Transgenerational analysis of transcriptional silencing in zebrafish

• Published in: Developmental biology 2011-04, Vol.352 (2), p.191-201
• Main Authors: Akitake, Courtney M., Macurak, Michelle, Halpern, Marnie E., Goll, Mary G.
• Format: Article
• Language: English
• Subjects: Animals; Animals, Genetically Modified; Base Sequence; Danio rerio; DNA Methylation; DNA Primers - genetics; DNA-Binding Proteins - genetics; Drosophila; Epigenesis, Genetic; fluorescence; Freshwater; Gal4; gene expression; Gene Expression Regulation, Developmental; Gene Silencing; Genes, Reporter; genetically modified organisms; green fluorescent protein; Green Fluorescent Proteins - genetics; guidelines; larvae; Recombinant Proteins - genetics; reporter genes; Saccharomyces cerevisiae Proteins - genetics; transcription (genetics); Transcription Factors - genetics; Transcriptional Activation; Transgene silencing; transgenes; transposons; yeasts; Zebrafish - genetics; Zebrafish - growth & development
• ISSN: 0012-1606; 1095-564X
• DOI: 10.1016/j.ydbio.2011.01.002

The yeast Gal4/UAS transcriptional activation system is a powerful tool for regulating gene expression in Drosophila and has been increasing in popularity for developmental studies in zebrafish. It is also useful for studying the basis of de novo transcriptional silencing. Fluorescent reporter genes under the control of multiple tandem copies of the upstream activator sequence (UAS) often show evidence of variegated expression and DNA methylation in transgenic zebrafish embryos. To characterize this systematically, we monitored the progression of transcriptional silencing of UAS-regulated transgenes that differ in their integration sites and in the repetitive nature of the UAS. Transgenic larvae were examined in three generations for tissue-specific expression of a green fluorescent protein (GFP) reporter and DNA methylation at the UAS. Single insertions containing four distinct upstream activator sequences were far less susceptible to methylation than insertions containing fourteen copies of the same UAS. In addition, transgenes that integrated in or adjacent to transposon sequence exhibited silencing regardless of the number of UAS sites included in the transgene. Placement of promoter-driven Gal4 upstream of UAS-regulated responder genes in a single bicistronic construct also appeared to accelerate silencing and methylation. The results demonstrate the utility of the zebrafish for efficient tracking of gene silencing mechanisms across several generations, as well as provide useful guidelines for optimal Gal4-regulated gene expression in organisms subject to DNA methylation. ► Widely used 14X UAS is transcriptionally silenced in transgenic zebrafish. ► CpG methylation at the 14X UAS causes silencing of transgene expression. ► Transgenes integrated near transposons are rapidly silenced in the first generation. ► New, less repetitive 4X UAS reduces transgene silencing and methylation. ► High reporter expression persists for at least three generations using new 4X UAS.