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Transcriptional silencing and reactivation in transgenic zebrafish. Genetics

Department of Embryology, carnegie Institution for Science, Baltimore, Maryland 21218, USA.
Genetics (Impact Factor: 4.87). 06/2009; 182(3):747-55. DOI: 10.1534/genetics.109.102079
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ABSTRACT 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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    • "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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    • "However, the generation and evaluation of transgenic animals is labor intensive and requires fish husbandry for the months corresponding to a generation time. In addition, transgenes may be subject to position effects and silencing (Choo et al., 2006; Goll et al., 2009; Akitake et al., 2011), complicating both maintenance of stocks and analysis of the consequences of transgenic expression . Methods for the systematic down-regulation of maternal genes could also be achievable in principle through transgenic expression, however , RNA interference (RNAi) during oogenesis, which has been shown to be effective in the mouse (Roy and Matzuk, 2006), has not yet been reported in the zebrafish. "
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