Phylogenetic and DNA methylation analysis reveal novel regions of variable methylation in the mouse IAP class of transposons

Abstract

Background: Select retrotransposons in the long terminal repeat (LTR) class exhibit interindividual variation in DNA methylation that is altered by developmental environmental exposures. Yet, neither the full extent of variability at these "metastable epialleles," nor the phylogenetic relationship underlying variable elements is well understood. The murine metastable epialleles, Avy and CabpIAP, result from independent insertions of an intracisternal A particle (IAP) mobile element, and exhibit remarkably similar sequence identity (98.5%).

Results: Utilizing the C57BL/6 genome we identified 10802 IAP LTRs overall and a subset of 1388 in a family that includes Avy and CabpIAP. Phylogenetic analysis revealed two duplication and divergence events subdividing this family into three clades. To characterize interindividual variation across clades, liver DNA from 17 isogenic mice was subjected to combined bisulfite and restriction analysis (CoBRA) for 21 separate LTR transposons (7 per clade). The lowest and highest mean methylation values were 59% and 88% respectively, while methylation levels at individual LTRs varied widely, ranging from 9% to 34%. The clade with the most conserved elements had significantly higher mean methylation across LTRs than either of the two diverged clades (p = 0.040 and p = 0.017). Within each mouse, average methylation across all LTRs was not significantly different (71%-74%, p > 0.99).

Conclusions: Combined phylogenetic and DNA methylation analysis allows for the identification of novel regions of variable methylation. This approach increases the number of known metastable epialleles in the mouse, which can serve as biomarkers for environmental modifications to the epigenome.

Figure 1

Multiple sequence alignment. The sequence of the A^vy and Cabp^IAP LTR insertions are compared to the IAPLTR1_Mm consensus sequence. A^vy shares 98.5% sequence identity with Cabp^IAP (85% sequence identity over the length of Cabp^IAP). Colors indicate base changes from consensus (A = red, T = blue, C = yellow, G = green).

Figure 2

Neighbor-joining tree. Illustrated are 1388 IAPLTR_Mm elements drawn from the C57BL/6 genome and including A^vy and Cabp^IAP elements for a total of 1390. Subclades are highlighted in red (clade 1) and green (clade 2), with the remaining elements in black (clade 3). The bifurcation of subclades 1 and 2 demonstrates duplication and divergence events followed by rapid radiation of these subfamilies. Scale bar indicates number of nucleotide substitutions per site.

Figure 3

IAP element insertions. The 1388 insertions of IAPLTR1_Mm are plotted against their genomic location. The color of the clade corresponding to the insertion’s position on the phylogenetic tree is highlighted to the right of each chromosome. Elements from each clade in the tree are found dispersed throughout the genome. The color scale internal to the chromosomes corresponds to overall repetitive element density.

Figure 4

CoBRA design. For each candidate locus we amplify a portion of the antisense strand of the 5′ LTR and unique flanking sequence (arrows) containing a BceAI recognition site. (A) The product of IAP236 is shown with cut sites and CpGs indicated by numbers 1–4. Sites 2 and 3 are conserved in all candidate loci and cut. PCR primers are underlined. (B) Percent methylation is derived from relative intensity of the cut vs. uncut bands. This locus exhibits an 18% range in variable methylation. Controls are shown in lanes 1 and 2, indicating the specificity and completeness of the digestion.

Figure 5

Candidate loci with interindividual variation. DNA from 17 isogenic mouse livers at d21 was isolated and bisulfite converted. Amplification and digestion of 21 representative IAP elements show variable levels of methylation per locus and mouse. Upper bands indicate uncut PCR product corresponding to the unmethylated BceAI site. Lower bands indicate a fully methylated BceAI site consisting of CpGs 2 and 3 from Figure 4. Locus 281Y is located on the Y chromosome and is therefore amplified only from male animals.

Figure 6

Boxplot of methylation values. Interindividual variation in each IAP correlates with phylogenetic clade. Average methylation values are lower for red and green clades (70% and 68%) than for the black clade (79%) suggesting higher methylation for older insertions. The average range per clade, in parentheses, is also lower, however the standard deviation is similar across clades.