Maternal vitamin D depletion alters DNA methylation at imprinted loci in multiple generations

Abstract

BackgroundEnvironmental perturbation of epigenetic mechanisms is linked to a growing number of diseases. Characterizing the role environmental factors play in modifying the epigenome is important for disease etiology. Vitamin D is an essential nutrient affecting brain, bone, heart, immune and reproductive health. Vitamin D insufficiency is a global issue, and the role in maternal and child health remains under investigation.MethodsWe used Collaborative Cross (CC) inbred mice to characterize the effect of maternal vitamin D depletion on offspring phenotypic and epigenetic outcomes at imprinted domains (H19/Igf2, Snrpn, Dlk1/Gtl2, and Grb10) in the soma (liver) and germline (sperm). We assessed outcomes in two generations of offspring to determine heritability. We used reciprocal crosses between lines CC001/Unc and CC011/Unc to investigate parent of origin effects.ResultsMaternal vitamin D deficiency led to altered body weight and DNA methylation in two generations of offspring. Loci assayed in adult liver and sperm were mostly hypomethylated, but changes were few and small in effect size (<7 % difference on average). There was no change in total expression of genes adjacent to methylation changes in neonatal liver. Methylation changes were cell type specific such that changes at IG-DMR were present in sperm but not in liver. Some methylation changes were distinct between generations such that methylation changes at the H19ICR in second-generation liver were not present in first-generation sperm or liver. Interestingly, some diet-dependent changes in body weight and methylation were seemingly influenced by parent of origin such that reciprocal crosses exhibited inverse effects.ConclusionsThese findings demonstrate that maternal vitamin D status plays a role in determining DNA methylation state in the germline and soma. Detection of methylation changes in the unexposed second-generation demonstrates that maternal vitamin D depletion can have long-term effects on the epigenome of subsequent generations. Differences in vitamin D-dependent epigenetic state between cell types and generations indicate perturbation of the epigenetic landscape rather than a targeted, locus-specific effect. While the biological importance of these subtle changes remains unclear, they warrant an investigation of epigenome-wide effects of maternal vitamin D depletion.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0276-4) contains supplementary material, which is available to authorized users.

Impact of vitamin D depletion during development on mouse sperm DNA methylation

“…A large number of the genome-wide loss of methylation changes detected here localize to genes involved in Wnt (168 genes, 68 with effect size > 10%) and Cadherin (95 genes, 45 with effect size > 10%) signaling pathways, demonstrating enrichment of genes in these pathways, which could be indicative of perturbed sperm development [32,33] or perturbed development of the second generation which could inherit these changes. Any sperm developmental changes in these first generation animals would likely be subtle since our previous assessment of DVD in males showed no difference in sperm count or litter size [20]. We did show that the DVD-induced sperm methylation response at validated loci was only present in offspring that also exhibited an increase in bodyweight and testes weight.…”

“…Experimental male mice were generated by crossing Collaborative Cross inbred mice, CC001 dams and CC011 sires, as described previously [20]. Males were exposed to vitamin D depleted conditions during pre-and post-natal development via maternal diet of either AIN-93G diet (1000 IU/kg of vitamin D3, CON) or modified AIN-93G lacking vitamin D (0 IU/kg of vitamin D3, DVD).…”

“…Males were exposed to vitamin D depleted conditions during pre-and post-natal development via maternal diet of either AIN-93G diet (1000 IU/kg of vitamin D3, CON) or modified AIN-93G lacking vitamin D (0 IU/kg of vitamin D3, DVD). Adult (8wks) male sperm samples were selected from animals that differed in adult body weight and testes weight between the two diet groups, as previously reported [20]. Genome-wide DNA methylation was measured by bisulfite sequencing (bis-seq) using the Agilent SureSelect Methyl-seq target enrichment system.…”

“…Since sperm samples chosen for bis-seq were partly selected on the basis of the animals having a phenotypic response to DVD, we determined whether methylation response to DVD only coincided with phenotypic response. We defined phenotypic 'responders' and 'non-responders' based on DVD-induced changes in relative body weight and testes weight described previously [20] ( Figure 5(a,b)). Interestingly, none of the DVDinduced methylation changes validated by pyrosequencing in phenotypic responders were detected in sperm from phenotypic non-responders ( Figure 5(c)).…”

“…In particular, deficiency induced a significant increase in adult liver global methylation that was linked to changes in transcript levels of DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b) [17]. We showed previously that dietary vitamin D depletion during development (DVD) altered offspring bodyweight and adiposity and led to loss of DNA methylation at several imprinted loci in adult liver and sperm of first generation treated animals as well as the second generation offspring of treated males [20]. Effect sizes at these imprinted loci were small (< 10%) and unlikely to have physiological consequences, but the multigenerational phenotypes were indicative of a greater epigenetic response to DVD that we proposed could be propagated through the male germline.…”