BACKGROUND
across mammalian genomes, the retrotransposon class of repeat elements make up nearly half the nuclear dna, with long terminal repeats  alone accounting for 8-10%  <cit> .
many retrotransposons retain the ability to move  to new locations in the genome, a potentially deleterious phenomenon that can result in direct disruption to coding regions  <cit> .
therefore, the suppression of retrotransposons is vital to organismal survival, particularly through sensitive life stages, and is primarily accomplished via epigenetic mechanisms, including dna methylation  <cit> .
epigenetic suppression is typically established early in development at the blastocyst stage and maintained during differentiation of the primordial germ cells  <cit> .
importantly, in certain cases suppression is incomplete, leading to adverse consequences  <cit> .
alternatively, this incomplete suppression may be an evolutionarily selected method of fine-tuning gene expression  <cit> .
genome-wide, little is known about the extent to which interindividual variation of dna methylation at these elements is affected by environmental factors and/or their phylogenetic lineage  <cit> .
intracisternal a particles  are a class of murine retrotransposons named for the appearance of budding doughnut shaped particles in the cisternae of the endoplasmic reticulum  <cit> .
iaps are members of the endogenous retrovirus family  class ii  <cit> .
structurally, a full length iap consists of gag, pro, and pol genes capped at each end by a direct pair of ltrs, each approximately 350 bp in length  <cit> .
however, the largest majority of iap transcripts issue from truncated  <dig>  kb copies of iap of subtype iΔ <dig> rather than full length  <dig>  kb copies  <cit> .
previous work has identified only a subset of elements likely to be active, due to genomic position, sequence mutation, or methylation status  <cit> .
the number of paralogous iap ltrs in the genome has been uncertain with estimates ranging from less than  <dig> to over  <dig> with significant polymorphism among strains  <cit> .
this family of ervs is highly active and rapidly transposing, with estimates of 60% of elements being strain specific  <cit> .
it is advantageous for these mobile elements to be kept under tight control by heavy dna methylation and tight chromatin structure.
a small number of “metastable epialleles” have been identified in mice  in which variable methylation of the 5′ ltr of an antisense iap insertion results in dysregulation of gene expression concomitant with the level of methylation  <cit> .
metastable epialleles show variable expression among individuals with epigenetic profiles that are consistent across tissues and are thus likely to have been set prior to germ line differentiation  <cit> .
further, the distribution of variable expressivity has been shifted at these metastable epialleles following maternal exposure to nutritional and environmental factors  <cit> .
to date, no studies have determined the genetic relationship underlying the ability of these iap ltrs to be variably methylated.
two of the epialleles, avy and axinfu, were identified due to dramatic phenotypes induced by their insertion into nearby protein-coding genes  <cit> .
conversely, cabpiap was identified by a search of genbank cdna databases for chimeric sequence containing iap ltr and genic sequence  <cit> .
to systematically investigate the relationship of genetics and metastability we applied phylogenetic methods to identify candidate iap retrotransposons and validate their variable methylation status.
through this approach, we find the phylogeny of metastable loci sheds light on the evolutionary basis of metastability.
additionally, the validated analysis of epigenetic state across individual isogenic mice now increases the number of known metastable epialleles, which can serve as a test panel for environmental modifications to the epigenome.
RESULTS
iap distribution in the genome
initial pairwise alignment of the two most well-studied metastable epialleles, avy and cabpiap revealed that the ltrs of these two insertions exhibit  <dig> % sequence identity over the length of shared sequence .
repeatmasker was used to scan the c57bl/ <dig>  mouse genome and identified  <dig> iap ltr elements.
the majority of these ltrs were pairs found on the 5’ and 3’ end of the same iap insertion, however a fraction were present as solo ltrs.
since the iaps underlying both avy and cabpiap are of the class iΔ <dig>  type iapltr1_mm as identified in repbase, this subtype was used to filter the complete list of transposons, resulting in a total list of  <dig> iapltr1_mm elements after removing  <dig> unmapped, short, or non-alignable elements.
there was no bias in the orientation of insertion as  <dig> sense and  <dig> anti-sense iapltr1_mm elements were identified throughout the genome.
phylogenetic similarity of known metastable iaps
a neighbor-joining tree of these  <dig> elements revealed three distinct clades with avy and cabpiap clustering within clade  <dig> , more closely than 99% of all other elements .
the largest cluster, clade  <dig> , contains the most conserved elements, and consists of  <dig> sequences with an average sequence divergence of only  <dig> % from the consensus.
clade  <dig>  and clade  <dig>  contain  <dig> and  <dig> elements each, and are more divergent, with  <dig> % and  <dig> % average differences from the consensus respectively.
bias in location of these insertions was not identified, with members of each clade represented across numerous chromosomes and locations .
unique identifiers were assigned to each element in our list, in the format iap27-iap <dig>  used throughout the study.
characterization of interindividual metastability
from these candidates  <dig> from each clade were randomly selected to test for variable methylation using combined bisulfite and restriction analysis .
primers for cobra were designed with the forward primer internal to the iap sequence and the reverse primer located in the flanking sequence , providing an amplicon specific to an insertion and containing homologous iap element sequence .
bceai enzyme was chosen since each element contains two cpg sites as part of a bceai restriction site, acggcg.
loss of methylation at either or both would prevent bceai activity.
consequently, the relative intensity of cut vs. uncut bands gives a semi-quantitative measurement of methylation at these sites .
uncut bands correspond to unmethylated dna while cut bands correspond to methylated dna.
the two cpg sites examined here exhibited interindividual variation in previous studies from our group  <cit> , and are identified as sites  <dig> and  <dig> in this study.
a quantitated cobra gel image of iap <dig> is depicted in figure 4b, revealing an 18% range of methylation at the cut site.
a group of  <dig> isogenic avy/a genotype mice of both genders  from  <dig> litters was sacrificed at weaning and dna was extracted from the liver.
following bisulfite conversion, candidate regions were amplified via pcr and digested with bceai .
a total of  <dig> representative loci,  <dig> from the red clade including cabpiap, and  <dig> each from the green and black clades were evaluated.
the 0% control  was uncut in all loci, while the high percent methylated control was nearly completely digested in all loci  indicating sufficient enzyme activity and specificity.
the mean methylation across all mice for all iap insertions was  <dig> % and no mouse deviated by more than 3% , showing no statistical difference  .
specific loci however, exhibited much greater variability.
we found that mean methylation of individual iaps fluctuated from a low of 59% to a high of 88% and standard deviations varied from  <dig>  to  <dig> , indicating that individual transposons can differ dramatically in both average value and standard deviation among mice.
thus, methylation variability ranged from 9% for the least variable element to greater than 34% for the most variable element.
there were no significant differences in methylation between sexes.
importantly, the average methylation was significantly lower for both red clade elements, 70% , and green clade elements, 68%  than for black clade elements, 79%.
interestingly, the range through which a particular element can be methylated is also correlated with its position within the phylogenetic tree .
the average range for red clade elements is 21%, 17% for green clade elements, and just 11% for black clade elements.
the element with the greatest range is in the red clade  while the element with the greatest dispersion as measured by interquartile range is found in the black clade .
* iap located on the y chromosome, only measured in male animals.
lod = limit of detection.
discussion
our bioinformatic analysis identified  <dig> iap ltrs, consistent with recent estimates of ~ <dig> 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  <cit> .
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 .
both the avy and cabpiap epialleles exhibit variable dna methylation at specific cpg sites, which can be influenced by developmental environmental exposure  <cit> .
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  <dig> 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 .
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  class and hypothesized this would be a general finding in ervs  <cit> .
though retrotransposons, including iaps, are generally highly methylated, important windows of vulnerability exist in which genomic methylation is globally reprogrammed  <cit> .
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  <cit> .
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  <cit> .
their genomic distribution also suggests that they retained retrotranspositional activity after divergence, a finding also supported previously  <cit> .
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  <cit> , bisulfite sequencing, northern blots for transcript quantity  <cit> , methylation specific pcr, and pyrosequencing  <cit> .
the method we use here, combined bisulfite and restriction analysis , 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  <cit> .
additionally, automation platforms such as the qiaxel system  would increase the speed of sample quantitation after enzyme digestion.
our candidate loci were also mined from the c57bl/ <dig> genome, and as such, they are more likely to be useful as epigenetic markers in both mutant and exposed mice than previous epialleles , 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   <cit> .
further, iaps, unlike other transposons, can induce changes in nearby chromatin marks, but no phylogenetic basis underlying epigenetic states has been explored  <cit> .
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  <cit> .
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.
CONCLUSIONS
combined phylogenetic and dna methylation analysis resulted in the identification of novel regions of variable methylation.
the phylogenetic analysis revealed two duplication and divergence events subdividing this family into three clades.
importantly, average methylation was significantly lower for both the red and green clade elements, compared to black clade elements, supporting the notion that phylogenetically older and more uniform elements tend to be more highly methylated than younger and diverged elements.
thus, through this endeavor the number of known metastable epialleles in the mouse has dramatically increased.
these sites can now serve as biomarkers for environmental modifications to the epigenome.
parallel approaches in humans are underway.

 across mammalian genomes, the retrotransposon class of repeat elements make up nearly half the nuclear dna, with long terminal repeats alone accounting for 8-10%. many retrotransposons retain the ability to move to new locations in the genome, a potentially deleterious phenomenon that can result in direct disruption to coding regions. therefore, the suppression of retrotransposons is vital to organismal survival, particularly through sensitive life stages, and is primarily accomplished via epigenetic mechanisms, including dna methylation. epigenetic suppression is typically established early in development at the blastocyst stage and maintained during differentiation of the primordial germ cells. importantly, in certain cases suppression is incomplete, leading to adverse consequences. alternatively, this incomplete suppression may be an evolutionarily selected method of fine-tuning gene expression. genome-wide, little is known about the extent to which interindividual variation of dna methylation at these elements is affected by environmental factors and/or their phylogenetic lineage. intracisternal a particles are a class of murine retrotransposons named for the appearance of budding doughnut shaped particles in the cisternae of the endoplasmic reticulum. iaps are members of the endogenous retrovirus family class ii. structurally, a full length iap consists of gag, pro, and pol genes capped at each end by a direct pair of ltrs, each approximately 350 bp in length. however, the largest majority of iap transcripts issue from truncated  kb copies of iap of subtype iΔ rather than full length  kb copies. previous work has identified only a subset of elements likely to be active, due to genomic position, sequence mutation, or methylation status. the number of paralogous iap ltrs in the genome has been uncertain with estimates ranging from less than to over with significant polymorphism among strains. this family of ervs is highly active and rapidly transposing, with estimates of 60% of elements being strain specific. it is advantageous for these mobile elements to be kept under tight control by heavy dna methylation and tight chromatin structure. a small number of “metastable epialleles” have been identified in mice in which variable methylation of the 5′ ltr of an antisense iap insertion results in dysregulation of gene expression concomitant with the level of methylation. metastable epialleles show variable expression among individuals with epigenetic profiles that are consistent across tissues and are thus likely to have been set prior to germ line differentiation. further, the distribution of variable expressivity has been shifted at these metastable epialleles following maternal exposure to nutritional and environmental factors. to date, no studies have determined the genetic relationship underlying the ability of these iap ltrs to be variably methylated. two of the epialleles, avy and axinfu, were identified due to dramatic phenotypes induced by their insertion into nearby protein-coding genes. conversely, cabpiap was identified by a search of genbank cdna databases for chimeric sequence containing iap ltr and genic sequence. to systematically investigate the relationship of genetics and metastability we applied phylogenetic methods to identify candidate iap retrotransposons and validate their variable methylation status. through this approach, we find the phylogeny of metastable loci sheds light on the evolutionary basis of metastability. additionally, the validated analysis of epigenetic state across individual isogenic mice now increases the number of known metastable epialleles, which can serve as a test panel for environmental modifications to the epigenome.  iap distribution in the genome initial pairwise alignment of the two most well-studied metastable epialleles, avy and cabpiap revealed that the ltrs of these two insertions exhibit % sequence identity over the length of shared sequence. repeatmasker was used to scan the c57bl/ mouse genome and identified iap ltr elements. the majority of these ltrs were pairs found on the 5’ and 3’ end of the same iap insertion, however a fraction were present as solo ltrs. since the iaps underlying both avy and cabpiap are of the class iΔ type iapltr1_mm as identified in repbase, this subtype was used to filter the complete list of transposons, resulting in a total list of iapltr1_mm elements after removing unmapped, short, or non-alignable elements. there was no bias in the orientation of insertion as sense and anti-sense iapltr1_mm elements were identified throughout the genome. phylogenetic similarity of known metastable iaps a neighbor-joining tree of these elements revealed three distinct clades with avy and cabpiap clustering within clade, more closely than 99% of all other elements. the largest cluster, clade, contains the most conserved elements, and consists of sequences with an average sequence divergence of only % from the consensus. clade and clade contain and elements each, and are more divergent, with % and % average differences from the consensus respectively. bias in location of these insertions was not identified, with members of each clade represented across numerous chromosomes and locations. unique identifiers were assigned to each element in our list, in the format iap27-iap used throughout the study. characterization of interindividual metastability from these candidates from each clade were randomly selected to test for variable methylation using combined bisulfite and restriction analysis. primers for cobra were designed with the forward primer internal to the iap sequence and the reverse primer located in the flanking sequence, providing an amplicon specific to an insertion and containing homologous iap element sequence. bceai enzyme was chosen since each element contains two cpg sites as part of a bceai restriction site, acggcg. loss of methylation at either or both would prevent bceai activity. consequently, the relative intensity of cut vs. uncut bands gives a semi-quantitative measurement of methylation at these sites. uncut bands correspond to unmethylated dna while cut bands correspond to methylated dna. the two cpg sites examined here exhibited interindividual variation in previous studies from our group, and are identified as sites and in this study. a quantitated cobra gel image of iap is depicted in figure 4b, revealing an 18% range of methylation at the cut site. a group of isogenic avy/a genotype mice of both genders from litters was sacrificed at weaning and dna was extracted from the liver. following bisulfite conversion, candidate regions were amplified via pcr and digested with bceai. a total of representative loci, from the red clade including cabpiap, and each from the green and black clades were evaluated. the 0% control was uncut in all loci, while the high percent methylated control was nearly completely digested in all loci indicating sufficient enzyme activity and specificity. the mean methylation across all mice for all iap insertions was % and no mouse deviated by more than 3%, showing no statistical difference. specific loci however, exhibited much greater variability. we found that mean methylation of individual iaps fluctuated from a low of 59% to a high of 88% and standard deviations varied from to, indicating that individual transposons can differ dramatically in both average value and standard deviation among mice. thus, methylation variability ranged from 9% for the least variable element to greater than 34% for the most variable element. there were no significant differences in methylation between sexes. importantly, the average methylation was significantly lower for both red clade elements, 70%, and green clade elements, 68% than for black clade elements, 79%. interestingly, the range through which a particular element can be methylated is also correlated with its position within the phylogenetic tree. the average range for red clade elements is 21%, 17% for green clade elements, and just 11% for black clade elements. the element with the greatest range is in the red clade while the element with the greatest dispersion as measured by interquartile range is found in the black clade. * iap located on the y chromosome, only measured in male animals. lod = limit of detection. discussion our bioinformatic analysis identified iap ltrs, consistent with recent estimates of ~ 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. 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. 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 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. 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 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. 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, 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 would increase the speed of sample quantitation after enzyme digestion. our candidate loci were also mined from the c57bl/ genome, and as such, they are more likely to be useful as epigenetic markers in both mutant and exposed mice than previous epialleles, 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. 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.  combined phylogenetic and dna methylation analysis resulted in the identification of novel regions of variable methylation. the phylogenetic analysis revealed two duplication and divergence events subdividing this family into three clades. importantly, average methylation was significantly lower for both the red and green clade elements, compared to black clade elements, supporting the notion that phylogenetically older and more uniform elements tend to be more highly methylated than younger and diverged elements. thus, through this endeavor the number of known metastable epialleles in the mouse has dramatically increased. these sites can now serve as biomarkers for environmental modifications to the epigenome. parallel approaches in humans are underway. 
phylogenetic analysis revealed two duplication and divergence events subdividing this family into three clades. 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. select retrotransposons in the long terminal repeat 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 mobile element, and exhibit remarkably similar sequence identity. to characterize interindividual variation across clades, liver dna from isogenic mice was subjected to combined bisulfite and restriction analysis for separate ltr transposons. within each mouse, average methylation across all ltrs was not significantly different.
 select retrotransposons in the long terminal repeat 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 mobile element, and exhibit remarkably similar sequence identity.  utilizing the c57bl/ genome we identified iap ltrs overall and a subset of 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 isogenic mice was subjected to combined bisulfite and restriction analysis for separate ltr transposons. 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. within each mouse, average methylation across all ltrs was not significantly different.  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. epigeneticsdna methylationmetastable epiallelephylogenytransposonmouse 