Defective folate metabolism causes germline epigenetic instability and distinguishes Hira as a phenotype inheritance biomarker

Abstract

The mechanism behind transgenerational epigenetic inheritance is unclear, particularly through the maternal grandparental line. We previously showed that disruption of folate metabolism in mice by the Mtrr hypomorphic mutation results in transgenerational epigenetic inheritance of congenital malformations. Either maternal grandparent can initiate this phenomenon, which persists for at least four wildtype generations. Here, we use genome-wide approaches to reveal genetic stability in the Mtrr model and genome-wide differential DNA methylation in the germline of Mtrr mutant maternal grandfathers. We observe that, while epigenetic reprogramming occurs, wildtype grandprogeny and great grandprogeny exhibit transcriptional changes that correlate with germline methylation defects. One region encompasses the Hira gene, which is misexpressed in embryos for at least three wildtype generations in a manner that distinguishes Hira transcript expression as a biomarker of maternal phenotypic inheritance.

Disruption of Folate Metabolism Causes Poor Alignment and Spacing of Mouse Conceptuses for Multiple Generations

“…Since Mtrr gt/gt conceptuses at E10.5 derived from Mtrr gt/gt intercrosses showed a similar frequency of skewing (5.8% of conceptuses, p < 0.001) as F2 Mtrr +/+ conceptuses from the MGM pedigree (Figure 8B versus Figure 8C), the mechanism was likely due to Mtrr deficiency in one (or both) maternal grandparent. Notably, the frequency of skewing was greater in F2 Mtrr +/+ progeny when derived from an Mtrr +/gt MGM compared to an Mtrr +/gt MGF (Figure 8C versus Figure 8H) suggesting that the causative mechanism might be different when initiated via the F0 oocyte versus the F0 sperm, as was previously hypothesized (Blake et al, 2021). In further support of this hypothesis, we observed that severe conceptus misalignment occurred until at least until the F4 wildtype generation when derived from an F0 Mtrr +/gt female (Figures 8E,F) yet only until the F3 wildtype generation when derived from an F0 Mtrr +/gt male (Figures 8J,K).…”

“…This nonconventional mode of inheritance is typically caused by exposure to an environmental stressor or metabolic disruption only in the initiating F0 generation and occurs independent of DNA basesequence mutations (Blake and Watson, 2016). The Mtrr gt mouse line is a known model of transgenerational epigenetic inheritance as several embryonic phenotypes are inherited by the wildtype grandprogeny and great grandprogeny when either maternal grandparent is a carrier for the Mtrr gt mutation (Padmanabhan et al, 2013;Blake et al, 2021). While the mechanism of epigenetic inheritance of phenotypes is unknown in the Mtrr gt model and other mammalian multigenerational models, it is particularly difficult to explore when the phenomenon occurs via the maternal lineage.…”

“…Besides neural tube closure defects, other developmental phenotypes observed in Mtrr gt/gt conceptuses at E10.5 are growth restriction, developmental delay, cardiac malformations, and placenta phenotypes (Padmanabhan et al, 2013). While the molecular mechanism and developmental timing of these phenotypes is currently unclear, alterations in genome-wide DNA methylation associated with gene misexpression are apparent in Mtrr gt/gt conceptuses at E10.5 (Padmanabhan et al, 2013;Bertozzi et al, 2021;Blake et al, 2021). This result implicates an epigenetic mechanism.…”

“…Pedigree key: circle, female; squares, males; blue outline, C57Bl/6J mouse strain; red outline, B6D2F1 mouse strain; black outline, Mtrr gt mouse strain; white fill, Mtrr +/+ ; black fill, Mtrr gt/gt ; half black/half white, Mtrr +/gt . Mtrr gt mouse line is known for its multigenerational effects on development specifically through the maternal grandparental lineage (Padmanabhan et al, 2013;Blake et al, 2021). To this end, we reanalyzed our published phenotype data set at E10.5 whereby several genetic pedigrees were established to rigorously assess the phenotypic effects of a maternal grandparental Mtrr gt allele on the subsequent wildtype generations (Padmanabhan et al, 2013;Padmanabhan et al, 2017).…”

“…It is hypothesized that an epigenetic factor(s) is inherited via the germline in a manner that is independent of DNA mutation (Blake and Watson, 2016), yet it is difficult to tease apart the effects of the uterine environment from those intrinsic to the germline. In the case of the Mtrr gt mouse line, an Mtrr gt allele in either maternal grandparent (i.e., the F0 generation) is sufficient to disrupt germ cell DNA methylation (Blake et al, 2021) and cause growth defects and congenital malformations at E10.5 in the wildtype grandprogeny, in some cases up to the F4 wildtype generation (Padmanabhan et al, 2013). This phenomenon occurs when the F1 generation and all subsequent generations are wildtype for the Mtrr gene and display normal folate metabolism (Padmanabhan et al, 2013;Padmanabhan et al, 2018).…”