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Células Madre Pluripotentes Inducidas/metabolismo , Trastorno del Espectro Autista/diagnóstico , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/terapia , Biomarcadores , Técnicas de Cultivo de Célula , Estudios de Asociación Genética/métodos , Humanos , Células Madre Pluripotentes Inducidas/citología , Terapia Molecular Dirigida , Herencia Multifactorial , FenotipoRESUMEN
Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome-wide silencing. Whilst recent studies have defined candidate silencing factors, their relative contribution to repressing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in mouse embryonic stem cell-based models. Using a machine learning approach we identify distance to the Xist locus and prior gene expression levels as key determinants of silencing efficiency. We go on to show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of weakly expressed genes. Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin. LBR and the Rbm15/m6A-methyltransferase complex make only minor contributions to gene silencing. Together our results provide a comprehensive model for Xist-mediated chromosome silencing.
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ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Inactivación del Cromosoma X , Cromosoma X/genética , Animales , Línea Celular , Proteínas de Unión al ADN , Técnicas de Inactivación de Genes , Silenciador del Gen , Histonas/genética , Ratones , Células Madre Embrionarias de Ratones , Proteínas del Grupo Polycomb/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), A/B compartments and formation of two mega-domains. Here we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 mutant cells reveals the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including A/B compartments and partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred following SmcHD1 knockout in a somatic cell model, but in this case, independent of Xi gene derepression. We conclude that SmcHD1 is a key factor in defining the unique chromosome architecture of Xi.
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Proteínas Cromosómicas no Histona/genética , Metilación de ADN/genética , Activación Transcripcional/genética , Inactivación del Cromosoma X , Alelos , Animales , Sistemas CRISPR-Cas , Línea Celular , Proteínas Cromosómicas no Histona/metabolismo , Islas de CpG , Exones/genética , Femenino , Fibroblastos , Técnicas de Inactivación de Genes , Histonas/genética , Histonas/metabolismo , Masculino , Ratones , Mutación Puntual , Proteínas del Grupo Polycomb/metabolismoRESUMEN
We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist-HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation.
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Proteínas Cromosómicas no Histona/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Complejo Represivo Polycomb 1/metabolismo , ARN Largo no Codificante/metabolismo , Inactivación del Cromosoma X/genética , Animales , Secuencia de Bases , Diferenciación Celular , Femenino , Genoma , Histonas/metabolismo , Lisina/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Oligonucleótidos/metabolismo , Transporte de ProteínasRESUMEN
The Polycomb-repressive complexes PRC1 and PRC2 play a key role in chromosome silencing induced by the non-coding RNA Xist. Polycomb recruitment is initiated by the PCGF3/5-PRC1 complex, which catalyzes chromosome-wide H2A lysine 119 ubiquitylation, signaling recruitment of other PRC1 complexes, and PRC2. However, the molecular mechanism for PCGF3/5-PRC1 recruitment by Xist RNA is not understood. Here we define the Xist RNA Polycomb Interaction Domain (XR-PID), a 600 nt sequence encompassing the Xist B-repeat element. Deletion of XR-PID abolishes Xist-dependent Polycomb recruitment, in turn abrogating Xist-mediated gene silencing and reversing Xist-induced chromatin inaccessibility. We identify the RNA-binding protein hnRNPK as the principal XR-PID binding factor required to recruit PCGF3/5-PRC1. Accordingly, synthetically tethering hnRNPK to Xist RNA lacking XR-PID is sufficient for Xist-dependent Polycomb recruitment. Our findings define a key pathway for Polycomb recruitment by Xist RNA, providing important insights into mechanisms of chromatin modification by non-coding RNA.
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Células Madre Embrionarias/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/metabolismo , ARN Largo no Codificante/metabolismo , Ribonucleoproteínas/metabolismo , Inactivación del Cromosoma X , Cromosoma X/metabolismo , Animales , Sitios de Unión , Línea Celular , Ribonucleoproteína Heterogénea-Nuclear Grupo K , Histonas/metabolismo , Lisina/metabolismo , Ratones , Complejo Represivo Polycomb 1/genética , Proteínas del Grupo Polycomb/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , ARN Largo no Codificante/genética , Ribonucleoproteínas/genética , Transcripción Genética , Transfección , Ubiquitinación , Cromosoma X/genéticaRESUMEN
Recruitment of the Polycomb repressive complexes PRC1 and PRC2 by Xist RNA is an important paradigm for chromatin regulation by long noncoding RNAs. Here, we show that the noncanonical Polycomb group RING finger 3/5 (PCGF3/5)-PRC1 complex initiates recruitment of both PRC1 and PRC2 in response to Xist RNA expression. PCGF3/5-PRC1-mediated ubiquitylation of histone H2A signals recruitment of other noncanonical PRC1 complexes and of PRC2, the latter leading to deposition of histone H3 lysine 27 methylation chromosome-wide. Pcgf3/5 gene knockout results in female-specific embryo lethality and abrogates Xist-mediated gene repression, highlighting a key role for Polycomb in Xist-dependent chromosome silencing. Our findings overturn existing models for Polycomb recruitment by Xist RNA and establish precedence for H2AK119u1 in initiating Polycomb domain formation in a physiological context.
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Células Madre Embrionarias/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Inactivación del Cromosoma X , Animales , Femenino , Ratones , Proteínas del Grupo Polycomb/genética , ARN Largo no Codificante/metabolismoRESUMEN
The nuclear matrix protein Cip1-interacting zinc finger protein 1 (CIZ1) promotes DNA replication in association with cyclins and has been linked to adult and pediatric cancers. Here we show that CIZ1 is highly enriched on the inactive X chromosome (Xi) in mouse and human female cells and is retained by interaction with the RNA-dependent nuclear matrix. CIZ1 is recruited to Xi in response to expression of X inactive-specific transcript (Xist) RNA during the earliest stages of X inactivation in embryonic stem cells and is dependent on the C-terminal nuclear matrix anchor domain of CIZ1 and the E repeats of Xist CIZ1-null mice, although viable, display fully penetrant female-specific lymphoproliferative disorder. Interestingly, in mouse embryonic fibroblast cells derived from CIZ1-null embryos, Xist RNA localization is disrupted, being highly dispersed through the nucleoplasm rather than focal. Focal localization is reinstated following re-expression of CIZ1. Focal localization of Xist RNA is also disrupted in activated B and T cells isolated from CIZ1-null animals, suggesting a possible explanation for female-specific lymphoproliferative disorder. Together, these findings suggest that CIZ1 has an essential role in anchoring Xist to the nuclear matrix in specific somatic lineages.
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Regulación de la Expresión Génica , Trastornos Linfoproliferativos/patología , Proteínas Nucleares/fisiología , ARN Largo no Codificante/metabolismo , Inactivación del Cromosoma X , Cromosoma X/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/patología , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , ARN Largo no Codificante/genética , Caracteres Sexuales , Cromosoma X/genéticaRESUMEN
Recent experimental evidence indicates that lncRNAs can act as regulatory molecules in the context of development and disease. Xist, the master regulator of X chromosome inactivation, is a classic example of how lncRNAs can exert multi-layered and fine-tuned regulatory functions, by acting as a molecular scaffold for recruitment of distinct protein factors. In this review, we discuss the methodologies employed to define Xist RNA structures and the tight interplay between structural clues and functionality of lncRNAs. This model of modular function dictated by structure, can be also generalized to other lncRNAs, beyond the field of X chromosome inactivation, to explain common features of similarly folded RNAs.
RESUMEN
In female mammals, one of the two X chromosomes in each cell is transcriptionally silenced in order to achieve dosage compensation between the genders in a process called X chromosome inactivation. The master regulator of this process is the long non-coding RNA Xist. During X-inactivation, Xist accumulates in cis on the future inactive X chromosome, triggering a cascade of events that provoke the stable silencing of the entire chromosome, with relatively few genes remaining active. How Xist spreads, what are its binding sites, how it recruits silencing factors and how it induces a specific topological and nuclear organization of the chromatin all remain largely unanswered questions. Recent studies have improved our understanding of Xist localization and the proteins with which it interacts, allowing a reappraisal of ideas about Xist function. We discuss recent advances in our knowledge of Xist-mediated silencing, focusing on Xist spreading, the nuclear organization of the inactive X chromosome, recruitment of the polycomb complex and the role of the nuclear matrix in the process of X chromosome inactivation.
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ARN Largo no Codificante/metabolismo , Inactivación del Cromosoma X , Animales , Núcleo Celular/genética , Femenino , Silenciador del Gen , Ratones , Proteínas del Grupo Polycomb/metabolismo , ARN Largo no Codificante/análisisRESUMEN
X chromosome inactivation (XCI) is the dosage compensation mechanism that evolved in female mammals to correct the genetic imbalance of X-linked genes between sexes. X chromosome inactivation occurs in early development when one of the two X chromosomes of females is nearly-completely silenced. Differentiating Embryonic Stem cells (ESC) are regarded as a useful tool to study XCI, since they recapitulate many events occurring during early development. In this review we aim to summarise the advances in the field and to discuss the close connection between cell differentiation and X chromosome inactivation, with a particular focus on mouse ESCs.
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Diferenciación Celular/genética , Células Madre Embrionarias de Ratones/citología , Inactivación del Cromosoma X/genética , Cromosoma X/genética , Animales , Humanos , RatonesRESUMEN
X-chromosome inactivation is the process that evolved in mammals to equalize levels of X-linked gene expression in XX females relative to XY males. Silencing of a single X chromosome in female cells is mediated by the non-coding RNA Xist. Although progress has been made toward identifying factors that function in the maintenance of X inactivation, the primary silencing factors are largely undefined. We developed an shRNA screening strategy to produce a ranked list of candidate primary silencing factors. Validation experiments performed on several of the top hits identified the SPOC domain RNA binding proteins Rbm15 and Spen and Wtap, a component of the m6A RNA methyltransferase complex, as playing an important role in the establishment of Xist-mediated silencing. Localization analysis using super-resolution 3D-SIM microscopy demonstrates that these factors co-localize with Xist RNA within the nuclear matrix subcompartment, consistent with a direct interaction.
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Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Silenciador del Gen , Proteínas Nucleares/metabolismo , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/metabolismo , Transporte Activo de Núcleo Celular , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Ratones , Proteínas Nucleares/química , Proteínas Nucleares/genética , Unión Proteica , Estructura Terciaria de Proteína , Factores de Empalme de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
Transgenerational effects have wide-ranging implications for human health, biological adaptation, and evolution; however, their mechanisms and biology remain poorly understood. Here, we demonstrate that a germline nuclear small RNA/chromatin pathway can maintain stable inheritance for many generations when triggered by a piRNA-dependent foreign RNA response in C. elegans. Using forward genetic screens and candidate approaches, we find that a core set of nuclear RNAi and chromatin factors is required for multigenerational inheritance of environmental RNAi and piRNA silencing. These include a germline-specific nuclear Argonaute HRDE1/WAGO-9, a HP1 ortholog HPL-2, and two putative histone methyltransferases, SET-25 and SET-32. piRNAs can trigger highly stable long-term silencing lasting at least 20 generations. Once established, this long-term memory becomes independent of the piRNA trigger but remains dependent on the nuclear RNAi/chromatin pathway. Our data present a multigenerational epigenetic inheritance mechanism induced by piRNAs.