Identification Characterization and Heritability of Murine Metastable Epialleles: Implications for Non-genetic Inheritance

摘要

class="head no_bottom_margin" id="sec1title">IntroductionMost interindividual phenotypic variation is explained by genetic variation. However, studies in plant and animal models indicate that non-genetic mechanisms can contribute to phenotypic variability, and such phenotypes can be inherited over multiple generations (, , ). Epigenetic changes in the absence of genetic effects have been reported to have long-lasting phenotypic outcomes over multiple generations in non-mammalian organisms. In mammals, such non-genetic effects are difficult to explain mechanistically, and it has been challenging to define the regulatory processes underlying the observed phenomena ().Two of the best-characterized paradigms of non-genetic inheritance in mammals occur at the murine Agouti viable yellow (A^vy) and Axin Fused (Axin^Fu) loci (, ). In these naturally occurring mutant mice, genetically identical individuals exhibit quantifiable phenotypic variability in coat color or tail morphology due to the insertion of an endogenous retrovirus (ERV) of the intracisternal A particle (IAP) class into the Agouti or the Fused loci, respectively. The range of phenotypes correlates reproducibly with interindividual differences in the level of DNA methylation at a long terminal repeat (LTR) promoter of the IAP, driving abnormal expression of the genes (, ). The consistency in methylation level observed within an individual is in contrast to the variation of methylation levels and phenotypic outcomes observed between individuals, defining A^vy and Axin^Fu as so-called “metastable epialleles” (). Transgenerational inheritance of the methylation pattern at these metastable epialleles has been observed, whereby the distribution of phenotypes in the offspring was shown to be dependent on parental phenotype (, ). Furthermore, A^vy is susceptible to environmental influence impacting methylation and phenotype (, , , ). Using genetic screens, proteins with epigenetic function associated with the maintenance of A^vy have been identified (href="#bib9" rid="bib9" class=" bibr popnode">Daxinger et al., 2013). In another study, a C57BL/6J endogenous IAP insertion at Cdk5rap1 regulates transcriptional dosage via promoter methylation; however, an association with phenotype has not been reported (href="#bib14" rid="bib14" class=" bibr popnode">Druker et al., 2004). Together, these studies suggest that ERVs of the IAP subclass have the potential to be variably methylated, here referred to as variably methylated IAPs (VM-IAPs).The properties and underlying mechanisms governing the establishment, behavior, and inheritance of VM-IAPs remain elusive, as does the extent to which they represent a genome-wide phenomenon. 45% of the murine genome is made up of repetitive sequences, with ERVs comprising about 12% of the genome. In the C57BL/6J genome, there are approximately 12,000 ERVs of the IAP subclass (href="#bib49" rid="bib49" class=" bibr popnode">Smit et al., 2015). The degree to which this substantial fraction of the repeat genome might modulate phenotype is unclear, and the total number of naturally existing murine VM-IAPs is unknown to date.Previous studies have searched for metastable epialleles with limited success. Strategies have included surveying expression microarray data for within-strain interindividual expression patterns, screening for retrotransposons that neighbor promoters marked by the active histone modification H3K4me3, and conducting a phylogenetic analysis on IAP elements (href="#bib57" rid="bib57" class=" bibr popnode">Weinhouse et al., 2011, href="#bib16" rid="bib16" class=" bibr popnode">Ekram et al., 2012, href="#bib65" rid="bib65" class=" bibr popnode">Faulk et al., 2013). A recent more extensive screen used comparative whole-genome bisulfite sequencing (WGBS) data and described 55 ERV regions exhibiting some interindividual differential methylation, with validation in two tissues shown for four (href="#bib36" rid="bib36" class=" bibr popnode">Oey et al., 2015). This study confirmed that naturally occurring germline mutations and interindividual genetic differences do not underlie the epigenetic variation observed at the identified regions. While individually informative, there is little or no overlap between the results of these screens. The more challenging task of identifying human metastable epialleles has been tackled before, but the genetic heterogeneity associated with human cohorts remains a significant hindrance in such studies (href="#bib48" rid="bib48" class=" bibr popnode">Silver et al., 2015).Here, we report a novel high-stringency genome-wide approach to comprehensively identify VM-IAPs. Using WGBS and RNA sequencing (RNA-seq) datasets generated from pure non-cycling populations of ex vivo purified naive B and T cells, we identified individual elements possessing features of metastable epialleles. After extensive validation, we have characterized their relationship to each other and to the vast majority of IAPs in the genome that are fully and stably modified. Furthermore, we determined their patterns of inheritance from one generation to the next. Our study identifies a repertoire of loci with the potential to act as markers of normal and compromised environmental contexts and as tools to uncover mechanisms of non-genetic inheritance and, more generally, to provide insights into the mechanisms of silencing at repeats and the impact of mammalian repetitive elements on genome function and phenotype.