Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes

Markstein, Michele; Pitsouli, Chrysoula; Villalta, Christians; Celniker, S; Perrimon, Norbert

doi:10.1038/ng.101

Cited by 508 publications

(543 citation statements)

References 47 publications

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“…1A). We used FlyC31 site-specific recombination technology to introduce the slicer-deficient Myc-Piwi transgene ("DUO," which means "two" in Latin, herein refers to two-point mutations) or a wild-type Myc-Piwi control transgene (WT) into a well-characterized genomic site called attP2 to generate transgenic Drosophila strains (21). The WT Myc-Piwi gene has been shown to be fully functional compared with the endogenous piwi gene (22).…”

Section: Resultsmentioning

confidence: 99%

Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity

Darricarrère

Liu

Watanabe

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The Piwi protein subfamily is essential for Piwi-interacting RNA (piRNA) biogenesis, transposon silencing, and germ-line development, all of which have been proposed to require Piwi endonuclease activity, as validated for two cytoplasmic Piwi proteins in mice. However, recent evidence has led to questioning of the generality of this mechanism for the Piwi members that reside in the nucleus. Drosophila offers a distinct opportunity to study the function of nuclear Piwi proteins because, among three Drosophila Piwi proteins-called Piwi, Aubergine, and Argonaute 3-Piwi is the only member of this subfamily that is localized in the nucleus and expressed in both germ-line and somatic cells in the gonad, where it is responsible for piRNA biogenesis and regulatory functions essential for fertility. In this study, we demonstrate beyond doubt that the slicer activity of Piwi is not required for any known functions in vivo. We show that, in transgenic flies with the DDX catalytic triad of PIWI mutated, neither primary nor secondary piRNA biogenesis is detectably affected, transposons remain repressed, and fertility is normal. Our observations demonstrate that the mechanism of Piwi is independent of its in vitro endonuclease activity. Instead, it is consistent with the alternative mode of Piwi function as a molecule involved in the piRNA-directed guidance of epigenetic factors to chromatin.small RNA | RNase H fold | transposable element | sterility

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Section: Resultsmentioning

confidence: 99%

Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity

Darricarrère

Liu

Watanabe

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Luciferase was measured using the Promega Steady-Glo Luciferase Assay Kit, as described in ref. 32. See SI Text for experimental details.…”

Section: Dna Construction and Generation Of Transgenic Fliesmentioning

confidence: 99%

Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy

Storkebaum

Leitão-Gonçalves

Godenschwege

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

161

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“…Dhalo AJ is a deletion of ;19 kb, encompassing the entire halo gene, made using FLP/ FRT-mediated recombination between Exilixis insertions PBac{WH}f04301 and PBac{WH}f07557. Transgenic flies for enhancer assays were established with the landing site line attP40 (w P{nos-phiC31\int.NLS};P{CaryP}attP40) (Markstein et al 2008) for CycE_401, Mef2_401, and CG1488_400-lacZ or attP51C (y w M{eGFP.vas-int.Dm}ZH-2A; M{RFP.attP}ZH-51C) (Bischof et al 2007) for Mef2 I-D[L] and tin B-374. Transgenes for enhancer assays were recombined with the loss-of-function EMS-induced allele sna 18 (sna IIG05 ) (Grau et al 1984) and the hypomorphic sna V2 allele.…”

Section: Drosophila Strainsmentioning

confidence: 99%

A conserved role for Snail as a potentiator of active transcription

et al. 2014

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The transcription factors of the Snail family are key regulators of epithelial–mesenchymal transitions, cell morphogenesis, and tumor metastasis. Since its discovery in Drosophila ∼25 years ago, Snail has been extensively studied for its role as a transcriptional repressor. Here we demonstrate that Drosophila Snail can positively modulate transcriptional activation. By combining information on in vivo occupancy with expression profiling of hand-selected, staged snail mutant embryos, we identified 106 genes that are potentially directly regulated by Snail during mesoderm development. In addition to the expected Snail-repressed genes, almost 50% of Snail targets showed an unanticipated activation. The majority of “Snail-activated” genes have enhancer elements cobound by Twist and are expressed in the mesoderm at the stages of Snail occupancy. Snail can potentiate Twist-mediated enhancer activation in vitro and is essential for enhancer activity in vivo. Using a machine learning approach, we show that differentially enriched motifs are sufficient to predict Snail's regulatory response. In silico mutagenesis revealed a likely causative motif, which we demonstrate is essential for enhancer activation. Taken together, these data indicate that Snail can potentiate enhancer activation by collaborating with different activators, providing a new mechanism by which Snail regulates development.

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Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes

Cited by 508 publications

References 47 publications

Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity

Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity

Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy

A conserved role for Snail as a potentiator of active transcription

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