RESUMEN
Chromatin modifications are linked with regulating patterns of gene expression, but their causal role and context-dependent impact on transcription remains unresolved. Here we develop a modular epigenome editing platform that programs nine key chromatin modifications, or combinations thereof, to precise loci in living cells. We couple this with single-cell readouts to systematically quantitate the magnitude and heterogeneity of transcriptional responses elicited by each specific chromatin modification. Among these, we show that installing histone H3 lysine 4 trimethylation (H3K4me3) at promoters can causally instruct transcription by hierarchically remodeling the chromatin landscape. We further dissect how DNA sequence motifs influence the transcriptional impact of chromatin marks, identifying switch-like and attenuative effects within distinct cis contexts. Finally, we examine the interplay of combinatorial modifications, revealing that co-targeted H3K27 trimethylation (H3K27me3) and H2AK119 monoubiquitination (H2AK119ub) maximizes silencing penetrance across single cells. Our precision-perturbation strategy unveils the causal principles of how chromatin modification(s) influence transcription and dissects how quantitative responses are calibrated by contextual interactions.
Asunto(s)
Cromatina , Epigenoma , Edición Génica , Histonas , Cromatina/metabolismo , Cromatina/genética , Histonas/metabolismo , Histonas/genética , Humanos , Edición Génica/métodos , Epigénesis Genética , Regiones Promotoras Genéticas , Ensamble y Desensamble de Cromatina , Ubiquitinación , Código de Histonas , Transcripción Genética , Análisis de la Célula Individual/métodosRESUMEN
Microsatellite expansions of CCTG repeats in the cellular nucleic acid-binding protein (CNBP) gene leads to accumulation of toxic RNA and have been associated with myotonic dystrophy type 2 (DM2). However, it is still unclear whether the dystrophic phenotype is also linked to CNBP decrease, a conserved CCHC-type zinc finger RNA-binding protein that regulates translation and is required for mammalian development. Here, we show that depletion of Drosophila CNBP in muscles causes ageing-dependent locomotor defects that are correlated with impaired polyamine metabolism. We demonstrate that the levels of ornithine decarboxylase (ODC) and polyamines are significantly reduced upon dCNBP depletion. Of note, we show a reduction of the CNBP-polyamine axis in muscles from DM2 patients. Mechanistically, we provide evidence that dCNBP controls polyamine metabolism through binding dOdc mRNA and regulating its translation. Remarkably, the locomotor defect of dCNBP-deficient flies is rescued by either polyamine supplementation or dOdc1 overexpression. We suggest that this dCNBP function is evolutionarily conserved in vertebrates with relevant implications for CNBP-related pathophysiological conditions.
Asunto(s)
Proteínas de Drosophila/metabolismo , Actividad Motora/genética , Actividad Motora/fisiología , Poliaminas/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular , Regulación hacia Abajo/fisiología , Proteínas de Drosophila/genética , Drosophila melanogaster , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Músculo Esquelético/metabolismo , Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , Biosíntesis de Proteínas , Putrescina/farmacología , Interferencia de ARN , Proteínas de Unión al ARN/genética , Espermidina/farmacologíaRESUMEN
The nuclear pore complex (NPC) is the principal gateway between nucleus and cytoplasm that enables exchange of macromolecular cargo. Composed of multiple copies of ~30 different nucleoporins (Nups), the NPC acts as a selective portal, interacting with factors which individually license passage of specific cargo classes. Here we show that two Nups of the inner channel, Nup54 and Nup58, are essential for transposon silencing via the PIWI-interacting RNA (piRNA) pathway in the Drosophila ovary. In ovarian follicle cells, loss of Nup54 and Nup58 results in compromised piRNA biogenesis exclusively from the flamenco locus, whereas knockdowns of other NPC subunits have widespread consequences. This provides evidence that some Nups can acquire specialised roles in tissue-specific contexts. Our findings consolidate the idea that the NPC has functions beyond simply constituting a barrier to nuclear/cytoplasmic exchange as genomic loci subjected to strong selective pressure can exploit NPC subunits to facilitate their expression.
Transposons are genetic sequences, which, when active, can move around the genome and insert themselves into new locations. This can potentially disrupt the information required for cells to work properly: in reproductive organs, for example, transposon activity can lead to infertility. Many organisms therefore have cellular systems that keep transposons in check. Animal cells comprise two main compartments: the nucleus, which contains the genetic information, and the cytosol, where most chemical reactions necessary for life take place. Molecules continually move between nucleus and cytosol, much as people go in and out of a busy train station. The connecting 'doors' between the two compartments are called Nuclear Pore Complexes (NPCs), and their job is to ensure that each molecule passing through reaches its correct destination. Recent research shows that the individual proteins making up NPCs (called nucleoporins) may play other roles within the cell. In particular, genetic studies in fruit flies suggested that some nucleoporins help to control transposon activity within the ovary but how they did this was still unclear. Munafò et al. therefore set out to determine if the nucleoporins were indeed actively silencing the transposons, or if this was just a side effect of altered nuclear-cytosolic transport. Experiments using cells grown from fruit fly ovaries revealed that depleting two specific nucleoporins, Nup54 and Nup58, re-activated transposons with minimal effects on most genes or the overall health of the cells. This suggests that Nup54 and Nup58 play a direct role in transposon silencing. Further, detailed analysis of gene expression in Nup54- and Nup58-lacking cells revealed that the product of one gene, flamenco, was indeed affected. Normally, flamenco acts as a 'master switch' to turn off transposons. Without Nup54 and Nup58, the molecule encoded by flamenco could not reach its dedicated location in the cytosol, and thus could not carry out its task. These results show that, far from being mere 'doorkeepers' for the nucleus, nucleoporins play important roles adapted to individual tissues in the body. Further research will help determine if the same is true for other organisms, and if these mechanisms can help understand human diseases.
Asunto(s)
Elementos Transponibles de ADN , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Ovario/metabolismo , Interferencia de ARN , Animales , Animales Modificados Genéticamente , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Regulación de la Expresión Génica , Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ovario/citología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
In animal gonads, the PIWI-interacting RNA (piRNA) pathway guards genome integrity in part through the co-transcriptional gene silencing of transposon insertions. In Drosophila ovaries, piRNA-loaded Piwi detects nascent transposon transcripts and instructs heterochromatin formation through the Panoramix-induced co-transcriptional silencing (PICTS) complex, containing Panoramix, Nxf2 and Nxt1. Here, we report that the highly conserved dynein light chain LC8/Cut-up (Ctp) is an essential component of the PICTS complex. Loss of Ctp results in transposon de-repression and a reduction in repressive chromatin marks specifically at transposon loci. In turn, Ctp can enforce transcriptional silencing when artificially recruited to RNA and DNA reporters. We show that Ctp drives dimerisation of the PICTS complex through its interaction with conserved motifs within Panoramix. Artificial dimerisation of Panoramix bypasses the necessity for its interaction with Ctp, demonstrating that conscription of a protein from a ubiquitous cellular machinery has fulfilled a fundamental requirement for a transposon silencing complex.
Asunto(s)
Elementos Transponibles de ADN , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Dineínas/genética , Silenciador del Gen , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Dineínas/metabolismoRESUMEN
The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We found that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Precursores del ARN/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Elementos Transponibles de ADN/genética , Drosophila/genética , Proteínas de Drosophila/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Unión al ARN/genéticaRESUMEN
The PIWI-interacting RNA (piRNA) pathway is a small RNA-based immune system that controls the expression of transposons and maintains genome integrity in animal gonads. In Drosophila, piRNA-guided silencing is achieved, in part, via co-transcriptional repression of transposons by Piwi. This depends on Panoramix (Panx); however, precisely how an RNA binding event silences transcription remains to be determined. Here we show that Nuclear Export Factor 2 (Nxf2) and its co-factor, Nxt1, form a complex with Panx and are required for co-transcriptional silencing of transposons in somatic and germline cells of the ovary. Tethering of Nxf2 or Nxt1 to RNA results in silencing of target loci and the concomitant accumulation of repressive chromatin marks. Nxf2 and Panx proteins are mutually required for proper localization and stability. We mapped the protein domains crucial for the Nxf2/Panx complex formation and show that the amino-terminal portion of Panx is sufficient to induce transcriptional silencing.
Asunto(s)
Silenciador del Gen , Proteínas Nucleares/metabolismo , ARN Interferente Pequeño/metabolismo , Transcripción Genética , Transporte Activo de Núcleo Celular , Animales , Cromatina/metabolismo , Drosophila melanogaster/genética , Unión Proteica , Estabilidad ProteicaRESUMEN
Ribosomal RNA (rRNA) biogenesis is a multistep process requiring several nuclear and cytoplasmic exonucleases. The exact processing steps for mammalian 5.8S rRNA remain obscure. Here, using loss-of-function approaches in mouse embryonic stem cells (mESCs) and deep sequencing of rRNA intermediates, we investigate the requirements of exonucleases known to be involved in 5.8S maturation at nucleotide resolution and explore the role of the Perlman syndrome-associated 3'-5' exonuclease Dis3l2 in rRNA processing. We uncover a novel cytoplasmic intermediate that we name '7SB' rRNA that is generated through sequential processing by distinct exosome complexes. 7SB rRNA can be oligoadenylated by an unknown enzyme and/or oligouridylated by TUT4/7 and subsequently processed by Dis3l2 and Eri1. Moreover, exosome depletion triggers Dis3l2-mediated decay (DMD) as a surveillance pathway for rRNAs. Our data identify previously unknown 5.8S rRNA processing steps and provide nucleotide-level insight into the exonuclease requirements for mammalian rRNA processing.
Asunto(s)
Exorribonucleasas/metabolismo , ARN Ribosómico 5.8S/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/metabolismo , Macrosomía Fetal/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Transporte de ARN , Ribosomas/metabolismo , Uridina/metabolismo , Tumor de Wilms/metabolismoRESUMEN
The Piwi-interacting RNA (piRNA) pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialized machinery that converts long single-stranded precursors into small RNAs of â¼25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and the correct localization of the indispensable piRNA biogenesis factor Armitage (Armi). We found that Gasz and Daed interact with each other and likely provide a mitochondrial "anchoring platform" to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb bodies in a manner that depends on Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing; namely, how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , ARN Helicasas/metabolismo , ARN Interferente Pequeño/biosíntesis , Animales , Línea Celular , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Técnicas de Silenciamiento del Gen , Unión Proteica , Transporte de Proteínas , ARN Interferente Pequeño/metabolismoRESUMEN
PIWI-interacting RNAs (piRNAs) and their associated PIWI clade Argonaute proteins constitute the core of the piRNA pathway. In gonadal cells, this conserved pathway is crucial for genome defense, and its main function is to silence transposable elements. This is achieved through posttranscriptional and transcriptional gene silencing. Precursors that give rise to piRNAs require specialized transcription and transport machineries because piRNA biogenesis is a cytoplasmic process. The ping-pong cycle, a posttranscriptional silencing mechanism, combines the cleavage-dependent silencing of transposon RNAs with piRNA production. PIWI proteins also function in the nucleus, where they scan for nascent target transcripts with sequence complementarity, instructing transcriptional silencing and deposition of repressive chromatin marks at transposon loci. Although studies have revealed numerous factors that participate in each branch of the piRNA pathway, the precise molecular roles of these factors often remain unclear. In this review, we summarize our current understanding of the mechanisms involved in piRNA biogenesis and function.
Asunto(s)
Proteínas Argonautas/genética , Elementos Transponibles de ADN/genética , ARN Interferente Pequeño/genética , Transcripción Genética , Animales , Drosophila melanogaster/genética , Silenciador del Gen , Gónadas/crecimiento & desarrollo , ARN Interferente Pequeño/biosíntesisRESUMEN
Mutations in the 3'-5' exonuclease DIS3L2 are associated with Perlman syndrome and hypersusceptibility to Wilms tumorigenesis. Previously, we found that Dis3l2 specifically recognizes and degrades uridylated pre-let-7 microRNA. However, the widespread relevance of Dis3l2-mediated decay of uridylated substrates remains unknown. Here, we applied an unbiased RNA immunoprecipitation strategy to identify Dis3l2 targets in mouse embryonic stem cells. The disease-associated long noncoding RNA (lncRNA) Rmrp, 7SL, as well as several other Pol III-transcribed noncoding RNAs (ncRNAs) were among the most highly enriched Dis3l2-bound RNAs. 3'-Uridylated Rmrp, 7SL, and small nuclear RNA (snRNA) species were highly stabilized in the cytoplasm of Dis3l2-depleted cells. Deep sequencing analysis of Rmrp 3' ends revealed extensive oligouridylation mainly on transcripts with imprecise ends. We implicate the terminal uridylyl transferases (TUTases) Zcchc6/11 in the uridylation of these ncRNAs, and biochemical reconstitution assays demonstrate the sufficiency of TUTase-Dis3l2 for Rmrp decay. This establishes Dis3l2-mediated decay (DMD) as a quality-control pathway that eliminates aberrant ncRNAs.