Transgenerational epigenetic effects of the Apobec1 cytidine deaminase deficiency on testicular germ cell tumor susceptibility and embryonic viability

Abstract

Environmental agents and genetic variants can induce heritable epigenetic changes that affect phenotypic variation and disease risk in many species. These transgenerational effects challenge conventional understanding about the modes and mechanisms of inheritance, but their molecular basis is poorly understood. The Deadend1 (Dnd1) gene enhances susceptibility to testicular germ cell tumors (TGCTs) in mice, in part by interacting epigenetically with other TGCT modifier genes in previous generations. Sequence homology to A1cf, the RNA-binding subunit of the ApoB editing complex, raises the possibility that the function of Dnd1 is related to Apobec1 activity as a cytidine deaminase. We conducted a series of experiments with a genetically engineered deficiency of Apobec1 on the TGCT-susceptible 129/Sv inbred background to determine whether dosage of Apobec1 modifies susceptibility, either alone or in combination with Dnd1, and either in a conventional or a transgenerational manner. In the paternal germ-lineage, Apobec1 deficiency significantly increased susceptibility among heterozygous but not wild-type male offspring, without subsequent transgenerational effects, showing that increased TGCT risk resulting from partial loss of Apobec1 function is inherited in a conventional manner. By contrast, partial deficiency in the maternal germ-lineage led to suppression of TGCTs in both partially and fully deficient males and significantly reduced TGCT risk in a transgenerational manner among wild-type offspring. These heritable epigenetic changes persisted for multiple generations and were fully reversed after consecutive crosses through the alternative germ-lineage. These results suggest that Apobec1 plays a central role in controlling TGCT susceptibility in both a conventional and a transgenerational manner.

Fig. 1.

Effects of Apobec1 deficiency on TGCTs. Parental and offspring Apobec1^ko/ko homozygosity (test 1). *P < 0.05.

Fig. 2.

Effects of partial deficiency of Apobec1 on TGCTs. (A) Parental Apobec1^ko/+ heterozygosity (test 2). (B) Parental Apobec1^ko/ko homozygosity and offspring Apobec1^ko/+ heterozygosity (test 3). *P < 0.05.

Fig. 3.

Effects of switching parent of origin on TGCTs. χ² contingency tests were used to determine whether the two groups were derived from populations with similar properties, whereas χ² goodness-of-fit tests were used to determine whether a given result was consistent with the baseline prevalence of 7%. (A) Ancestral genotype and switched transmission from ancestral females to males (test 4). Details for comparisons: 0% before switch versus 0% one generation after switch: not significant; 0% one generation after switch versus 9% two generations after switch: χ² = 6.6, P < 0.02; 0% one generation after switch versus 7% baseline expectation: χ² = 6.4, P < 0.02; 9% two generations after switch versus 7% baseline expectation: χ² = 0.8, not significant. (B) Ancestral genotype and switched transmission from ancestral males to females (test 4). Details for comparisons: 14% before switch versus 13% one generation after switch: not significant; 13% one generation after switch versus 0% two generations after switch: χ² = 3.3, P < 0.07; 13% one generation after switch versus 7% baseline expectation: χ² = 5.7, P < 0.02; 0% two generations after switch versus 7% baseline expectation: χ² = 0.1, not significant. * indicates a significant difference relative to the baseline TGCT prevalence in 129 (+/+)_p control males.

Fig. 4.

Ancestral Apobec1 genotype controls TGCT susceptibility across multiple generations. (A) Female germ-lineage transmission of Apobec1 deficiency before switching the direction of the cross. (B) Male germ-lineage transmission of Apobec1 deficiency before switching the direction of the cross. Red text indicates crosses where the ko allele was maternally transmitted, and blue text indicates crosses where the ko allele was paternally transmitted. *P < 0.05. ‘+/+’ indicates a genotypically wild-type mouse that had at least one ancestor that was ko; this designation is used to distinguish these mice from genotypically similar mice from a conventional wild-type (+/+) strain.

Fig. P1.

Contrasting patterns of conventional, parent-of-origin, and transgenerational inheritance for testicular cancer risk. (A) Parent-of-origin and conventional inheritance in which the occurrence of affected males depends on the sex of the parent. Maternal ko/+ heterozygosity significantly reduces risk among both ko/+ and ^+/+ male offspring (parent-of-origin inheritance), whereas paternal ko/+ heterozygosity affects risk only among ko/+ but not ^+/+ male offspring (conventional inheritance). (B) Transgenerational inheritance in which an individual’s risk depends on genetic effects in ancestral generations. Cancer risk is reduced among ^+/+ male offspring when the great-grandmaternal genotype is ko/+ but not when the great-grandpaternal genotype is ko/+. The ko symbol denotes mutated Apobec1; ko/+ are carriers of the mutation; and ^+/+ are wild type (nonmutant). The baseline frequency of affected males is ∼7% in TGCT-susceptible inbred strains. The letter “N” denotes the count of sequential generations. By convention, the genotype of females precedes that of males.