Fig. 1: Identification and characterization of Mendelian and non-Mendelian epigenetic inheritance patterns.<\/b>![\"Fig.](\"https:\/\/www.europesays.com\/us\/wp-content\/uploads\/2026\/05\/41588_2026_2604_Fig1_HTML.png\")
The alternative text for this image may have been generated using AI.<\/p>\n
a<\/b>, Long-read ONT sequencing was performed on DNA from inbred samples of two genetically divergent CC strains and their F1 crosses. Allele-specific methylation patterns were analyzed to identify and map both Mendelian and non-Mendelian patterns of epigenetic inheritance. Matched RNA was also sequenced and used to associate the identified epigenetic inheritance patterns with gene expression. Additionally, F2s from the same CC strains were sequenced to further investigate the genetic and nongenetic factors regulating the identified epigenetic inheritance patterns. b<\/b>, Schematic of a cis-acting meQTL in an F2 mouse, indicating that the local haplotype of the DMR will define the identity of the meQTL, as recombination is unlikely to occur between the SNP and the CpGs over which it regulates methylation when they are in close proximity (that is, <50\u2009cm apart). A cis-acting regulatory mechanism will establish an F2 population in which the local haplotype of the DMR is associated with the methylation level (for example, CC019 alleles are unmethylated while CC037 alleles are methylated), as shown in a<\/b>. c<\/b>, Schematic of a dominant trans-acting meQTL in an F2 mouse, indicating that the local haplotype of the DMR will not necessarily define the identity of the meQTL, as there is a high likelihood that independent assortment of chromosomes or meiotic recombination will occur between the SNP and the CpGs over which it regulates methylation if they are >50\u2009cM apart. A trans-acting regulatory mechanism will establish an F2 population in which the local haplotype over the DMR is not associated with the methylation level (that is, there is no clear methylation pattern associated with the CC019 or CC037 alleles), as shown for a dominant trans-acting meQTL in a<\/b>. Schematics in a<\/b> created in BioRender; Feinberg, A. https:\/\/biorender.com\/xobyc15<\/a> (2026).<\/p>\n Within the CC019 and CC037 reference genomes, there are ~19 million CpGs, of which ~4.8 million overlap regions identical by descent (IBD), that is, lacking genetic variation between the two strains. Using our experimental and computational approach, we analyzed ~12 million autosomal CpGs and ~350,000 X-chromosomal CpGs in both tissues, accounting for ~90% and ~85% of the CpGs present in both strains, mappable to the intermediate genome and within non-IBD regions.<\/p>\n To assess the functional implications of these methylation patterns, we sequenced liver RNA from a subset of the inbred and F1 samples that were also profiled using ONT sequencing. Outputs were evaluated for allele specific and total expression in the context of each identified epigenetic inheritance pattern, as detailed in the Methods. Only 9.5% of genes (4,092\/42,880 genetic elements considered; 3,956\/19,655 protein-coding genes) were analyzable, that is, they contained transcribed polymorphisms that differ between CC019 and CC037 with expression levels exceeding a minimum threshold, thereby limiting allele-specific expression comparisons to a subset of genes.<\/p>\n We defined and searched for 12 distinct patterns of epigenetic inheritance. Representations of each epigenetic inheritance pattern identified on the autosomes, as well as the number of regions exhibiting each pattern, are shown in Fig. 2<\/a>. A comprehensive list of these regions is included in Supplementary Tables 2<\/a> and 3<\/a>, and the top ten regions for each pattern are shown in Supplementary Figs. 1<\/a>\u201324<\/a>. Each pattern is discussed in detail below and in Supplementary Notes<\/a>.<\/p>\n Fig. 2: Autosomal epigenetic inheritance patterns.<\/b> Definitions, representative patterns and violations to Mendelian inheritance of each autosomal epigenetic inheritance pattern identified, as well as the number of each pattern identified in both the liver and muscle and the number common to both tissues.<\/p>\n Cis-acting meQTLs (pattern 1)<\/p>\n Among the observed epigenetic inheritance patterns, the most prevalent in both tissues were regions of the genome in which methylation stratifies by genotype and allele (Fig. 3a<\/a>). These methylation patterns are established by cis-acting meQTLs, defined as genetic variants that regulate DNA methylation levels of proximal CpGs located on the same allele23<\/a>. In total, 7,081 genomic regions were identified that follow this pattern in at least one tissue, accounting for ~93% of autosomal intergenerational epigenetic inheritance patterns (Figs. 2<\/a> and 3b,c<\/a>, Supplementary Figs. 1<\/a> and 2<\/a>, and Supplementary Tables 2<\/a> and 3<\/a>). We find that 16% and 50% of the cis-acting meQTLs identified in liver and muscle, respectively, are present in both tissues. Examples of tissue-independent and tissue-specific cis-acting meQTLs are shown in Fig. 3b,c<\/a>.<\/p>\n Fig. 3: Cis-acting meQTLs.<\/b> a<\/b>, Generic representation of methylation in the context of a cis-acting meQTL in inbred CC019 and CC037 samples and their F1 crosses. b<\/b>, Cis-acting meQTL identified in both the liver (left) and muscle (right) over Loxhd1. c<\/b>, Methylation pattern of the tissue-specific cis-acting meQTL identified over Tpcn1 in the liver (left) and not in the muscle (right). d<\/b>, Cis-acting meQTL identified in the liver over Vasp and Opa3, shown for inbred and F1 samples (left) and F2 samples (right). e<\/b>, Allele-specific expression of Haao in the liver, exhibiting regulation by a cis-acting eQTL and overlapping a cis-acting meQTL in both the liver and muscle. Expression was analyzed in 6 CC019 inbred, 9 CC037 inbred and 14 F1 mice. For the inbred samples (CC019 and CC037), the expression levels of the absent alleles are provided as technical controls confirming accurate allelic assignment. For the methylation plots shown in b<\/b>\u2013d<\/b>, bold lines represent coverage-weighted mean methylation of the respective group and CpG sites included in the final analysis are denoted by tick marks on the x axis. CPM, counts per million; hom., homozygous; het., heterozygous.<\/p>\n To confirm that these differentially methylated regions (DMRs) are controlled by cis-acting regulatory elements, we performed targeted ONT sequencing on 20 candidate regions in F2 mice. As expected, all 20 regions exhibit methylation levels that segregate with the local haplotype (Fig. 3d<\/a> and Supplementary Data 1<\/a>), thereby validating that these methylation patterns are established by cis-acting meQTLs.<\/p>\n To assess the effects of these cis-acting meQTLs on gene expression, we examined genes whose promoters or enhancers overlap with, or are located within 100\u2009kb of, the corresponding DMR. Specifically, we searched for genes with allelic imbalance in the F1s which mirrors the expression difference observed between the two parental strains (Fig. 3e<\/a>) with no limitation on which allele is more highly expressed. Allele-specific expression could be assessed for only a subset of these genes (2,610\/13,013; Supplementary Table 4<\/a>). We identified 700 genes showing cis-acting regulation of both expression and DNA methylation, suggesting that many cis-acting meQTLs may also function as expression quantitative trait loci (Supplementary Tables 4<\/a> and 5<\/a>).<\/p>\n Nondominant trans-acting meQTLs (pattern 2)<\/p>\n We also identified regions in which methylation stratifies by genotype only in the inbred samples, whereas both alleles of the F1s exhibit intermediate methylation levels (Fig. 4a,b<\/a>). These patterns are likely caused by trans-acting meQTLs, defined as genetic variants that influence methylation levels of distal CpGs and can regulate both alleles. We identified 51 genomic regions that exhibit this methylation pattern in at least one tissue (Figs. 2<\/a> and 4c<\/a>, Supplementary Figs. 3<\/a> and 4<\/a>, and Supplementary Tables 2<\/a> and 3<\/a>). While there were fewer nondominant trans-acting meQTLs identified in the muscle (n\u2009=\u20099) than in the liver (n\u2009=\u200944), only 22% (2\/9) of the muscle nondominant trans-acting meQTLs were also observed in the liver (Fig. 4c,d<\/a>), suggesting that these nondominant trans-acting meQTLs may be more tissue-specific than their cis-acting counterparts.<\/p>\n Fig. 4: Nondominant trans-acting meQTLs.<\/b> a<\/b>, Generic representation of methylation in the context of an incomplete dominant trans-acting meQTL in inbred CC019 and CC037 samples and their F1 crosses. b<\/b>, Generic representation of methylation in the context of a codominant trans-acting meQTL. Methylation patterns in a<\/b> and b<\/b> indicate that read-level methylation analyses are required to distinguish incomplete dominant from codominant trans-acting meQTLs, as their methylation patterns will appear identical at the sample level. c<\/b>, Nondominant trans-acting meQTL identified in both the liver (left) and the muscle (right) over Fggy. d<\/b>, Tissue-specific nondominant trans-acting meQTL identified over Ctdspl in the liver (left) and not in the muscle (right). e<\/b>, Nondominant trans-acting meQTL identified in the liver over Isoc2b, shown for inbred and F1 samples (left) and F2 samples (right). f<\/b>, Allele-specific expression of Socs5 in the liver, exhibiting regulation by a tissue-specific nondominant trans-acting eQTL identified in the liver. Expression was analyzed in 6 CC019 inbred, 9 CC037 inbred and 14 F1 mice. For the inbred samples (CC019 and CC037), the expression levels of the absent alleles are provided as technical controls confirming accurate allelic assignment. For the methylation plots shown in c<\/b>\u2013e<\/b>, bold lines represent coverage-weighted mean methylation of the respective group and CpG sites included in the final analysis are denoted by tick marks on the x axis.<\/p>\n Nondominant trans-acting meQTLs comprise two distinct subpatterns\u2014incomplete dominance and codominance. While each of these subpatterns establishes identical methylation patterns at the sample level, they can be distinguished using long-read sequencing data by further assessing the variability of DNA methylation at both the cellular and CpG levels. In regions regulated by an incomplete dominant trans-acting meQTL, each sequenced strand of DNA from both parental alleles of the F1 crosses exhibit intermediate methylation levels which are between the two inbred parental strains, indicating CpG-level methylation variability within each cell (Fig. 4a<\/a> and Extended Data Fig. 2<\/a>). Conversely, in regions regulated by codominant trans-acting meQTLs, each phased F1 alleles exhibits two clear epialleles, groups of reads that are either fully methylated or fully unmethylated, indicating variability at the cellular level (Fig. 4b<\/a> and Extended Data Fig. 3<\/a>). Thus, methylation differences between the inbred strains and F1 crosses in these regions reflect shifts in the distributions of these epialleles.<\/p>\n Five candidate regions regulated by nondominant trans-acting meQTLs were chosen for subsequent targeted analysis in the F2s. Three of these regions exhibited no clear relationship between local haplotype and observed methylation states (Fig. 4e<\/a> and Supplementary Data 1<\/a>), further supporting regulation by trans-acting genetic variants. The remaining two DMRs exhibited genotype-specific and allele-specific methylation patterns in the F2 generation that were smaller than observed in the inbred generation and roughly the same as in the F1 generation (Supplementary Data 1<\/a>).<\/p>\n We subsequently analyzed allele-specific and total expression levels of genes associated with these DMRs via direct promoter\/enhancer overlap or by genomic proximity to identify instances in which expression levels are consistent with regulation by nondominant trans-acting meQTLs. These genes are expected to exhibit expression levels that stratify by genotype in inbred samples but lack significant allelic imbalance in the F1s. We identified nine such instances (Fig. 4f<\/a> and Supplementary Tables 4<\/a> and 5<\/a>), suggesting that these nondominant trans-acting meQTLs may also function as expression quantitative trait loci (eQTL), thereby providing an additional link between these regulatory mechanisms.<\/p>\n Dominant trans-acting meQTLs, transvection and paramutation (patterns 3\u20135)<\/p>\n Our analysis further revealed epigenetic inheritance patterns in which methylation stratifies by genotype in the inbred samples, while methylation on both alleles of the F1s resembles only one of the parental strains (Fig. 5a,b<\/a>). We identified 23 such regions present in at least one tissue (Fig. 2<\/a>, Supplementary Figs. 5<\/a> and 6<\/a>, and Supplementary Tables 2<\/a> and 3<\/a>). Of these, four were identified in both tissues, representing 17% and 67% of the regions identified in the liver and muscle, respectively. Included among these four tissue-independent DMRs is a region overlapping the gene Vps37c (Fig. 5b<\/a>), involved in the vesicular sorting of endocytic cargo24<\/a> and subject to further investigation below.<\/p>\n Fig. 5: Dominant trans-acting meQTLs, transvection and paramutation.<\/b> a<\/b>, Generic representation of methylation in the context of a dominant trans-acting meQTL, transvection or paramutation in inbred CC019 and CC037 samples and their F1 crosses. b<\/b>, Dominant trans-acting meQTL\/transvection\/paramutation methylation pattern identified in both the liver (left) and muscle (right) over Vps37c. c<\/b>, Paramutation methylation pattern identified in the liver over Capn11, shown for inbred and F1 samples (left) and F2 samples (right). d<\/b>, Generic representation of methylation in the context of paramutation driven by the presence of a strain-specific IAP element in the inbred (left), F1 (middle) and F2 (right) generations. e<\/b>, Methylation over the CC037-specific Vps37c IAP is shown for the inbred and F1 samples of the liver (left), inbred and F1 samples of the muscle (center), and F2 samples of the liver (left). For the liver F2s, only those samples\/alleles with an average coverage of at least 1\u00d7 over the region shown are included. The IAP is highlighted in blue and the original DMR (identified without inclusion of the IAP in the reference genome) is highlighted in red. As these plots no longer conform to the original pseudohybrid intermediate coordinate system due to the addition of the IAP, a single anchor coordinate indicating the start of the IAP is shown. f<\/b>, Methylation over the CC037-specific Capn11 IAP shown for the inbred and F1 samples of the liver (left), inbred and F1 samples of muscle (center), and F2 samples of the liver (left). The IAP is highlighted in blue. g<\/b>, Total expression of Drap1 in the liver, exhibiting a dominant trans-acting eQTL\/transvection\/paramutation expression pattern and proximal to a dominant trans-acting meQTL\/transvection\/paramutation methylation pattern in the liver. Expression was analyzed in 6 CC019 inbred, 9 CC037 inbred and 14 F1 mice. For the methylation plots shown in b<\/b>, c<\/b>, e<\/b> and f<\/b>, bold lines represent coverage-weighted mean methylation of the respective groups and CpG sites included in the final analysis are denoted by tick marks on the x axis.<\/p>\n This pattern may conceivably arise from three distinct underlying mechanisms\u2014dominant trans-acting meQTLs, transvection or paramutation (Supplementary Notes 1<\/a> and 2<\/a>), and additional data from the F2 generation are required to distinguish these potential mechanisms (Extended Data Fig. 4<\/a>).<\/p>\n To differentiate genomic regions regulated by these three mechanisms, we profiled a set of three candidate regions in the F2 generation using targeted ONT sequencing on liver DNA from 19 F2 mice selected from a set of 58 based on their genotyping such that, at each region in the genome, there is at least one sample which is homozygous for each of the two parental alleles\u2014CC019 and CC037 (Extended Data Fig. 5a,b<\/a>). This ensures that, if the methylation of a candidate region is driven by a dominant trans-acting meQTL, at least one sample will be homozygous for the nondominant allele and will therefore exhibit the nondominant methylation pattern, allowing distinction from paramutation.<\/p>\n We observed that the candidate region overlapping the gene Capn11 exhibits only one methylation pattern in the F2s, matching that of both F1 alleles (Fig. 5c<\/a>, Extended Data Fig. 6<\/a>, Supplementary Fig. 25<\/a> and Supplementary Data 1<\/a>). This indicates that this region exhibits intergenerational paramutation, representing a form of non-Mendelian epigenetic inheritance observed across generations previously reported in engineered and transgenic mice. Notably, Capn11 is a calcium-dependent protease that is predominantly expressed in the testis during later stages of meiosis in both mice and humans25<\/a>,26<\/a>,27<\/a>, and decreased expression in humans has been associated with infertility and azoospermia28<\/a>.<\/p>\n Additionally, within the aforementioned DMR overlapping Vps37c (Fig. 5b<\/a>), we identified a ~5-kb insertion inside the DMR relative to the CC037 reference genome. Investigation of this insertion using the Dfam database29<\/a> reveals that it is a transposable element (TE) belonging to the IAP family of ERVs and is present only in the CC037 strain. We suspected that this DMR may be an additional example of intergenerational paramutation, in which the highly methylated pattern observed in the CC037 alleles of the F1 generation in both liver and muscle would be subsequently inherited in all CC037 alleles of the F2 generation. While this region was not included in the F2 sequencing target regions, there exists another IAP ~13\u2009kb downstream of the Capn11 paramutation region30<\/a>, which was included as a target region. The Capn11 IAP shows sufficiently high sequence homology to the Vps37c IAP such that the CC037 allele was captured by adaptive sampling with enough frequency for a regional methylation analysis (average coverage of the CC037 allele with the IAP, ~8\u00d7; average coverage of the CC019 allele without the IAP, ~0.5\u00d7). As such, we reanalyzed methylation of the CC037 samples and alleles in this region using a new CC037 reference genome that includes the IAP sequence. Methylation over the Vps37c IAP is much lower for the inbred CC037 samples than the CC037 alleles of the heterozygous F1s in both the muscle and liver (Fig. 5d,e<\/a> and Extended Data Fig. 7a<\/a>). Furthermore, we observe a methylation pattern in all CC037 alleles of the F2 generation that more closely matches the F1\u2013CC037 alleles than the inbred CC037 samples, independent of the zygosity of the F2 samples in this region, suggesting that this region is highly likely to be an additional example of intergenerational paramutation. However, as not all 19 F2 mice contain at least one copy of this IAP, there remains the possibility that this methylation pattern is driven by a dominant trans-acting meQTL (Supplementary Notes 1<\/a> and 2<\/a>).<\/p>\n Given this observation, we further analyzed DNA methylation at an IAP ~13\u2009kb downstream of the Capn11 DMR, which is also present only in the CC037 genome. Methylation at this IAP shows a pattern similar to the Vps37c IAP, with inbred CC037 samples exhibiting lower methylation than CC037 alleles of the F1 generation (Fig. 5f<\/a> and Extended Data Fig. 7b<\/a>). Furthermore, all CC037 alleles in the F2 generation exhibit a methylation pattern nearly identical to that of the F1\u2013CC037 alleles, strongly suggesting this is another example of intergenerational paramutation (Supplementary Notes 1<\/a> and 2<\/a>).<\/p>\n To investigate the link between these methylation patterns and gene expression, we analyzed genes associated with these DMRs via direct promoter or enhancer overlap or genomic proximity to identify those genes whose total F1 expression levels match that of only one parental strain. This analysis revealed 14 such genes (Fig. 5g<\/a> and Supplementary Tables 4<\/a> and 5<\/a>), suggesting a potential role for these methylation patterns in the regulation of gene expression.<\/p>\n Emergent epigenetic inheritance patterns (patterns 6\u201310)<\/p>\n We also searched for five distinct sets of emergent epigenetic inheritance patterns, in which at least one allele of the F1 generation exhibits a new methylation pattern not observed in either of the parental strains (for example, Fig. 6a\u2013c<\/a>). This analysis revealed 54 instances of such emergent epigenetic inheritance patterns present in at least one tissue (Fig. 2<\/a> and Supplementary Tables 2<\/a> and 3<\/a>), of which one was overdominant (Fig. 6d<\/a> and Supplementary Fig. 7<\/a>), 22 were allele-specific overdominant or allele-specific underdominant (Fig. 6e<\/a> and Supplementary Figs. 8<\/a> and 9<\/a>), and 31 were biallelic dominant (Fig. 6f<\/a> and Supplementary Figs. 10<\/a> and 11<\/a>). This represents a substantial increase over what has been previously reported in mammals in the literature\u2014seven examples identified in mice31<\/a>,32<\/a>,33<\/a> in addition to the callipyge locus in sheep13<\/a>. Notably, only a single emergent epigenetic inheritance pattern is found in both liver and muscle, suggesting that these patterns are primarily regulated in a tissue-specific manner.<\/p>\n Fig. 6: Emergent epigenetic inheritance patterns.<\/b> a<\/b>, Generic representation of methylation in the context of overdominance and underdominance in inbred CC019 and CC037 samples and their F1 crosses. b<\/b>, Generic representation of methylation in the context of allele-specific overdominance and allele-specific underdominance in inbred CC019 and CC037 samples and their F1 crosses. c<\/b>, Generic representation of methylation in the context of biallelic dominance in inbred CC019 and CC037 samples and their F1 crosses. d<\/b>, Overdominant methylation pattern identified in the liver over Usp18. e<\/b>, Allele-specific underdominant methylation pattern identified in the liver over Jak3. f<\/b>, Biallelic dominant methylation pattern identified in the liver over Ccdc85c. g<\/b>, Allele-specific expression of Ccdc85c in the liver, exhibiting a biallelic dominant expression pattern. The promoter of Ccdc85c overlaps the biallelic dominant methylation pattern shown in f<\/b>. Expression was analyzed in 6 CC019 inbred, 9 CC037 inbred and 14 F1 mice. For the inbred samples (CC019 and CC037), the expression levels of the absent alleles are provided as technical controls confirming accurate allelic assignment. For the methylation plots shown in d<\/b>\u2013f<\/b>, bold lines represent coverage-weighted mean methylation of the respective groups and CpG sites included in the final analysis are denoted by tick marks on the x axis.<\/p>\n To further explore the functional role of these emergent epigenetic inheritance patterns, we analyzed the expression of genes associated with these DMRs via promoter or enhancer overlap or genomic proximity and identified 19 that exhibit expression patterns consistent with biallelic dominance (Fig. 6g<\/a> and Supplementary Tables 4<\/a> and 5<\/a>).<\/p>\n Genomic imprinting (pattern 11)<\/p>\n Parent-of-origin-specific methylation, or genomic imprinting (Fig. 7a<\/a>), is the most thoroughly studied example of non-Mendelian inheritance of DNA methylation in mammals. We identified 111 autosomal regions exhibiting parent-of-origin-specific methylation patterns in at least one tissue (Figs. 2<\/a> and 7b<\/a>, Supplementary Figs. 12<\/a> and 13<\/a>, and Supplementary Tables 2<\/a> and 3<\/a>). We also identified a single region with parent-of-origin-specific methylation on the X chromosome in females, which is discussed further alongside the other X-chromosomal patterns. We compared this list to a curated set of known DNA methylation-regulated imprinting control regions (ICRs)34<\/a> to evaluate our analysis approach on known regions. Of the 13 analyzable ICRs (non-IBD, mappable), we detected imprinted methylation patterns in at least one tissue within 1\u2009kb of all 13 regions.<\/p>\n Fig. 7: Genomic imprinting.<\/b> a<\/b>, Generic representation of methylation in the context of genomic imprinting in inbred CC019 and CC037 samples and their F1 crosses in both cross directions (denoted as maternal strain x paternal strain). b<\/b>, Imprinted methylation pattern identified over the promoter of Slc38a4 in the liver. Inbred samples are shown on both plots and F1 samples have been split by cross direction, CC019 x CC037 (top) and CC037 x CC019 (bottom). c<\/b>, Parent-of-origin-specific methylation pattern found exclusively in liver, identified over Zswim9, shown in both the liver (left) and muscle (right). Inbred samples are shown on both plots and F1 samples have been split by cross direction, CC019 x CC037 (top) and CC037 x CC019 (bottom). d<\/b>, Parent-of-origin-specific methylation pattern found exclusively within the muscle identified over Asb4, shown in both the liver (left) and muscle (right). Inbred samples are shown on both plots and F1 samples have been split by cross direction, CC019 x CC037 (top) and CC037 x CC019 (bottom). e<\/b>, Novel parent-of-origin-specific methylation pattern identified over Scn8a in the liver. Inbred samples are shown on both plots and F1 samples have been split by cross direction, CC019 x CC037 (top) and CC037 x CC019 (bottom). f<\/b>, Allele-specific expression of Sgce in the liver, exhibiting a parent-of-origin-specific expression pattern and overlapping a parent-of-origin-specific methylation pattern. Expression was analyzed in six CC019 inbred, nine CC037 inbred, six CC019 x CC037 F1 and eight CC037 x CC019 F1 mice. For the inbred samples (CC019 and CC037), the expression levels of the absent alleles are provided as technical controls confirming accurate allelic assignment. For the methylation plots shown in b<\/b>\u2013e<\/b>, bold lines represent coverage-weighted mean methylation of the respective groups and CpG sites included in the final analysis are denoted by tick marks on the x axis.<\/p>\n In addition, we identified parent-of-origin-specific DMRs overlapping four autosomal genes that, to our knowledge, have not previously been identified as imprinted\u2014Scn8a, Pcdhb4, Fry (identified in the liver) and Socs5 (identified in the muscle). Furthermore, we identified parent-of-origin-specific methylation near four autosomal genes, which are variably reported as imprinted in the literature\u2014Zswim9 (ref. 35<\/a>), Nav2 (ref. 36<\/a>), Casc1 (ref. 35<\/a>) and Cntnap1 (refs. 37<\/a>,38<\/a>). Methylation over these imprinted genes is shown in Supplementary Fig. 26<\/a>. Additionally, although not statistically significant, we observe a parent-of-origin-specific methylation pattern in muscle over Scn8a, which was identified in the liver, as well as a parent-of-origin-specific methylation pattern in the liver over Socs5, which was identified in the muscle (Supplementary Fig. 27<\/a>).<\/p>\n Our analysis also revealed parent-of-origin-specific expression in two genes associated with these parent-of-origin-specific DMRs via promoter or enhancer overlap or genomic proximity, including the known imprinted gene Sgce (Fig. 7<\/a> and Supplementary Tables 4<\/a> and 5<\/a>). However, another well-documented imprinted gene, Slc38a4, did not exhibit parent-of-origin-specific expression (Supplementary Fig. 28<\/a>) despite clear parent-of-origin-specific methylation over its promoter (Fig. 7b<\/a>). This finding, previously reported in adult mouse liver39<\/a>, indicates that methylation over an ICR, while necessary to establish imprinting of certain genes, may be insufficient to cause parent-of-origin-specific expression in all cases. This suggests that additional regulatory mechanisms are involved in imprinting.<\/p>\n Sex-specific DNA methylation (pattern 12)<\/p>\n We also identified 305 autosomal regions exhibiting sex-specific methylation patterns (Figs. 2<\/a> and 8a,b<\/a>, Supplementary Fig. 14<\/a>, and Supplementary Tables 2<\/a> and 3<\/a>). Notably, methylation differences in all but one region (304; 99.7%) show female hypermethylation relative to males. All 305 sex-specific patterns were detected in the liver, whereas none were observed in muscle. Sex-specific DNA methylation and gene expression patterns in the liver have been reported in both humans40<\/a>,41<\/a> and mice42<\/a> and, as such, were expected to be less abundant in muscle.<\/p>\n Fig. 8: Sex-specific DNA methylation and X-chromosomal epigenetic inheritance patterns.<\/b> a<\/b>, Generic representation of sex-specific methylation in male and female inbred samples and F1 crosses. b<\/b>, Sex-specific methylation pattern found exclusively within the liver identified over Aox3, shown in both the liver (left) and muscle (right). c<\/b>, Sex-specific DMR identified in the liver upstream of Rps14, shown for inbred and F1 samples (left) and F2 samples (right). d<\/b>, Total expression of Aox3 in the liver, split by sex, exhibiting a sex-specific expression pattern and overlapping the sex-specific methylation pattern shown in b<\/b>. Expression was analyzed in 16 female and 13 male mice. e<\/b>, Generic representation of methylation in the context of skewed XCI in inbred CC019 and CC037 samples and their F1 crosses. f<\/b>, Skewed XCI methylation pattern identified in the liver over the promoter of Efnb1. g<\/b>, Allele-specific expression of Efnb1 in the liver, exhibiting skewed XCI and overlapping the skewed XCI methylation pattern shown in b<\/b>. Expression was analyzed in 6 CC019 inbred, 9 CC037 inbred and 14 F1 mice. For the inbred samples (CC019 and CC037), the expression levels of the absent alleles are provided as technical controls confirming accurate allelic assignment. h<\/b>, New parent-of-origin-specific methylation pattern identified in the liver over Zfp92. Inbred samples are shown on both plots and F1 samples have been split by cross direction, CC019 x CC037 (left) and CC037 x CC019 (right). For the methylation plots shown in b<\/b>, c<\/b>, f<\/b> and h<\/b>, bold lines represent coverage-weighted mean methylation of the respective group and CpG sites included in the final analysis are denoted by tick marks on the x axis. M, male; F, female.<\/p>\n To validate the sex specificity of these methylation patterns, we examined 20 candidate regions in the F2 generation. Sex-specific methylation is established in a manner independent of the identity of local and distal autosomal genetic variants, although an effect of X-dosage or Y chromosome cannot be ruled out. As anticipated, all 20 regions exhibited methylation patterns that segregated by sex in the F2s (Fig. 8c<\/a> and Supplementary Data 1<\/a>), confirming a sex-dependent regulatory mechanism.<\/p>\n Our analysis also identified 233 genes associated with these sex-specific DMRs that exhibit concordant sex-biased gene expression (Fig. 8d<\/a> and Supplementary Tables 4<\/a> and 5<\/a>). Notably, 182 genes (78.1%) showed higher expression in males, consistent with the observed methylation patterns.<\/p>\n X-chromosomal epigenetic inheritance patterns<\/p>\n DMR finding was performed separately for the autosomes and the X chromosome. Due to expected differences between the male and female X-chromosomal methylation patterns resulting from X chromosome inactivation (XCI), two separate analyses were performed for the X chromosome\u2014one using only female samples and the other using only the maternal alleles of male samples. XCI is a dosage compensation mechanism in which females inactivate one copy of the X chromosome in each cell. In mice, XCI has been shown to have a genetic contribution that maps to a region (QTL) on the X chromosome, within which local genetics contribute to the determination of which parental copy of the X chromosome is inactivated43<\/a>,44<\/a>. This can lead to skewed XCI, in which one X-chromosomal allele is preferentially inactivated and, thus, more highly methylated than the other allele (Fig. 8e<\/a>).<\/p>\n For both liver and muscle, we identified few DMRs on the maternal alleles of the male samples (Supplementary Figs. 15<\/a>\u201318<\/a> and Supplementary Tables 6<\/a> and 7<\/a>), whereas we found many DMRs in the female samples, the majority of which were categorized as skewed XCI (Supplementary Figs. 19<\/a>\u201324<\/a> and Supplementary Tables 8<\/a> and 9<\/a>). Of the 218 and 226 regions indicating skewed XCI in liver and muscle, respectively, 209 (95.9% and 92.5% for liver and muscle) exhibit hypermethylation of the CC037 allele relative to CC019 (Fig. 8f<\/a>, Supplementary Figs. 22<\/a> and 23<\/a>, and Supplementary Tables 8<\/a> and 9<\/a>). This indicates skewing of XCI toward the CC037 allele in crosses of these strains, consistent with the expected preferential inactivation of the CC037 allele relative to the CC019 allele.<\/p>\n This pattern of skewed XCI is evident in the read-level methylation data, in which the CC037 allele of female F1 samples typically exhibits a much higher proportion of the inactive methylation pattern (hypermethylated) than the CC019 allele (Extended Data Fig. 8<\/a>). Additionally, we observe hypermethylation of the active CC019 allele over Xist and its antisense RNA Tsix (Extended Data Fig. 9<\/a>), key regulators of XCI known to be expressed exclusively from the inactive X chromosome45<\/a>. Expression analysis of genes associated with these DMRs revealed that all 40 genes showing expression patterns consistent with skewed XCI exhibit higher expression from the CC019 allele, further supporting the preferential inactivation of the CC037 X-chromosomal allele (Fig. 8g<\/a> and Supplementary Tables 4<\/a> and 5<\/a>).<\/p>\n Our analysis also identified a previously unreported region with parent-of-origin-specific methylation on the X chromosome in female liver samples. This region overlaps a CpG island within the gene Zfp92 (Fig. 8h<\/a> and Supplementary Figs. 24<\/a> and 26<\/a>), which is involved in the suppression of TEs30<\/a> and implicated in X-linked intellectual disability46<\/a>,47<\/a> and nonobstructive azoospermia48<\/a>. Analyses of disorders including Turner syndrome and autism spectrum disorder have suggested the existence of an imprinted locus on the X chromosome affecting cognitive function49<\/a>,50<\/a>, although such a locus has not previously been identified. Furthermore, while not statistically significant, we also observe parent-of-origin-specific methylation in this region within female muscle samples (Supplementary Fig. 27<\/a>).<\/p>\n","protected":false},"excerpt":{"rendered":"Integrated genetic and epigenetic analysis of the intergenerational inheritance of DNA methylation patterns We designed a combined genetic…\n","protected":false},"author":3,"featured_media":811685,"comment_status":"","ping_status":"","sticky":false,"template":"","format":"standard","meta":{"footnotes":"","_share_on_mastodon":"0"},"categories":[8],"tags":[2906,21939,15576,6958,27360,21938,834,152578,15577,159,67,132,68],"class_list":["post-811684","post","type-post","status-publish","format-standard","has-post-thumbnail","category-science","tag-agriculture","tag-animal-genetics-and-genomics","tag-biomedicine","tag-cancer-research","tag-epigenomics","tag-gene-function","tag-general","tag-genome-informatics","tag-human-genetics","tag-science","tag-united-states","tag-unitedstates","tag-us"],"share_on_mastodon":{"url":"https:\/\/pubeurope.com\/@us\/116611364715341727","error":""},"_links":{"self":[{"href":"https:\/\/www.europesays.com\/us\/wp-json\/wp\/v2\/posts\/811684","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.europesays.com\/us\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.europesays.com\/us\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.europesays.com\/us\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/www.europesays.com\/us\/wp-json\/wp\/v2\/comments?post=811684"}],"version-history":[{"count":0,"href":"https:\/\/www.europesays.com\/us\/wp-json\/wp\/v2\/posts\/811684\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.europesays.com\/us\/wp-json\/wp\/v2\/media\/811685"}],"wp:attachment":[{"href":"https:\/\/www.europesays.com\/us\/wp-json\/wp\/v2\/media?parent=811684"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.europesays.com\/us\/wp-json\/wp\/v2\/categories?post=811684"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.europesays.com\/us\/wp-json\/wp\/v2\/tags?post=811684"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}![\"Fig.](\"https:\/\/www.europesays.com\/us\/wp-content\/uploads\/2026\/05\/41588_2026_2604_Fig2_HTML.png\")
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