Transgenerational analysis of transcriptional silencing in zebrafish

Abstract

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.

Figure 1. Silencing across Gal4FF bicistronic transgenic insertions

(A) Lateral views of GFP and mCherry labeling in 3 dpf larvae from two transgenic lines in which GFP is regulated by the 4Xnr UAS. GFP expression recapitulates the pattern of mCherry in independently derived F1 larvae, which can be variable in their fluorescence. Representative sibling larvae in the F2 generation show widespread, highly mosaic, or largely absent mCherry and GFP labeling. (B) Colocalization of variegated mCherry and GFP fluorescence in muscle fibers of a c347 F2 larva. (C) Comparison of approximate number of GFP labeled cells in F2 larvae from lines carrying the 4Xnr UAS (c342, c345, c347) and the 14X UAS (c350). (D) Schematic of Gal4FF bipartite reporter construct and analysis of CpG methylation in c347 F2 larva from DNA bisulfite sequencing. Methylation at eleven CpGs within the EF1α promoter and the 4Xnr UAS are indicated on the horizontal axis, with black circles indicating methylated CpGs and open circles representing unmethylated CpGs. Patterns from eight different representative clones from one larva are shown on the vertical axis.

Figure 2. Assay of UAS constructs in binary transgenic system

(A) Experimental scheme using the Tg(ptf1a:Gal4-VP16)^jh16 driver line for transcriptional activation of 14X and 4Xnr UAS:GFP reporter transgenes. (B) Pattern of tissue-specific fluorescence in Tg(ptf1a:GFP)^jh1/+ larvae at 2 dpf (Pisharath et al., 2007). (C) In the presence of the ptf1a driver, larvae from independently derived lines that carry either 14X UAS:GFP or 4Xnr UAS:GFP transgenes show a similar pattern of GFP labeling.

Figure 3. Transgenerational analysis of UAS-regulated GFP expression

(A) Scoring of representative c364 transgenic larvae carrying the ptf1a driver and 14X UAS:GFP reporter transgenes as GFP^high, GFP^med, or GFP^low, based on the extent of GFP labeling within the ptf1a expression domain. (B) Transgenerational analysis of GFP fluorescence in independently derived lines. All F2 larvae were obtained from matings between single F1 adults and AB fish. After F2 larvae were evaluated for their GFP labeling, GFP^med and GFP^high individuals were raised, mated to AB, and their progeny scored in the F3 generation. Only GFP positive F3 larvae were included in the pie charts, which represent the cumulative data from at least five different GFP^med and GFP^high F2 parents for each line.

Figure 4. Correlation between variegated expression and CpG methylation

(A) In the c368 line, variegation in GFP labeling was observed in F1 larvae (not shown) and was more frequently observed in F2 and F3 larvae compared to other 4Xnr UAS lines (compare with Fig. 3B). Few c368 GFP^high larvae were found in either generation. (B) Fluorescence images and representative DNA bisulfite sequencing data for individual GFP^med and GFP^low c368 F2 larvae at 2 dpf. The upper larvae possess both the c368a and c368b transgenic insertions (Table 2), whereas the bottom two larvae have only the c368a transgene. GFP-labeled individuals carrying only the c368b insertion were not detected.

Figure 5. Reduced CpG methylation at the 4Xnr UAS

(A) Fluorescence images and corresponding DNA bisulfite sequencing data for representative 2 dpf F2 larvae. Methylation at the 33 CpGs in the 14X UAS or the 11 CpGs in the 4Xnr UAS promoter are indicated on the horizontal axis, with black circles indicating methylated CpGs and open circles unmethylated CpGs. Patterns from eight different clones are shown on the vertical axis. (B) Fluorescence images and corresponding DNA bisulfite sequencing data for GFP^high (top) and GFP^low (bottom) F3 larvae from 14X UAS lines and GFP^high larvae from 4Xnr UAS lines. (C) Quantification of DNA bisulfite methylation data. Solid bars and striped bars indicate the average percentage of methylation of 14X UAS GFP^high and GFP^low individuals, respectively. Percent methylation corresponds to the number of methylated CpG residues divided by the total number of CpG residues. Error bars represent standard error of the mean, P-values were calculated using the Fisher’s exact test and Mann-Whitney U test, with *p<0.01.