Transcriptional Silencing and Reactivation in Transgenic Zebrafish

Department of Embryology, carnegie Institution for Science, Baltimore, Maryland 21218, USA.
Genetics (Impact Factor: 5.96). 06/2009; 182(3):747-55. DOI: 10.1534/genetics.109.102079
Source: PubMed


Epigenetic regulation of transcriptional silencing is essential for normal development. Despite its importance, in vivo systems for examining gene silencing at cellular resolution have been lacking in developing vertebrates. We describe a transgenic approach that allows monitoring of an epigenetically regulated fluorescent reporter in developing zebrafish and their progeny. Using a self-reporting Gal4-VP16 gene/enhancer trap vector, we isolated tissue-specific drivers that regulate expression of the green fluorescent protein (GFP) gene through a multicopy, upstream activator sequence (UAS). Transgenic larvae initially exhibit robust fluorescence (GFP(high)); however, in subsequent generations, gfp expression is mosaic (GFP(low)) or entirely absent (GFP(off)), despite continued Gal4-VP16 activity. We find that transcriptional repression is heritable and correlated with methylation of the multicopy UAS. Silenced transgenes can be reactivated by increasing Gal4-VP16 levels or in DNA methyltransferase-1 (dnmt1) mutants. Strikingly, in dnmt1 homozygous mutants, reactivation of gfp expression occurs in a reproducible subset of cells, raising the possibility of different sensitivities or alternative silencing mechanisms in discrete cell populations. The results demonstrate the power of the zebrafish system for in vivo monitoring of epigenetic processes using a genetic approach.

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Available from: Marnie E Halpern, Jan 31, 2014
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    • "Tg(tpma:Gal4;UAS:EGFP) adults in which the UAS-linked gene was transcriptionally silent (Fig. 4c). Similar results were Mar Biotechnol observed in previous work in which DNA methylation occurred in both 14 tandem copies of the same upstream activator sequence (14xUAS) (Goll et al. 2009) and four distinct upstream activator sequences arrayed in tandem (4xnr UAS) (Akitake et al. 2011) in zebrafish. Interestingly, the silent egfp was reactivated in the later generation, and the increased transcriptional activity of the egfp gene was accompanied by decreased DNA methylation of the 4xUAS sequence (Fig. 4b, c). "
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    ABSTRACT: The Gal4/upstream activating sequence (UAS) system is a powerful genetic tool for the temporal and spatial expression of target genes. In this study, the dynamic activity of the Gal4/UAS system was monitored in zebrafish throughout the entire lifespan and during germline transmission, using an optimized Gal4/UAS, KalTA4/4xUAS, which is driven by two muscle-specific regulatory sequences. We found that UAS-linked gene expression was transcriptionally amplified by Gal4/UAS during early developmental stages and that the amplification effects tended to weaken during later stages and even disappear in subsequent generations. In the F2 generation, the transcription of a UAS-linked enhanced green fluorescent protein (EGFP) reporter was transcriptionally silent from 16 days post-fertilization (dpf) into adulthood, yet offspring of this generation showed reactivation of the EGFP reporter in some strains. We further show that the transcriptional silencing and reactivation of UAS-driven EGFP correlated with the DNA methylation levels of the UAS regulatory sequences. Notably, asymmetric DNA methylation of the 4xUAS occurred in oocytes and sperm. Moreover, the paternal and maternal 4xUAS sequences underwent different DNA methylation dynamics after fertilization. Our study suggests that the Gal4/UAS system may represent a powerful tool for tracing the DNA methylation dynamics of paternal and maternal loci during zebrafish development and that UAS-specific DNA methylation should be seriously considered when the Gal4/UAS system is applied in zebrafish.
    Marine Biotechnology 05/2015; 17(5). DOI:10.1007/s10126-015-9641-0 · 3.27 Impact Factor
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    • "A more global analysis of zygotic transcription initiation in embryos in which maternal Dnmt1 has been abolished would lend support to this factor being the elusive transcriptional repressor. Early embryonic development is normal in zebrafish dnmt1 mutants, suggesting that zygotic dnmt1 has little if any role in controlling the MZT (Anderson et al., 2009; Goll et al., 2009). However, the MZT has not been studied in zebrafish embryos in which both maternal and zygotic dnmt1 function has been disrupted. "
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    ABSTRACT: The initial phases of embryonic development occur in the absence of de novo transcription and are instead controlled by maternally inherited mRNAs and proteins. During this initial period, cell cycles are synchronous and lack gap phases. Following this period of transcriptional silence, zygotic transcription begins, the maternal influence on development starts to decrease, and dramatic changes to the cell cycle take place. Here, we discuss recent work that is shedding light on the maternal to zygotic transition and the interrelated but distinct mechanisms regulating the onset of zygotic transcription and changes to the cell cycle during early embryonic development.
    Development 10/2014; 141(20):3834-3841. DOI:10.1242/dev.102368 · 6.46 Impact Factor
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    • "Despite of loss of GFP expression, these gene trap lines can still be used as Gal4 drivers, as they successfully transactivate UAS:mRFP. Replacing the 14XUAS with a less repetitive variant such as nrUAS [66] may be worthwhile if sensitivity for low expression levels can be retained. "
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    ABSTRACT: External development and optical transparency of embryos make zebrafish exceptionally suitable for in vivo insertional mutagenesis using fluorescent proteins to visualize expression patterns of mutated genes. Recently developed Gene Breaking Transposon (GBT) vectors greatly improve the fidelity and mutagenicity of transposon-based gene trap vectors. We constructed and tested a bipartite GBT vector with Gal4-VP16 as the primary gene trap reporter. Our vector also contains a UAS:eGFP cassette for direct detection of gene trap events by fluorescence. To confirm gene trap events, we generated a UAS:mRFP tester line. We screened 270 potential founders and established 41 gene trap lines. Three of our gene trap alleles display homozygous lethal phenotypes ranging from embryonic to late larval: nsftpl6, atp1a3atpl10 and flrtpl19. Our gene trap cassette is flanked by direct loxP sites, which enabled us to successfully revert nsftpl6, atp1a3atpl10 and flrtpl19 gene trap alleles by injection of Cre mRNA. The UAS:eGFP cassette is flanked by direct FRT sites. It can be readily removed by injection of Flp mRNA for use of our gene trap alleles with other tissue-specific GFP-marked lines. The Gal4-VP16 component of our vector provides two important advantages over other GBT vectors. The first is increased sensitivity, which enabled us to detect previously unnoticed expression of nsf in the pancreas. The second advantage is that all our gene trap lines, including integrations into non-essential genes, can be used as highly specific Gal4 drivers for expression of other transgenes under the control of Gal4 UAS. The Gal4-containing bipartite Gene Breaking Transposon vector presented here retains high specificity for integrations into genes, high mutagenicity and revertibility by Cre. These features, together with utility as highly specific Gal4 drivers, make gene trap mutants presented here especially useful to the research community.
    BMC Genomics 09/2013; 14(1):619. DOI:10.1186/1471-2164-14-619 · 3.99 Impact Factor
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