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.
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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.
    Full-text · Article · Oct 2014 · Development
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    • "Owing to its usefulness, this system has been adopted in several other model organisms, such as Arabidopsis (Engineer et al., 2005), Xenopus (Hartley et al., 2002), Medaka (Grabher and Wittbrodt, 2004), zebrafish (Asakawa et al., 2008; Scheer and Campos-Ortega, 1999; Scott et al., 2007), mouse (Hu et al., 2004; Ornitz et al., 1991; Rowitch et al., 1999) and human cell culture (Webster et al., 1988). A serious disadvantage of the Gal4/gUAS system is that the UAS is silenced in subsequent generations in vertebrates due to methylation at CpG nucleotides (Akitake et al., 2011; Goll et al., 2009) (Fig. 1B). This leads to the silencing of the UAS-regulated effector/reporter gene as early as the first (F1) generation and necessitates continual reestablishment of these lines. "
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    ABSTRACT: The ability to visualize and manipulate cell fate and gene expression in specific cell populations has made gene expression systems valuable tools in developmental biology studies. Here, we describe a new system that uses the E. coli tryptophan repressor and its upstream activation sequence (TrpR/tUAS) to drive gene expression in stable zebrafish transgenic lines and in mammalian cells. We show that TrpR/tUAS transgenes are not silenced in subsequent generations of zebrafish, which is a major improvement over some of the existing systems, such as Gal4/gUAS and the Q-system. TrpR transcriptional activity can be tuned by mutations in its DNA-binding domain, or silenced by Gal80 when fused to the Gal4 activation domain. In cases in which more than one cell population needs to be manipulated, TrpR/tUAS can be used in combination with other, existing systems.
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