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
Source: PubMed

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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    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 · 3.15 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 · 6.27 Impact Factor
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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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    ABSTRACT: In animals, females deposit gene products into developing oocytes, which drive early cellular events in embryos immediately after fertilization. As maternal gene products are present before fertilization, the functional manipulation of maternal genes is often challenging to implement, requiring gene expression or targeting during oogenesis. Maternal expression can be achieved through transgenesis, but transgenic approaches are time consuming and subject to undesired epigenetic effects. Here, we have implemented in vitro culturing of experimentally manipulated immature oocytes to study maternal gene contribution to early embryonic development in the zebrafish. We demonstrate phenotypic rescue of a maternal-effect mutation by expressing wild-type product in cultured oocytes. We also generate loss-of-function phenotypes in embryos through either the expression of a dominant-negative transcript or injection of translation-blocking morpholino oligonucleotides. Finally, we demonstrate subcellular localization during the early cell divisions immediately after fertilization of an exogenously provided maternal product fused to a fluorescent protein. These manipulations extend the potential to carry out genetic and imaging studies of zebrafish maternal genes during the egg-to-embryo transition. Developmental Dynamics 242:44-52, 2013. © 2012 Wiley Periodicals, Inc.
    Developmental Dynamics 02/2013; 242(2):1. DOI:10.1002/dvdy.23930 · 2.67 Impact Factor
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