An N-ethyl-N-nitrosourea screen for genes involved in variegation in the mouse

Abstract

We have developed a sensitized screen to identify genes involved in gene silencing, using random N-ethyl-N-nitrosourea mutagenesis on mice carrying a variegating GFP transgene. The dominant screen has produced six mutant lines, including both suppressors and enhancers of variegation. All are semidominant and five of the six are homozygous embryonic lethal. In one case, the homozygous lethality depends on sex: homozygous females die at midgestation and display abnormal DNA methylation of the X chromosome, whereas homozygous males are viable. Linkage analysis reveals that the mutations map to unique chromosomal locations. We have studied the effect of five of the mutations on expression of an endogenous allele known to be sensitive to epigenetic state, agouti viable yellow. In all cases, there is an effect on penetrance, and in most cases, parent of origin and sex-specific effects are detected. This screen has identified genes that are involved in epigenetic reprogramming of the genome, and the behavior of the mutant lines suggests a common mechanism between X inactivation and transgene and retrotransposon silencing. Our findings raise the possibility that the presence or absence of the X chromosome in mammals affects the establishment of the epigenetic state at autosomal loci by acting as a sink for proteins involved in gene silencing. The study demonstrates the power of sensitized screens in the mouse not only for the discovery of novel genes involved in a particular process but also for the elucidation of the biology of that process.

Fig. 1.

GFP expression profiles in Momme Ds. Erythrocytes from 3-week-old mice from six different mutant lines were analyzed by flow cytometry. In each case, the expression profile of phenotypically mutant offspring in a litter were averaged (red) and overlaid with the average of the WT littermates (black). The x axis represents the erythrocyte fluorescence on a logarithmic scale, and the y axis is the number of cells detected at each fluorescence level. The charts are representative of what occurs in at least 50 litters tested for each mutant line, but because of day-to-day variation in the flow cytometer readings, litters from different days cannot be pooled. For quantitative analysis and statistical significance see Table 1. The GFP-positive gate is indicated as GFP⁺.

Fig. 2.

Momme D1 phenotypes. (a) Erythrocytes from 3-week-old mice from a Momme D1 heterozygous intercross were analyzed. The expression profile of two homozygote mutant offspring were averaged (blue) and overlaid with the average of the three heterozygote (red) and two WT littermates (black). The homozygotes are significantly different from the heterozygotes (P < 0.01). The x axis represents the erythrocyte fluorescence on a logarithmic scale, and the y axis is the number of cells detected at each fluorescence level. The charts are representative of the 29 litters tested, but because of day-to-day variation in the flow cytometer readings, litters from different days cannot be pooled. (b) Embryonic day 10.5 embryos derived from an FVB/C57 F₁ heterozygous intercross were genotyped and sexed. Samples were bisulfite-converted, and the hprt promoter region was amplified. Clones from five homozygous, five heterozygous, and five WT females and from two WT males are shown. A gray box represents a methylated CpG, and a white box indicates unmethylated CpG. The homozygous females completely lack methylation at this locus. Where there is a separation between clones from the same embryo, clones were derived from different bisulfite conversions.

Fig. 3.

Momme D1, D2, and D4 shift penetrance at the A^vy allele. A^vy/a C57BL/6J mice of the indicated phenotype were mated with Momme D^–/+ FVB/NJ mice. Data were produced from at least five different mating pairs. Offspring not carrying the A^vy allele have been omitted from the pedigrees. (a) Paternal inheritance of Momme D1. Pedigrees from A^vy/a dams mated with Momme D1^–/+ FVB/NJ sires. The Momme D1^–/+ mutants shift toward yellow when the A^vy allele is inherited from a pseudoagouti dam (P < 0.0001). The data shown have been reanalyzed according to the sex of the offspring. There is no significant shift in the proportion of phenotypes observed between mutant and WT male littermates. In the female offspring, there is a significant shift toward yellow, removing the mottled class entirely (P < 0.0001). (b) Maternal inheritance of Momme D2. Pseudoagouti A^vy/a C57BL/6J sires were mated with Momme D2^–/+ FVB/NJ dams. When the data were analyzed according to the sex of the offspring, there was a significant shift in the mutant female offspring toward a yellow phenotype (P = 0.01). There is no shift in the proportion of phenotypes observed between mutant and WT male littermates. (c) Paternal inheritance of Momme D4. Yellow A^vy/a C57BL/6J dams were mated with Momme D4^–/+ FVB/NJ sires. There was a significant shift toward the mottled phenotype in both male and female offspring.