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A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila

ABSTRACT: A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and novel components of the piRNA pathway, we carried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several novel piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE46009 | GEO | 2013/05/09

SECONDARY ACCESSION(S): PRJNA198429

REPOSITORIES: GEO

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A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila

Project description:A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and novel components of the piRNA pathway, we carried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several novel piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels. We sequenced small RNAs, RNA-seq and ChIP-seq from either tj-Gal4 driven hpRNA knockdown flies or P-element insertion flies

2013-05-09 | E-GEOD-46009 | biostudies-arrayexpress

eCLIP of Drosophila Asterix/Gtsf1 (endogenous promoter) in Drosophila ovarian somatic sheath (OSS) cells

Project description:Asterix/Gtsf1 has been previously been shown to be involved in the silencing of retrotransposons in animal germ cells through the piRNA pathway. Using eCLIP and a custom informatic pipeline, we found that Asterix/Gtsf1 binds directly to RNAs and preferentially interacts with tRNAs as a class. Given the role of tRNAs as primers for Long Terminal Repeat (LTR) transposons, this work implicates Asterix/Gtsf1 as exploiting tRNA dependence to identify transposon transcripts and promote piRNA silencing.

2021-02-20 | GSE151109 | GEO

eCLIP of mouse Asterix/Gtsf1 transfected in mouse P19 embryonal teratoma cells

2021-02-20 | GSE151108 | GEO

eCLIP of Drosophila Asterix/Gtsf1 transfected in Drosophila ovarian somatic sheath (OSS) cells

2021-02-20 | GSE151107 | GEO

RIP-seq of mouse Asterix/Gtsf1 expressed in Sf9 cells

Project description:Asterix/Gtsf1 has been previously been shown to be involved in the silencing of retrotransposons in animal germ cells through the piRNA pathway. During early stages of purification of mouse Gtsf1 protein in Sf9 cells for structural studies, we found that the protein was co-purifying with endogenous nucleic acids. We first characterized those RNAs by next-generation sequencing (this dataset) and then later investigated its RNA-binding in more relevant cell culture systems. In each case, we found Asterix/Gtsf1 was binding tRNAs. Given the role of tRNAs as primers for Long Terminal Repeat (LTR) transposons, this work implicates Asterix/Gtsf1 as exploiting tRNA dependence to identify transposon transcripts and promote piRNA silencing.

2021-02-20 | GSE151110 | GEO

Maternally inherited piRNAs silence transposons during Drosophila embryogenesis

Project description:The piRNA pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. piRNAs are maternally deposited and required for proper transposon silencing in adult offspring. However, a long-standing question in the field is the precise function of maternally deposited piRNAs and its associated factors during embryogenesis. Here, we probe the spatio-temporal expression patterns of several piRNA pathway components during early stages of development. Amongst those, factors required for transcriptional gene silencing (TGS) showed ubiquitous abundance in somatic and pole cells throughout the first half of embryogenesis. We further analysed the transcriptomes of various embryo stages and correlated these with the presence of selected chromatin marks. We found that a number of transposon families show bursts of transcription during early embryonic stages. Transposons heavily targeted by maternally deposited piRNAs accumulated repressive chromatin marks following their spike in expression. Furthermore, depletion of maternally deposited Piwi protein in early embryos resulted in increased expression of transposons targeted by inherited piRNAs and was accompanied by a strong loss of repressive chromatin marks at coding sequences. Overall, our data suggests a pivotal role for the piRNA pathway in transposon defence during Drosophila embryogenesis in somatic cells.

2021-07-26 | PXD022409 | Pride

Minotaur is critical for primary piRNA biogenesis [RNA-Seq]

Project description:Piwi proteins and their associated small RNAs are essential for fertility in animals. This is due, in part, to their roles in guarding germ cell genomes against the activity of mobile genetic elements. piRNA populations direct Piwi proteins to silence transposon targets and as such form a molecular code that discriminates transposons from endogenous genes. Information ultimately carried by piRNAs is encoded within genomic loci, termed piRNA clusters. These give rise to long, single-stranded, primary transcripts that are processed into piRNAs. Despite the biological importance of this pathway, neither the characteristics that define a locus as a source of piRNAs nor the mechanisms that catalyze primary piRNA biogenesis are well understood. We searched an EMS-mutant collection annotated for fertility phenotypes for genes involved in the piRNA pathway. Twenty-seven homozygous-sterile strains showed transposon-silencing defects. One of these, which strongly impacted primary piRNA biogenesis, harbored a causal mutation in CG5508, a member of the Drosophila glycerol-3-phosphate O-acetyltransferase (GPAT) family. These enzymes catalyze the first acylation step on the path to the production of phosphatidic acid (PA). Though this pointed strongly to a function for phospholipid signaling in the piRNA pathway, a mutant form of CG5508, which lacks the GPAT active site, still functions in piRNA biogenesis. We have named this new biogenesis factor Minotaur. Examination of transcriptom profile in heterozygous and homozygous CG5508 mutant ovaries

2013-04-27 | E-GEOD-46423 | biostudies-arrayexpress

Adaptation to transposon invasion in Drosophila melanogaster

Project description:Transposons evolve rapidly and can mobilize and trigger genetic instability. piRNAs silence these genome pathogens, but it is unclear how the piRNA pathway adapts to new transposons. In Drosophila piRNAs, encoded by heterochromatic clusters are maternally deposited in the embryo. Paternally inherited P-element transposons thus escape silencing and trigger a genetic instability and sterility. We show that this syndrome, termed P-M hybrid dysgenesis, also disrupts the piRNA biogenesis machinery and activates resident transposons. As dysgenic hybrids age, however, fertility is restored, P-elements are silenced, and P-element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery is restored and resident elements are silenced. Significantly, new resident transposons insertions accumulate in piRNA clusters, and these new insertions are transmitted to progeny with high fidelity, produce novel piRNAs, and are associated with reduced transposition. P-M hybrid dysgenesis thus leads to heritable changes in chromosome structure that appear to enhance transposon silencing. 3 replicates of each sample (Har 2-4 days, w1 x Har 2-4 days, w1 x Har 21 days), total RNA samples hybridized to tiling array.

2011-12-23 | E-GEOD-31813 | biostudies-arrayexpress

C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germline

Project description:In the male mouse germline, PIWI-interacting RNAs (piRNAs), bound by the PIWI protein MIWI2 (PIWIL4), guide DNA methylation of young active transposons through SPOCD1. However, the underlying mechanisms of SPOCD1-mediated piRNA-directed transposon methylation and whether this pathway functions to protect the human germline remains unknown. We identified loss-of-function variants in human SPOCD1 that cause defective transposon silencing and male infertility. Through the analysis of one of these pathogenic alleles, we discovered that the uncharacterised protein C19ORF84 interacts with SPOCD1. DNMT3C, the DNA methyltransferase responsible for transposon methylation, associates with SPOCD1 and C19ORF84 in foetal gonocytes. Furthermore, C19ORF84 is essential for piRNA-directed DNA methylation and male mouse fertility. Finally, C19ORF84 mediates the in vivo association of SPOCD1 with the de novo methylation machinery. In summary, we have discovered a conserved role for the human piRNA pathway in transposon silencing and C19ORF84, an uncharacterised protein essential for orchestrating piRNA-directed DNA methylation.

2024-02-14 | PXD047331 | Pride

Transcriptional silencing of transposable elements by Piwi and its impact on chromatin and gene expression

Project description:Eukaryotic genomes are colonized by transposable elements whose uncontrolled activity results in genomic instability. The piRNA pathway silences transposons in animal gonads, yet how this is achieved molecularly remains controversial. We assign an essential role to the HMG protein Maelstrom in the process of 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 for establishing heterochromatic H3K9me3 marks on transposons and their genomic surrounding. In contrast, loss of Maelstrom impacts transposon H3K9me3 patterns only marginally yet leads to increased heterochromatin spreading, suggesting that Maelstrom acts downstream of or in parallel to H3K9me3. Our work uncovers the widespread influence of transposons and the piRNA pathway on chromatin patterns and gene expression programs. RNA Polymerase II and H3K9me3 occupancy, and steady-state and nascent RNA levels in wild-type ovarian somatic cells (OSC) and RNAi knock-downs of the piRNA pathway components. RNA Polymerase II occupancy in tissue-specific knockdowns of tejas (control) and armi in somatic cells of Drosophila ovary.

2012-11-26 | E-GEOD-41729 | biostudies-arrayexpress

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