De novo piRNA cluster formation in the Drosophila germline triggered by transgenes containing a transcribed transposon fragment
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE41780 | GEO | 2013/04/25
SECONDARY ACCESSION(S): PRJNA178167
REPOSITORIES: GEO
Similar Datasets
Project description:PIWI interacting RNAs (piRNAs) provide defense against transposable element (TE) expansion in the germline of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the LINE-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions which normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germline. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences — not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs. The fractions of small RNAs (19-29 nt) from ovaries of wild type and 11 transgenic lines of Drosophila melanogaster were sequenced using Illumina HiSeq 2000.
2013-04-25 | E-GEOD-41780 | biostudies-arrayexpress
Project description:Piwi-interacting RNAs (piRNAs) control transposable element (TE) activity in the germline. piRNAs are produced from single-stranded precursors transcribed from distinct genomic loci, enriched by TE fragments and termed piRNA clusters. The specific chromatin organization and transcriptional regulation of Drosophila germline-specific piRNA clusters ensure transcription and processing of piRNA precursors. TEs harbour various regulatory elements that could affect piRNA cluster integrity. One of such elements is the suppressor-of-hairy-wing (Su(Hw))-mediated insulator, which is harboured in the retrotransposon gypsy. To understand how insulators contribute to piRNA cluster activity, we studied the effects of transgenes containing gypsy insulators on local organization of endogenous piRNA clusters. We show that transgene insertions interfere with piRNA precursor transcription, small RNA production and the formation of piRNA cluster-specific chromatin, a hallmark of which is Rhino, the germline homolog of the heterochromatin protein 1 (HP1). The mutations of Su(Hw) restored the integrity of piRNA clusters in transgenic strains. Surprisingly, Su(Hw) depletion enhanced the production of piRNAs by the domesticated telomeric retrotransposon TART, indicating that Su(Hw)-dependent elements protect TART transcripts from piRNA processing machinery in telomeres. A genome-wide analysis revealed that Su(Hw)-binding sites are depleted in endogenous germline piRNA clusters, suggesting that their functional integrity is under strict evolutionary constraints.
2019-03-15 | GSE125173 | GEO
Project description:PIWI-interacting RNAs (piRNAs) are genomically-encoded small RNAs that regulate germ cell development and guarantee germline integrity. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. To date, piRNA biogenesis mechanisms are still unclear. Here, we show that the RNA Polymerase II subunit RPB-9 is required to promote transcription elongation at piRNA loci. Through genetic and biochemical experiments, we demonstrate that rpb-9-mediated piRNA production is needed to repress two DNA transposon families and a subset of somatic genes in the C. elegans germline.
2020-12-07 | PXD018657 | Pride
Project description:Transgenes containing a fragment of I transposon represent a powerful model of piRNA cluster de novo formation in the Drosophila germline. We revealed that the same transgenes located at different genomic loci form piRNA clusters with various capacity of small RNA production. Transgenic piRNA clusters are not established in piRNA pathway mutants. However, in wild-type context, the endogenous ancestral I-related piRNAs are sufficient to heterochromatinize and convert the I-containing transgenes into piRNA-producing loci. Here, we address how the quantitative level of piRNAs influences the heterochromatinization and piRNA production. We show that neither the piRNAs mediated by active I-element copies nor inheritance of abundant maternal I-derived piRNAs enable to stimulate additional changes of transgenes chromatin state or piRNA production from them. Therefore, chromatin changes and piRNA production are initiated by a minimum threshold level of complementary piRNAs suggesting a selective advantage of prompt cell response to the lowest level of piRNAs. Noteworthy, the weak piRNA clusters do not transform into strong ones after being targeted by a larger amount of I-specific piRNAs, indicating the importance of the genomic context for piRNA cluster establishment. Analysis of ovarian transcription profiles suggests that regions facilitating convergent transcription favor formation of transgenic piRNA clusters .
2020-04-10 | GSE138886 | GEO
Project description:PIWI-interacting RNAs (piRNAs) are animal gonad-specific small RNAs that control the activity of transposable elements. Long single stranded RNAs from a variety of sources are substrates for the nebulous primary processing pathway that converts these into thousands of 24-30 nucleotide (nt) piRNAs. How these transcripts are selected as precursors is not known. Here we show that targeting a transcript with PIWI slicer activity of cysosolic Ago3 is sufficient to trigger ~30-nt waves of non-overlapping primary piRNAs in the fly ovarian germline. The generated primary piRNAs are almost exclusively loaded into the nuclear PIWI protein, Piwi. In the fly ovarian somatic environment we find that an RNA fragment from the 5? end of a piRNA cluster is able to direct a heterologous sequence into primary processing. This piRNA trigger sequence (PTS) element drives generation of overlapping piRNAs from the transcript. Both mechanisms proceed with general 5?-3? directionality. We propose that the former pathway serves to link cytoplasmic silencing of a target to nuclear transcriptional repression, while the latter extracts silencing information from a wide variety of genomic sources including piRNA clusters, select protein coding and transposon transcripts. Total or immunoprecipitated small RNAs were purified from transfected BmN4 cells, Drosophila ovarian somatic cells (OSC) and from fly ovaries and high-throughput sequencing libraries were prepared. The mouse testicular RNAs were purified after ribozero treatment.
2015-07-09 | E-GEOD-69102 | biostudies-arrayexpress
Project description:PIWI-clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ~23-30nt piRNAs that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endo-nuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the inter-dependencies between these piRNA biogenesis pathways in the developing Drosophila ovary. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and that defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear, transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways. To understand the hierarchical order of primary versus secondary piRNA biogenesis in Drosophila ovaries, we sequenced piRNAs bound to total-Piwi, germline-Piwi, Aubergine and Argonaute3 from ovaries of germline specific knockdowns of control, piwi, aub, ago3 single knockdowns and aub/ago3 double knockdowns. To determine changes in Transposable Element (TE) transcription or TE RNA steady state in perturbed piRNA pathway conditions, we performed Pol2-ChIP-sequencing and polyA bound RNA-sequencing from ovaries of multiple germline knockdown genotypes. We also sequenced genomic DNA from ovaries of control knockdowns to experimentally estimate the TE copy number in our genetic background. Finally, we used CAP-seq from germline specific Piwi depletions to identify the Transcriptional Start Sites (TSS) in TEs in a deregulated background. Replicates are labeled with R1, R2, R3, R4 where indicated.
2015-08-31 | E-GEOD-71775 | biostudies-arrayexpress
Project description:PIWI-interacting RNAs (piRNAs) are animal gonad-specific small RNAs that control the activity of transposable elements. Long single stranded RNAs from a variety of sources are substrates for the nebulous primary processing pathway that converts these into thousands of 24-30 nucleotide (nt) piRNAs. How these transcripts are selected as precursors is not known. Here we show that targeting a transcript with PIWI slicer activity of cysosolic Ago3 is sufficient to trigger ~30-nt waves of non-overlapping primary piRNAs in the fly ovarian germline. The generated primary piRNAs are almost exclusively loaded into the nuclear PIWI protein, Piwi. In the fly ovarian somatic environment we find that an RNA fragment from the 5′ end of a piRNA cluster is able to direct a heterologous sequence into primary processing. This piRNA trigger sequence (PTS) element drives generation of overlapping piRNAs from the transcript. Both mechanisms proceed with general 5′-3′ directionality. We propose that the former pathway serves to link cytoplasmic silencing of a target to nuclear transcriptional repression, while the latter extracts silencing information from a wide variety of genomic sources including piRNA clusters, select protein coding and transposon transcripts.
2015-07-09 | GSE69102 | GEO
Project description:The maintenance of genome integrity is an essential trait to the successful transmission of genetic information. In animal germ cells, piRNAs guide PIWI proteins to silence transposable elements (TEs) in order to maintain genome integrity. In insects, most of TE silencing in the germline is achieved by secondary piRNAs that are produced by a feed-forward loop (the ping-pong cycle), which requires the piRNA-directed cleavages of two types of RNAs: mRNAs of functional euchromatic TEs and heterochromatic transcripts that contain defective TE sequences. The first cleavage which initiates such amplification loop remains poorly understood. Taking advantage of the existence of strains that are devoid of functional copies of the LINE-like I-element, we report that in such Drosophila ovaries, the initiation of a ping-pong cycle is achieved only by secondary I-element piRNAs that are produced in the ovary and deposited in the embryonic germline. This unusual secondary piRNA biogenesis, detected in the absence of functional I-element copies, results from the processing of sense and antisense transcripts of several different defective I-elements. Once acquired, for instance after ancestor aging, this capacity to produce heterochromatic-only secondary piRNAs is partially transmitted through generations via maternal piRNAs. Furthermore, such piRNAs acting as ping-pong initiators in a chromatin-independent manner confer to the progeny a high capacity to repress the I-element mobility. Our study explains at the molecular level the basis for epigenetic memory of maternal immunity that protects females from hybrid dysgenesis caused by transposition of paternally inherited functional I-elements. Comparison of Drosophila small RNA populations in ovaries and/or eggs from 3-day-old or 25-day-old females.
2012-04-13 | E-GEOD-34506 | biostudies-arrayexpress
Project description:Piwi Argonautes and Piwi-interacting RNAs (piRNAs) mediate genome defense by targeting transposons. However, many piRNA species lack obvious sequence complementarity to transposons or other loci. For example, only one C. elegans transposon is a known piRNA target. Here we show that, in mutants lacking the Piwi Argonaute PRG-1 and associated piRNAs (21U-RNAs), many silent loci in the germline exhibit increased levels of mRNA expression and depletion of an amplified RNAdependent RNA polymerase (RdRP)-derived species of small RNA termed 22G-RNAs. Sequences depleted of 22G-RNAs are enriched nearby potential target sites that base pair imperfectly but extensively to 21U-RNAs. We show that PRG-1 is required to initiate, but not to maintain, silencing of transgenes engineered to contain complementarity to endogenous 21U-RNAs. Our findings support a model in which C. elegans piRNAs utilize their enormous repertoire of targeting capacity to scan the germline transcriptome for foreign sequences, while endogenous germline-expressed genes are actively protected from piRNA-induced silencing. Examine small RNA population changes in prg-1 and rescued strains
2012-09-01 | E-GEOD-38723 | biostudies-arrayexpress
Project description:The maintenance of genome integrity is an essential trait to the successful transmission of genetic information. In animal germ cells, piRNAs guide PIWI proteins to silence transposable elements (TEs) in order to maintain genome integrity. In insects, most of TE silencing in the germline is achieved by secondary piRNAs that are produced by a feed-forward loop (the ping-pong cycle), which requires the piRNA-directed cleavages of two types of RNAs: mRNAs of functional euchromatic TEs and heterochromatic transcripts that contain defective TE sequences. The first cleavage which initiates such amplification loop remains poorly understood. Taking advantage of the existence of strains that are devoid of functional copies of the LINE-like I-element, we report that in such Drosophila ovaries, the initiation of a ping-pong cycle is achieved only by secondary I-element piRNAs that are produced in the ovary and deposited in the embryonic germline. This unusual secondary piRNA biogenesis, detected in the absence of functional I-element copies, results from the processing of sense and antisense transcripts of several different defective I-elements. Once acquired, for instance after ancestor aging, this capacity to produce heterochromatic-only secondary piRNAs is partially transmitted through generations via maternal piRNAs. Furthermore, such piRNAs acting as ping-pong initiators in a chromatin-independent manner confer to the progeny a high capacity to repress the I-element mobility. Our study explains at the molecular level the basis for epigenetic memory of maternal immunity that protects females from hybrid dysgenesis caused by transposition of paternally inherited functional I-elements.
2012-04-13 | GSE34506 | GEO