Isogenic mice exhibit sexually-dimorphic DNA methylation patterns across multiple tissues

- Isogenic mice exhibit sexually-dimorphic DNA methylation patterns across multiple tissues.
- a proportion of these differences were independent of the presence of testosterone in males.
- Conclusions: Our findings suggest that gender is underwritten in the epigenome in a tissue-specific and potentially sex hormone-independent manner.
- Gender-specific epigenetic states are likely to have important implications for understanding sexually dimorphic phenotypes in health and disease..
- There is evidence for gender differences in DNA methylation in various tissues in eutherian mammals..
- A re- cent meta-analysis of Infinium 450 K array data from 76 individual studies identified gender-specific methylation at about 200 autosomal CpG sites in peripheral blood [4].
- Gender-specific methylation differences have also been reported using 450 K array in human prefrontal cortex [5], saliva [6], and pancreatic islets [7], and by re- duced representation bisulphite sequencing (RRBS) in mouse liver [8].
- A notable recent study used RRBS to find gender-specific methylation at 160 autosomal loci in mouse liver [9].
- Full list of author information is available at the end of the article.
- 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- This study suggests that there is an inter- action between sex hormones and epigenetic marks, but whether gender-specific marks might exist independent of hormonal or other postnatal factors is unknown..
- Here we have explored this possibility by performing RRBS on male and female mouse tissues derived from each of the three germ layers: liver (endoderm), heart (mesoderm), and brain (ectoderm).
- By combining our analyses with publically available data, we find that in the liver, a proportion of loci exhibit gender-specific methylation in the absence of testosterone.
- Very few gender-specific differences are shared among tissues, even those from the same germ layer..
- Gender is predicted by DNA methylation patterns in the mouse liver.
- RRBS captures only ~1% of the mammalian genome, but the captured fraction is highly enriched for functional regions such as CpG islands, shores, and other gene regulatory elements..
- Around a third of the DMTs overlapped with ENCODE liver H3K4me1 and H3K27Ac peaks, a highly significant enrichment (both p <.
- This is consistent with many of the intergenic DMTs residing in active enhancers..
- All but one of the ten loci showed a difference in methylation levels between males and females as pre- dicted by the RRBS signal (Additional file 3: Figure S2)..
- This experimental validation indicates that our RRBS and informatics strategy detects gender-specific differ- ences with high confidence..
- Gender DMTs in the liver can be testosterone independent.
- [9] (GEO Accession GSE60012), who previously identified 160 gender DMTs in the adult mouse liver.
- Using our bio- informatics pipeline with their liver dataset, we identified 83 autosomal gender DMTs, of which 48 (~58%) over- lapped with the gender DMTs from our data (Fig.
- The difference in the num- ber of DMTs identified can be at least partially attributed to the difference in dataset size (our liver dataset con- tained 820,388 tiles, and Reizel et al’s contained 167,462 tiles).
- Like Reizel and colleagues, we found that the ma- jority of gender DMTs in the liver could be attributed to hypermethylation in females relative to males.
- Interestingly, the gender DMTs identified in the liver by Reizel et al.
- This implies that gender DMTs in the liver are testosterone- dependent.
- We extended these observations by perform- ing an unbiased comparison of the Reizel et al.
- In doing so we were able to identify 228 gender DMTs, despite the absence of testosterone in the males (Additional file 2: Table S3).
- These testosterone- independent gender DMTs were also heavily skewed towards hypermethylation in females (Fig.
- 2d), although almost all the tiles in the castrated male dataset were also represented in our dataset.
- This suggests that there are additional factors beyond testosterone that are able to specify gender-specific methylation patterns..
- Heart and brain also harbour gender DMTs.
- The robust gender differences observed in the mouse liver in this study and others, along with reports of gen- der bias in human non-liver tissues such as peripheral blood leukocytes, prompted us to ask whether the.
- gender DMTs might be conserved across tissues.
- Given that some DMTs in liver appear to be sex hormone- independent, it is possible that these differences in methylation arise in the germline, or very early in devel- opment.
- To address this possibility we interrogated the methylomes of brain and heart from three of the males and three of the females used for liver analysis.
- Unlike the liver methylome, analysis of the brain methylome by hierarchical clustering or PCA did not separate the genders, even when sex chromosomes were included (Fig.
- likewise in the heart we observed no distinct clustering, although the genders.
- Despite the lack of unsupervised cluster- ing by gender, both heart and brain harboured gender DMTs.
- Applying the same stringent parameters as used for the liver analysis, we identified 957 autosomal DMTs in the brain (Fig.
- Additional file 2: Table S4), and 145 in the heart (Fig.
- While the brain gender DMTs exhibited, like the liver, a clear bias towards hypermethylation in females (Fig.
- this bias was absent from the gender DMTs in the heart (Fig.
- The gender DMTs in both tissues were again mostly outside CpG islands, in intronic and intergenic regions (Fig.
- 1 Autosomal DNA methylation patterns in the liver distinguish gender.
- d Clustered heat map of differentially methylated tiles (DMTs) identified in the liver of males versus females.
- Gender DMTs are tissue specific.
- We then asked whether there was any overlap of the gender DMTs we identified among tissues of distinct germ layer origin that might suggest that they were in- born.
- Only two liver gender DMTs were common to the brain, and no liver DMT was common to the heart (although 11 of the 145 heart DMTs were also differentially methylated in the brain).
- it is interesting to note that methylation patterns in the brain are much more variable among isogenic individuals than the other tissues examined..
- While this analysis suggests that gender DMTs do not arise in the germline, we considered whether gender DMTs might be common to tissues of the same germ layer origin (i.e.
- We identified 354 and 48 gender DMTs in muscle and spleen respectively (Additional file 2: Tables S6 and S7), but these DMTs were exclusive to both each other and to the heart (Fig.
- Here we confirm the existence of sex-specific cytosine methylation in the mouse liver and find that tissues.
- 2 Gender-specific methylation in the liver does not require testosterone.
- a Venn diagram showing overlap of gender DMTs in the liver identified by this study and our reanalysis of Reizel et al.
- b Bar plot of gender DMTs from our liver RRBS data showing.
- c Bar plot of gender DMTs between normal females and castrated males showing % of tiles hypermethylated in females (red) and males (blue) across all autosomes.
- d Venn diagram showing overlap of all liver gender DMTs (both those identified here and those from Reizel et al.
- In the majority of in- stances, sex differences were manifest as a strong female bias towards hypermethylation, and this was the case in the liver even when the demethylating action of testos- terone in males was removed.
- Tissues of different origins displayed varying extents of gender-specific methylation: the brain harboured more than a thousand differentially methylated regions whereas the heart had only a few hundred.
- Sex-specific methylation in the brain was remarkable also because it occurred on a very high background of inter-individual variation..
- 3 Mouse heart and brain also harbour gender-specific methylation.
- a, b Pseudo-3D PCA plots of the first three principal components of RRBS data from brain (a) and heart (b).
- e Genomic annotations of brain and heart gender DMTs.
- f, g Bar plot of gender DMTs from brain (f) and heart (g) showing % of tiles hypermethylated in females (in red) and males (blue) across all autosomes.
- Extensive inter-individual variation in the methylome of the human brain has been recognised only recently, and attributed largely to a secondary influence of genetic vari- ation [12].
- However in our study all samples were derived from isogenic mice, indicating that at least some propor- tion of inter-individual variation in methylation patterns in the brain is purely epigenetic in nature, and sex-specific epigenetic signatures are overlaid on this.
- it is probable that thou- sands more sex-specific differences exist in the brain, albeit smaller in magnitude or restricted to specific cell subtypes..
- Taken together with the ontological analysis showing enrichment for tissue-specific pathways, this strongly suggests that gender-specific epigenetic differences are likely to have a tissue-specific function.
- This idea is sup- ported by our finding that gender-specific methylation differences overlap significantly with histone markers of enhancer regions.
- It is also consistent with the recent finding that gender-specific methylation in the liver correlates with hepatic gene expression, particularly when the methylation differences occur in regions corre- sponding to tissue-specific enhancers [9].
- The expression of more than a thousand genes in the mammalian liver is sex-dependent, which is not surprising given that sex- ual dimorphism in both steroid and drug metabolism underpin normal liver physiology [13].
- 4 Gender DMTs are largely tissue autonomous.
- a Venn diagram showing overlap of gender DMTs from liver, brain and heart.
- b Molecular functions overrepresented by regions harbouring gender DMTs in liver, brain and heart.
- d Venn diagram showing overlap of gender DMTs from spleen, heart, skeletal muscle and liver.
- The mechanisms underpinning gender-specific methy- lation are generally unexplored.
- However two recent studies have implicated testosterone in the process.
- In the mouse forebrain, male-specific methylation patterns can be induced in females by neonatal administration of exogenous testosterone [14].
- In the liver, testosterone can trigger male-specific demethylation of certain regions.
- However, not all gender- specific methylation can be attributed to the actions of testosterone: in an unbiased comparison we found more than 300 gender differences between the livers of females and castrated males.
- Such factors may in- volve early and indirect actions of the sex chromosomes, as has been shown for early embryonic gene expression [15].
- Furthermore, RRBS allows the interrogation of methylation levels at only a representative fraction of the genome, and while this proportion is enriched for functionally relevant regions, whole genome bisulphite sequencing would be required to capture the full extent of gender-specific DNA methylation.
- Gender differences in environmental epigenetic programming are pervasive [17] and our findings here suggest that at least some of the gender bias in induced phenotypes derives from baseline gender differences in epigenetic state..
- Our study of the DNA methylomes of multiple tissues of male and female mice indicates that sex significantly in- fluences DNA methylation patterns in a tissue-specific manner.
- DNA was extracted from a 5 mm coronal section of the rostral end of the brain, a 5 mm apical section of the heart, and the extreme caudal section of the left lobe of the liver..
- RRBS data from liver heart and brain generated for this study have been deposited in the Gene Expression Omni- bus (GEO) under accession number GSE84573.
- To combine statistical evidence of neighbouring CpGs, we calculated DNA methylation levels in tiles of 100 bp across the genome.
- 99.8th percentile of coverage values across all samples in the comparison..
- COBRA vadlidation of candidate liver gender-specific DMRs.
- The datasets generated in this study are available in the Gene Expression Omnibus (GEO) under accession number GSE84573.
- A study of the influence of sex on genome wide methylation.
- Sex differences in the genome-wide DNA methylation pattern and impact on gene expression, microRNA levels and insulin secretion in human pancreatic islets.
- Intergenerational genomic DNA methylation patterns in mouse hybrid strains.
- Gender-specific postnatal demethylation and establishment of epigenetic memory.
- methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles.
- Inter-individual variability contrasts with regional homogeneity in the human brain DNA methylome.
- Sex differences in the expression of hepatic drug metabolizing enzymes.
- The effects of perinatal testosterone exposure on the DNA methylome of the mouse brain are late-emerging.

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