RESUMO
Argonaute proteins of the PIWI clade are central to transposon silencing in animal gonads. Their target specificity is defined by 23-30 nt PIWI interacting RNAs (piRNAs), which mostly originate from discrete genomic loci termed piRNA clusters. Here, we show that a complex composed of Rhino, Deadlock, and Cutoff (RDC) defines dual-strand piRNA clusters genome-wide in Drosophila ovaries. The RDC is anchored to H3K9me3-marked chromatin in part via Rhino's chromodomain. Depletion of Piwi results in loss of the RDC and small RNAs at a subset of piRNA clusters, demonstrating a feedback loop between Piwi and piRNA source loci. Intriguingly, profiles of RNA polymerase II occupancy, nascent transcription, and steady-state RNA levels reveal that the RDC licenses noncanonical transcription of dual-strand piRNA clusters. Likely, this process involves 5' end protection of nascent RNAs and suppression of transcription termination. Our data provide key insight into the regulation and evolution of piRNA clusters.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Animais , Feminino , Estudo de Associação Genômica Ampla , Ovário/metabolismo , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/metabolismo , Terminação da Transcrição GenéticaRESUMO
Eukaryotic genomes are colonized by transposons whose uncontrolled activity causes genomic instability. The piRNA pathway silences transposons in animal gonads, yet how this is achieved molecularly remains controversial. Here, we show that the HMG protein Maelstrom is essential for Piwi-mediated silencing in Drosophila. Genome-wide assays revealed highly correlated changes in RNA polymerase II recruitment, nascent RNA output, and steady-state RNA levels of transposons upon loss of Piwi or Maelstrom. Our data demonstrate piRNA-mediated trans-silencing of hundreds of transposon copies at the transcriptional level. We show that Piwi is required to establish heterochromatic H3K9me3 marks on transposons and their genomic surroundings. In contrast, loss of Maelstrom affects transposon H3K9me3 patterns only mildly yet leads to increased heterochromatin spreading, suggesting that Maelstrom acts downstream of or in parallel to H3K9me3. Our work illustrates the widespread influence of transposons and the piRNA pathway on chromatin patterns and gene expression.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Inativação Gênica , RNA Interferente Pequeno/metabolismo , Animais , Montagem e Desmontagem da Cromatina , Elementos de DNA Transponíveis , Heterocromatina/metabolismo , Código das HistonasRESUMO
The repression of transposable elements in eukaryotes often involves their transcriptional silencing via targeted chromatin modifications. In animal gonads, nuclear Argonaute proteins of the PIWI clade complexed with small guide RNAs (piRNAs) serve as sequence specificity determinants in this process. How binding of nuclear PIWI-piRNA complexes to nascent transcripts orchestrates heterochromatin formation and transcriptional silencing is unknown. Here, we characterize CG9754/Silencio as an essential piRNA pathway factor that is required for Piwi-mediated transcriptional silencing in Drosophila. Ectopic targeting of Silencio to RNA or DNA is sufficient to elicit silencing independently of Piwi and known piRNA pathway factors. Instead, Silencio requires the H3K9 methyltransferase Eggless/SetDB1 for its silencing ability. In agreement with this, SetDB1, but not Su(var)3-9, is required for Piwi-mediated transcriptional silencing genome-wide. Due to its interaction with the target-engaged Piwi-piRNA complex, we suggest that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/metabolismo , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , DNA/metabolismo , Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Feminino , Inativação Gênica , Genoma de Inseto/genética , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Metilação , Ovário/fisiologia , Ligação Proteica , RNA/metabolismo , Proteínas de Ligação a RNA , Proteínas Repressoras/metabolismoRESUMO
Damaged DNA is an obstacle during DNA replication and a cause of genome instability and cancer. To bypass this problem, eukaryotes activate DNA damage tolerance (DDT) pathways that involve ubiquitylation of the DNA polymerase clamp proliferating cell nuclear antigen (PCNA). Monoubiquitylation of PCNA mediates an error-prone pathway by recruiting translesion polymerases, whereas polyubiquitylation activates an error-free pathway that utilizes undamaged sister chromatids as templates. The error-free pathway involves recombination-related mechanisms; however, the factors that act along with polyubiquitylated PCNA remain largely unknown. Here we report that the PCNA-related 9-1-1 complex, which is typically linked to checkpoint signaling, participates together with Exo1 nuclease in error-free DDT. Notably, 9-1-1 promotes template switching in a manner that is distinct from its canonical checkpoint functions and uncoupled from the replication fork. Our findings thus reveal unexpected cooperation in the error-free pathway between the two related clamps and indicate that 9-1-1 plays a broader role in the DNA damage response than previously assumed.
Assuntos
Dano ao DNA , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Exodesoxirribonucleases/metabolismo , Fase G2 , Testes Genéticos , Mitose , Modelos Biológicos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Moldes GenéticosRESUMO
The PIWI-interacting RNA (piRNA) pathway is a small RNA silencing system that keeps selfish genetic elements such as transposons under control in animal gonads. Several lines of evidence indicate that nuclear PIWI family proteins guide transcriptional silencing of their targets, yet the composition of the underlying silencing complex is unknown. Here we demonstrate that the double CHHC zinc finger protein gametocyte-specific factor 1 (Gtsf1) is an essential factor for Piwi-mediated transcriptional repression in Drosophila. Cells lacking Gtsf1 contain nuclear Piwi loaded with piRNAs, yet Piwi's silencing capacity is ablated. Gtsf1 interacts directly with a small subpool of nuclear Piwi, and loss of Gtsf1 phenocopies loss of Piwi in terms of deregulation of transposons, loss of H3K9 trimethylation (H3K9me3) marks at euchromatic transposon insertions, and deregulation of genes in proximity to repressed transposons. We propose that only a small fraction of nuclear Piwi is actively engaged in target silencing and that Gtsf1 is an essential component of the underlying Piwi-centered silencing complex.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Complexo Mediador/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Células Cultivadas , Feminino , Inativação Gênica , Ovário/metabolismoRESUMO
Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9's off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.
Assuntos
Pró-Proteína Convertase 9 , Translocação Genética , Animais , Camundongos , Pró-Proteína Convertase 9/genética , Sistemas CRISPR-Cas/genética , Mutação , Endonucleases/genética , Streptococcus pyogenes/genéticaRESUMO
Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing leads to increased levels of by-products, it can rescue pegRNAs that perform poorly with a nickase-based prime editor. We also present a small molecule approach that yields increased product purity of PEn editing. Next, we develop a homology-independent PEn editing strategy, which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Animais , Sistemas CRISPR-Cas , Reparo do DNA , Endonucleases/metabolismo , Edição de Genes , Mamíferos/genéticaRESUMO
Nuclear Argonaute proteins, guided by small RNAs, mediate sequence-specific heterochromatin formation. The molecular principles that link Argonaute-small RNA complexes to cellular heterochromatin effectors on binding to nascent target RNAs are poorly understood. Here, we explain the mechanism by which the PIWI-interacting RNA (piRNA) pathway connects to the heterochromatin machinery in Drosophila. We find that Panoramix, a corepressor required for piRNA-guided heterochromatin formation, is SUMOylated on chromatin in a Piwi-dependent manner. SUMOylation, together with an amphipathic LxxLL motif in Panoramix's intrinsically disordered repressor domain, are necessary and sufficient to recruit Small ovary (Sov), a multi-zinc-finger protein essential for general heterochromatin formation and viability. Structure-guided mutations that eliminate the Panoramix-Sov interaction or that prevent SUMOylation of Panoramix uncouple Sov from the piRNA pathway, resulting in viable but sterile flies in which Piwi-targeted transposons are derepressed. Thus, Piwi engages the heterochromatin machinery specifically at transposon loci by coupling recruitment of a corepressor to nascent transcripts with its SUMOylation.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sítios de Ligação/genética , Cromatina/genética , Cromatina/metabolismo , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Inativação Gênica , Genes de Insetos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Mutação , Proteínas Nucleares/química , Células-Tronco de Oogônios/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Sumoilação/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
The fatty acid composition of phosphatidylethanolamine (PE) determines cellular metabolism, oxidative stress, and inflammation. However, our understanding of how cells regulate PE composition is limited. Here, we identify a genetic locus on mouse chromosome 11, containing two poorly characterized genes Tlcd1 and Tlcd2, that strongly influences PE composition. We generated Tlcd1/2 double-knockout (DKO) mice and found that they have reduced levels of hepatic monounsaturated fatty acid (MUFA)-containing PE species. Mechanistically, TLCD1/2 proteins act cell intrinsically to promote the incorporation of MUFAs into PEs. Furthermore, TLCD1/2 interact with the mitochondria in an evolutionarily conserved manner and regulate mitochondrial PE composition. Lastly, we demonstrate the biological relevance of our findings in dietary models of metabolic disease, where Tlcd1/2 DKO mice display attenuated development of non-alcoholic steatohepatitis compared to controls. Overall, we identify TLCD1/2 proteins as key regulators of cellular PE composition, with our findings having broad implications in understanding and treating disease.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Fosfatidiletanolaminas , Animais , Ácidos Graxos/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fosfatidiletanolaminas/metabolismoRESUMO
The E4 allele of the apolipoprotein E gene (APOE) has been established as a genetic risk factor for many diseases including cardiovascular diseases and Alzheimer's disease (AD), yet its mechanism of action remains poorly understood. APOE is a lipid transport protein, and the dysregulation of lipids has recently emerged as a key feature of several neurodegenerative diseases including AD. However, it is unclear how APOE4 perturbs the intracellular lipid state. Here, we report that APOE4, but not APOE3, disrupted the cellular lipidomes of human induced pluripotent stem cell (iPSC)-derived astrocytes generated from fibroblasts of APOE4 or APOE3 carriers, and of yeast expressing human APOE isoforms. We combined lipidomics and unbiased genome-wide screens in yeast with functional and genetic characterization to demonstrate that human APOE4 induced altered lipid homeostasis. These changes resulted in increased unsaturation of fatty acids and accumulation of intracellular lipid droplets both in yeast and in APOE4-expressing human iPSC-derived astrocytes. We then identified genetic and chemical modulators of this lipid disruption. We showed that supplementation of the culture medium with choline (a soluble phospholipid precursor) restored the cellular lipidome to its basal state in APOE4-expressing human iPSC-derived astrocytes and in yeast expressing human APOE4 Our study illuminates key molecular disruptions in lipid metabolism that may contribute to the disease risk linked to the APOE4 genotype. Our study suggests that manipulating lipid metabolism could be a therapeutic approach to help alleviate the consequences of carrying the APOE4 allele.
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Apolipoproteínas E , Homeostase , Humanos , NeurogliaRESUMO
Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Mutação , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Células HCT116 , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , CamundongosRESUMO
The ε4 allele of apolipoprotein E (APOE4) is a genetic risk factor for many diseases, including late-onset Alzheimer's disease (AD). We investigate the cellular consequences of APOE4 in human iPSC-derived astrocytes, observing an endocytic defect in APOE4 astrocytes compared with their isogenic APOE3 counterparts. Given the evolutionarily conserved nature of endocytosis, we built a yeast model to identify genetic modifiers of the endocytic defect associated with APOE4. In yeast, only the expression of APOE4 results in dose-dependent defects in both endocytosis and growth. We discover that increasing expression of the early endocytic adaptor protein Yap1802p, a homolog of the human AD risk factor PICALM, rescues the APOE4-induced endocytic defect. In iPSC-derived human astrocytes, increasing expression of PICALM similarly reverses endocytic disruptions. Our work identifies a functional interaction between two AD genetic risk factors-APOE4 and PICALM-centered on the conserved biological process of endocytosis.
Assuntos
Doença de Alzheimer/genética , Apolipoproteína E4/metabolismo , Endocitose/fisiologia , Doença de Alzheimer/patologia , Humanos , Fatores de RiscoRESUMO
MicroRNAs serve to fine-tune gene expression and play an important regulatory role in tissue specific gene networks. The identification and validation of miRNA target genes in a tissue still poses a significant problem since the presence of a seed sequence in the 3'UTR of an mRNA and its expression modulation upon ectopic expression of the miRNA do not reliably predict regulation under physiological conditions. The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in Ewing sarcoma. MiR-17-92, one of the most potent oncogenic miRNAs, was recently reported to be among the top EWS-FLI1 activated miRNAs. Using a combination of AGO2 pull-down experiments by PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) and of RNAseq upon miRNA depletion by ectopic sponge expression, we aimed to identify the targetome of miR-17-92 in Ewing sarcoma. Intersecting both datasets we found an enrichment of PAR-CLIP hits for members of the miR-17-92 cluster in the 3'UTRs of genes up-regulated in response to mir-17-92 specific sponge expression. Strikingly, approximately a quarter of these genes annotate to the TGFB/BMP pathway, the majority mapping downstream of SMAD signaling. Testing for SMAD phosphorylation, we identify quiet but activatable TGFB signaling and cell autonomous activity of the BMP pathway resulting in the activation of the stemness regulatory transcriptional repressors ID1 and ID3. Taken together, our findings shed light on the complex miRegulatory landscape of Ewing Sarcoma pointing miR-17-92 as a key node connected to TGFB/BMP pathway.
Assuntos
Regiões 3' não Traduzidas/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Tumoral , Predisposição Genética para Doença/genética , Humanos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , RNA Longo não Codificante , Proteína EWS de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Análise de Sequência de RNA , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The CRISPR-associated RNA-guided nuclease Cas9 has emerged as a powerful tool for genome engineering in a variety of organisms. To achieve efficient gene targeting rates in Drosophila, current approaches require either injection of in vitro transcribed RNAs or injection into transgenic Cas9-expressing embryos. We report a simple and versatile alternative method for CRISPR-mediated genome editing in Drosophila using bicistronic Cas9/sgRNA expression vectors. Gene targeting with this single-plasmid injection approach is as efficient as in transgenic nanos-Cas9 embryos and allows the isolation of targeted knock-out and knock-in alleles by molecular screening within 2 months. Our strategy is independent of genetic background and does not require prior establishment of transgenic flies.