An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse

Abstract

Background: We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes).

Results: We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly ,abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance.

Conclusions: Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines.

Figure 1

An ENU mutagenesis/whole exome deep sequencing pipeline enables rapid identification of causative mutations in mice with defects in epigenetic gene silencing. A schematic overview of the ENU mutagenesis gene discovery pipeline is presented. The major components of the screen are described in the figure. Briefly, male FVB/NJ mice carrying an epigenetically sensitive GFP transgene (Line3) were treated with ENU and mated with female Line3 mice. Offspring were screened for a shift in the percentage of GFP expressing erythrocytes using flow cytometry. Putative mutants were mated with Line3 mice for four generations to test for heritability and reproducibility of the GFP expression, and to reduce the number of non-causative ENU mutations within the genomes. Linkage analysis was carried out on the offspring from two generations of backcrosses between putative mutants and C57BL/6J mice, which also carry the GFP transgene (Line3C). Causative mutations were identified by whole exome deep sequencing, or gene candidate sequencing on individuals that had been maintained on the Line3 background for at least seven generations. FACS, fluorescence-activated cell sorting.

Figure 2

Causative mutations in MommeD30, MommeD18, MommeD8, MommeD28 and MommeD34. (a)MommeD30 carries a 1 bp deletion in Wiz that leads to a frame-shift at amino acid 553. Western blot analysis of embryo heads at 12.5 days post-coitum (dpc) shows reduced levels of Wiz protein in Wiz^MommeD30 heterozygotes and no Wiz protein in homozygotes. Wiz protein was detected at approximately 120 and approximately 130 kDa. Each track represents a different animal. Tubulin was used as a loading control. (b)MommeD18 harbors a point mutation in Rif1 that introduces a premature stop codon at amino acid 1,669. Western analysis of testes tissue shows that Rif^MommeD18 heterozygotes have a reduced dosage of Rif1. No Rif1 protein was detected in a homozygote. Each track represents a different animal. Rif1 protein was detected at approximately 260 kDa and Tubulin was used as a loading control. (c) ENU point mutations in MommeD8, MommeD28 and MommeD34 occur in conserved regions of the Rlf protein. Asterisks indicate mutation. (d) Western blotting of Rlf in embryo head lysates from 14.5 dpc Rlf^+/+, RlfMomme^D8/D8, Rlf^D28/D28 and Rlf^D34/D34 revealed greatly reduced amounts of Rlf protein. Each track represents a different animal. Rlf protein was detected at approximately 280 kDa. Tubulin was used as a loading control. (e) Total, cytoplasmic and nuclear fractions of HeLa cells were isolated and protein concentration quantified. Equal amounts of each fraction were immunoblotted with anti-Rlf, GAPDH (cytoplasmic marker) and SMARCA5 (nuclear marker) antibodies, revealing nuclear localization of RLF. Rlf protein was detected at approximately 280 kDa, GAPDH protein at 37 kDa and SMARCA5 at approximately 120 kDa. (f) Coat color of offspring carrying the A^vy allele produced from a Rlf^MommeD8 heterozygous female crossed to a pseudoagouti A^vy male. Rlf^MommeD8 heterozygous offspring showed a shift in coat color towards pseudoagouti compared to wild-type littermates.

Figure 3

Causative mutations in MommeD19, MommeD27, MommeD39, MommeD42, MommeD13, MommeD33 and MommeD40. (a)MommeD19 carries a mutation at a splice site of Smarcc1. Real-time RT-PCR and immunoblotting of embryos at 12.5 days post-coitum (dpc) showed reduced levels of Smarcc1 mRNA and Smarcc1 protein in heterozygotes (n ≥ 5 mice). Each track represents a different animal. Smarcc1 protein was detected at approximately 160 kDa. (b)MommeD27 harbors a mutation in a bromodomain of Pbrm1. Western blot analysis of 14.5 dpc embryo heads showed reduced levels of Pbrm1 in homozygotes. Each track represents a different animal. Pbrm1 was detected at approximately 190 kDa. (c)MommeD39 carries a mutation at a splice site of Smarca4. Real-time RT-PCR showed reduced Smarca4 mRNA in testes of heterozygotes (n ≥ 4 mice). cDNA sequencing revealed that the mutation in Smarca4^MommeD39 results in use of an alternative splice donor site. (d) The mutation in MommeD42 introduces a premature stop codon at amino acid 411 of the Brd1 protein. (e) Mutations in MommeD13 and MommeD17 occur in the conserved SET domain of Setdb1. Real-time RT-PCR of Setdb1 mRNA from testes of heterozygotes and age-matched wild types (n = 4 mice). cDNA analysis revealed that the Setdb1^MommeD13 allele is associated with the use of an alternative splice donor site in exon 20, leading to a 62 bp truncation. (f)MommeD33 carries a mutation at a splice site in Suv39h1. Western blot analysis of Suv39h1 in adult thymus showed reduced Suv39h1 in hemizygous mutant males. Each track represents a different animal. Suv39h1 protein was detected at 48 kDa. (g) The mutation in MommeD40 introduces a premature stop codon at amino acid 778 of Uhrf1. Western blot analysis of Uhrf1 revealed greatly reduced levels in 9.5 dpc embryos homozygous for the Uhrf1^MommeD40 mutation. Each track represents a different animal. Uhrf1 protein was detected at approximately 90 kDa. Error bars indicate ± standard error of the mean. N.s., not significant. Asterisks indicate mutation.

Figure 4

MommeD genes involved in transgene silencing in the mouse. Using an ENU mutagenesis screen, 18 unique genes were identified to be involved in transgene silencing in the mouse. Factors are grouped by their broad mechanistic role in epigenetic regulation. TF, transcription factor; Me, methyl. Figure adapted from [2].

Figure 5

MommeD mutants show abnormal embryonic development. Timed matings and intercrosses of MommeD mutants. (a)Wiz^MommeD30 mice; data show the number of mice observed (and in brackets the percentage) at 10.5 days post-coitum (dpc), 12.5 dpc, 14.5 dpc and at weaning. Representative embryos are shown. (b)Rif1^MommeD18 mice; data show the number of mice observed (and in brackets the percentage) at 10.5 to 12.5 dpc, 14.5 dpc and at weaning. na, not applicable. (c)Rlf mutants; data show the number of mice observed (and in brackets the percentage). Embryonic weights were measured from intercrosses of Rlf^MommeD34 and Rlf^MommeD28 mice. Homozygous embryos from both had a significant increase in weight variation at 17.5 dpc or 16.5 dpc, respectively. Weights for each litter were normalized to the average weight of wild-type embryos in that litter. Each data point represents an individual. Homozygotes were smaller than wild-type littermates at one week after birth. ns, not significant. (d)Setdb1^MommeD13 and Setdb1^MommeD17 mice; data show the number of mice observed (and in brackets the percentage). (e)Smarcc1^MommeD19 and Smarcc1^MommeD19 mated to wild-type mice revealed incomplete penetrance of heterozygous lethality after birth. Data show the number of mice (and in brackets the percentage). Weights of 17.5 dpc Smarcc1^MommeD19 embryos and pups were, on average, less than that of wild-type littermates (T-test, P < 0.0001) and showed greater variation (F-test, P < 0.05). Weights in each litter were normalized to the average weight of wild-type embryos in that litter. Each data point represents an individual. (f)Smarca4^MommeD39 heterozygotes showed reduced viability. Data show the number of mice and in brackets the percentage. Heterozygotes were smaller than their wild-type littermates. Smarc4^MommeD39/+ weighed less (T-test, P < 0.0001) and weights were more variable (F-test, P<0.0001) than wild types. Weights in each litter were normalized to the average weight of wild-type embryos in that litter. Each data point represents an individual.

Figure 6

Bisulfite sequencing of the HS-40 enhancer region of the GFP transgene. (a) DNA methylation levels were reduced in Dnmt1^MommeD32 heterozygous mice compared to wild-type mice in adult spleens (n = 4 wild-type mice and n = 2 Dnmt1^MommeD32 mutant mice). Filled circles represent methylated CpG sites. (b) DNA methylation levels in adult spleens were unaffected in mice heterozygous for Wiz^MommeD30 and Rif^MommeD18 (n = 4 for Wiz^MommeD30 and n = 2 for Rif1^MommeD18). (c) DNA methylation levels in adult spleens of Rlf^MommeD8, Rlf^MommeD28 and Rlf^MommeD34 heterozygotes were increased compared to wild-type mice (n = 2 mice for all genotypes). (d)Rlf homozygotes showed significantly increased DNA methylation (T-test, P < 0.05) in adult male spleen (Rlf^MommeD8 and Rlf^MommeD34) and 16.5 dpc embryo (Rlf^MommeD28) compared to wild types (n = 3 for all genotypes).