Our bioinformatic analysis identified 10802 IAP LTRs, consistent with recent estimates of ~5400 IAP transposons in the mouse genome as the vast majority of detected insertions in our analysis were paired on either end of a single IAP insertion [15, 28]. To validate candidate LTRs as metastable, we focused primarily on sequences of the specific subtype of IAP known in two cases to be variably methylated (IAPLTR1_Mm). Both the Avy and CabpIAP epialleles exhibit variable DNA methylation at specific CpG sites, which can be influenced by developmental environmental exposure . Phylogenetic analysis revealed that both epialleles clustered in one of two divergent subclades in the IAPLTR1_Mm family. Combined bisulfite and restriction analysis performed on 20 additional loci found interindividual methylation to be a common feature of all elements in this family. Though the degree of variation ranged from 9% to 34% among elements within each mouse, average methylation across all LTRs varied only 3%, a confirmation that global methylation of repetitive loci does not necessarily reflect locus specific deviation. Interestingly, on average, IAP methylation was higher and less variable in the more numerous, phylogenetically conserved elements (black clade 3). While the standard method of dating LTR insertions by measuring accumulated mutations in the 5′ and 3′ LTRs is inapplicable in this case because of their young age, the percent divergence from consensus strongly suggests the two derived subclades are younger. The increased methylation of the older IAPs here fits with the evidence of Reiss et al. who found a positive correlation of age with methylation in another family of ERVs, the Early Transposon (ETn) class and hypothesized this would be a general finding in ERVs .
Though retrotransposons, including IAPs, are generally highly methylated, important windows of vulnerability exist in which genomic methylation is globally reprogrammed [4, 6, 21]. Reactivation during genomic reprogramming is likely responsible for the frequent germ line insertion in this lineage of IAPs in rodents, counterbalanced by selective pressure to maintain epigenetic silencing in somatic cells . The presence of two diverged clades in our phylogeny suggests that the ancestor of the Avy and CabpIAP alleles at two times collected advantageous mutations that allowed it to escape genomic control and proliferate successfully throughout the genome following the master gene model . Their genomic distribution also suggests that they retained retrotranspositional activity after divergence, a finding also supported previously .
Epigenomic studies are increasingly cataloging variation across tissues, disease states, ages, and environmental exposures. It is therefore important to understand the baseline naturally extant variation in a population of isogenic, unexposed, age-matched animals and to provide assays to detect shifts in variation mediated by other conditions. Determination of interindividual variation in DNA methylation has followed a number of methods, including coat color shifts in Avy mice , bisulfite sequencing, northern blots for transcript quantity , methylation specific PCR, and pyrosequencing . The method we use here, combined bisulfite and restriction analysis (CoBRA), exploits site-specific enzyme digestion to determine methylation status. Advantages of CoBRA include semi-quantitative analysis, and relatively inexpensive costs, thereby facilitating the high throughput necessary for epidemiological testing. Indeed, CoBRA has been used to assess the epigenotype of colorectal tumors, providing direct health implications . Additionally, automation platforms such as the QIAxel system (Qiagen Inc., Valencia, CA) would increase the speed of sample quantitation after enzyme digestion. Our candidate loci were also mined from the C57BL/6 genome, and as such, they are more likely to be useful as epigenetic markers in both mutant and exposed mice than previous epialleles (Avy and AxinFu), which are restricted to specific lines.
Previous studies utilizing non-phylogenetic methods of variably methylated candidate identification have identified retroelements co-located with active histone modification (H3K4me3) . Further, IAPs, unlike other transposons, can induce changes in nearby chromatin marks, but no phylogenetic basis underlying epigenetic states has been explored . The close sequence identity of Avy and CabpIAP insertions may be associated structurally with directed epigenetic modifications by targeting enhanced recruitment of trans factors governing their metastability. Alternatively, high sequence similarity in the divergent clades may have resulted from very recent mobilization and high activity of the parent element and its rapid proliferation. We speculate that variable DNA methylation, being a general property of elements we tested from these clades, explains both the success of this lineage in duplication, and their interindividual variation. This hypothesis is borne out by the average methylation being higher in the black clade, containing the largest number, most conserved, and likely oldest, elements. Though in general, new insertions are considered deleterious, some have been co-opted by the genome as a mechanism of driving gene expression, and epigenetically labile insertions may be under selection to “fine tune” that expression . Our analysis supports the notion that mutation to escape genomic control is correlated with both increased retrotranspositional activity and decreased methylation. Consequently, these data are a strong indication that interindividual variation in methylation at repetitive elements is the default condition, rather than a rare occurrence, and that shifts in methylation profiles could affect numerous transcripts in the genome. Thus, environmental exposures capable of altering the epigenome could potentially have widespread effects for gene regulation.