Drosophila rasiRNA Pathway Mutations Disrupt Embryonic Axis Specification through Activation of an ATR/Chk2 DNA Damage Response

Program in Molecular Medicine and Program in Cell Dynamics, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Developmental Cell (Impact Factor: 9.71). 02/2007; 12(1):45-55. DOI: 10.1016/j.devcel.2006.12.001
Source: PubMed


Small repeat-associated siRNAs (rasiRNAs) mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila rasiRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization as well as asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellate silencing. Furthermore, rasiRNA pathway mutations lead to germline-specific accumulation of gamma-H2Av foci characteristic of DNA damage. We conclude that rasiRNA-based gene silencing is not required for axis specification, and that the critical developmental function for this pathway is to suppress DNA damage signaling in the germline.

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Available from: Diana P Bratu, Oct 04, 2015
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    • "In animals, PIWI-interacting RNAs (piRNAs), a diverse class of 23À36 nucleotide (nt) small silencing RNAs, repress transposons in the germline, ensuring the faithful transfer of genomic information from generation to generation (Girard et al., 2006; Aravin et al., 2006; Grivna et al., 2006; Lau et al., 2006; Vagin et al., 2006). Disrupting the piRNA pathway activates transposon transcription , arresting germ cell development (Wilson et al., 1996; Lin and Spradling, 1997; Deng and Lin, 2002; Klattenhoff et al., 2007) and making one or both sexes infertile. Drosophila germline nurse cells produce piRNAs by at least two mechanisms: the de novo pathway and the ping-pong cycle. "
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    ABSTRACT: In Drosophila ovarian germ cells, PIWI-interacting RNAs (piRNAs) direct Aubergine and Argonaute3 to cleave transposon transcripts and instruct Piwi to repress transposon transcription, thereby safeguarding the germline genome. Here, we report that RNA cleavage by Argonaute3 initiates production of most Piwi-bound piRNAs. We find that the cardinal function of Argonaute3, whose piRNA guides predominantly correspond to sense transposon sequences, is to produce antisense piRNAs that direct transcriptional silencing by Piwi, rather than to make piRNAs that guide post-transcriptional silencing by Aubergine. We also find that the Tudor domain protein Qin prevents Aubergine's cleavage products from becoming Piwi-bound piRNAs, ensuring that antisense piRNAs guide Piwi. Although Argonaute3 slicing is required to efficiently trigger phased piRNA production, an alternative, slicing-independent pathway suffices to generate Piwi-bound piRNAs that repress transcription of a subset of transposon families. This alternative pathway may help flies silence newly acquired transposons for which they lack extensively complementary piRNAs. Copyright © 2015 Elsevier Inc. All rights reserved.
    Molecular cell 09/2015; 59(5):819-30. DOI:10.1016/j.molcel.2015.08.007 · 14.02 Impact Factor
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    • "Plasmids containing UASp-GFP-Nup93 * and UASp-GFP- Nup62 * (the asterisk indicates shRNA-resistant) or Walium22-vectors (Harvard Medical School) containing shRNA against Mel-28/ELYS were injected into w embryos carrying an attP40 or VK33 landing site by Genetic Research, Inc., for transgenesis. The meiotic recombination checkpoint was suppressed by a heterozygous mutation, mnk p6 (DmChk2) (Klattenhoff et al. 2007), or by feeding adults with medium containing spectral ice blue dye (Sugarflair Colours Ltd.) and 1 mg/mL caffeine (Sigma). spn A1 (Tearle and Nüsslein-Volhard 1987) was used as control. "
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    ABSTRACT: The nuclear pore complex (NPC) tethers chromatin to create an environment for gene regulation, but little is known about how this activity is regulated to avoid excessive tethering of the genome. Here we propose a negative regulatory loop within the NPC controlling the chromatin attachment state, in which Nup155 and Nup93 recruit Nup62 to suppress chromatin tethering by Nup155. Depletion of Nup62 severely disrupts chromatin distribution in the nuclei of female germlines and somatic cells, which can be reversed by codepleting Nup155. Thus, this universal regulatory system within the NPC is crucial to control large-scale chromatin organization in the nucleus. © 2015 Breuer and Ohkura; Published by Cold Spring Harbor Laboratory Press.
    Genes & Development 09/2015; 29(17). DOI:10.1101/gad.264341.115 · 10.80 Impact Factor
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    • "Generation of double-mutant mice between Spo11 and additional mutants of germline TE reactivation may shed light on the exact contribution of TE retrotransposition to meiotic disruption phenotypes. Notably, while increased DNA damage is a known consequence of TE reactivation in piR- NA mutants in Drosophila (Klattenhoff et al. 2007), this has not been formally linked to active retrotransposition. Our most striking findings are certainly related to the observation that the most dramatic consequence of TE hypomethylation and reactivation on meiosis occurs at the chromatin level. "
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    ABSTRACT: DNA methylation is essential for protecting the mammalian germline against transposons. When DNA methylation-based transposon control is defective, meiotic chromosome pairing is consistently impaired during spermatogenesis: How and why meiosis is vulnerable to transposon activity is unknown. Using two DNA methylation-deficient backgrounds, the Dnmt3L and Miwi2 mutant mice, we reveal that DNA methylation is largely dispensable for silencing transposons before meiosis onset. After this, it becomes crucial to back up to a developmentally programmed H3K9me2 loss. Massive retrotransposition does not occur following transposon derepression, but the meiotic chromatin landscape is profoundly affected. Indeed, H3K4me3 marks gained over transcriptionally active transposons correlate with formation of SPO11-dependent double-strand breaks and recruitment of the DMC1 repair enzyme in Dnmt3L(-/-) meiotic cells, whereas these features are normally exclusive to meiotic recombination hot spots. Here, we demonstrate that DNA methylation restrains transposons from adopting chromatin characteristics amenable to meiotic recombination, which we propose prevents the occurrence of erratic chromosomal events. © 2015 Zamudio et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & development 06/2015; 29(12). DOI:10.1101/gad.257840.114 · 10.80 Impact Factor
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