Early life exposure to vitamin D deficiency impairs molecular mechanisms that regulate liver cholesterol biosynthesis, energy metabolism, inflammation, and detoxification.

Introduction: Emerging data suggests liver disease may be initiated during development when there is high genome plasticity and the molecular pathways supporting liver function are being developed. Methods: Here, we leveraged our Collaborative Cross mouse model of developmental vitamin D deficiency (DVD) to investigate the role of DVD in dysregulating the molecular mechanisms underlying liver disease. We defined the effects on the adult liver transcriptome and metabolome and examined the role of epigenetic dysregulation. Given that the parental origin of the genome (POG) influences response to DVD, we used our established POG model [POG1-(CC011xCC001)F1 and POG2-(CC001xCC011)F1] to identify interindividual differences. Results: We found that DVD altered the adult liver transcriptome, primarily downregulating genes controlling liver development, response to injury/infection (detoxification & inflammation), cholesterol biosynthesis, and energy production. In concordance with these transcriptional changes, we found that DVD decreased liver cell membrane-associated lipids (including cholesterol) and pentose phosphate pathway metabolites. Each POG also exhibited distinct responses. POG1 exhibited almost 2X more differentially expressed genes (DEGs) with effects indicative of increased energy utilization. This included upregulation of lipid and amino acid metabolism genes and increased intermediate lipid and amino acid metabolites, increased energy cofactors, and decreased energy substrates. POG2 exhibited broader downregulation of cholesterol biosynthesis genes with a metabolomics profile indicative of decreased energy utilization. Although DVD primarily caused loss of liver DNA methylation for both POGs, only one epimutation was shared, and POG2 had 6.5X more differentially methylated genes. Differential methylation was detected at DEGs regulating developmental processes such as amino acid transport (POG1) and cell growth & differentiation (e.g., Wnt & cadherin signaling, POG2). Conclusions: These findings implicate a novel role for maternal vitamin D in programming essential offspring liver functions that are dysregulated in liver disease. Importantly, impairment of these processes was not rescued by vitamin D treatment at weaning, suggesting these effects require preventative measures. Substantial differences in POG response to DVD demonstrate that the parental genomic context of exposure determines offspring susceptibility.

Timing of standard chow exposure determines the variability of mouse phenotypic outcomes and gut microbiota profile.

Standard chow diet contributes to reproducibility in animal model experiments since chows differ in nutrient composition, which can independently influence phenotypes. However, there is little evidence of the role of timing in the extent of variability caused by chow exposure. Here, we measured the impact of diet (5V5M, 5V0G, 2920X, and 5058) and timing of exposure (adult exposure (AE), lifetime exposure (LE), and developmental exposure (DE)) on growth & development, metabolic health indicators, and gut bacterial microbiota profiles across genetically identical C57BL6/J mice. Diet drove differences in macro- and micronutrient intake for all exposure models. AE had no effect on measured outcomes. However, LE mice exhibited significant sex-dependent diet effects on growth, body weight, and body composition. LE effects were mostly absent in the DE model, where mice were exposed to chow differences from conception to weaning. Both AE and LE models exhibited similar diet-driven beta diversity profiles for the gut bacterial microbiota, with 5058 diet driving the most distinct profile. Diet-induced beta diversity profiles were sex-dependent for LE mice. Compared to AE, LE drove 9X more diet-driven differentially abundant genera, majority of which were the result of inverse effects of 2920X and 5058. Our findings demonstrate that lifetime exposure to different chow diets has the greatest impact on reproducibility of experimental measures that are common components of preclinical mouse model studies. Importantly, weaning DE mice onto a uniform diet is likely an effective way to reduce unwanted phenotypic variability among experimental models.

Sex-specific transgenerational effects of preconception exposure to arsenite: metabolic phenotypes of C57BL/6 offspring.

Chronic exposure to inorganic arsenic (iAs) has been linked to diabetes in both humans and mice, but the role of iAs exposure prior to conception and its transgenerational effects are understudied. The present study investigated transgenerational effects of preconception iAs exposure in C57BL/6J mice, focusing on metabolic phenotypes of G1 and G2 offspring. Body composition and diabetes indicators, including fasting blood glucose, fasting plasma insulin, glucose tolerance, and indicators of insulin resistance and beta cell function, were examined in both generations. The results suggest that the preconception iAs exposure in the parental (G0) generation induced diabetic phenotypes in G1 and G2 offspring in a sex-dependent manner. G1 females from iAs-exposed parents developed insulin resistance while no significant effects were found in G1 males. In the G2 generation, insulin resistance was observed only in males from iAs-exposed grandparents and was associated with higher bodyweights and adiposity. Similar trends were observed in G2 females from iAs-exposed grandparents, but these did not reach statistical significance. Thus, preconception iAs exposure altered metabolic phenotype across two generations of mouse offspring. Future research will investigate the molecular mechanisms underlying these transgenerational effects, including epigenomic and transcriptomic profiles of germ cells and tissues from G0, G1 and G2 generations.

Inference of putative cell-type-specific imprinted regulatory elements and genes during human neuronal differentiation.

Genomic imprinting results in gene expression bias caused by parental chromosome of origin and occurs in genes with important roles during human brain development. However, the cell-type and temporal specificity of imprinting during human neurogenesis is generally unknown. By detecting within-donor allelic biases in chromatin accessibility and gene expression that are unrelated to cross-donor genotype, we inferred imprinting in both primary human neural progenitor cells and their differentiated neuronal progeny from up to 85 donors. We identified 43/20 putatively imprinted regulatory elements (IREs) in neurons/progenitors, and 133/79 putatively imprinted genes in neurons/progenitors. Although 10 IREs and 42 genes were shared between neurons and progenitors, most putative imprinting was only detected within specific cell types. In addition to well-known imprinted genes and their promoters, we inferred novel putative IREs and imprinted genes. Consistent with both DNA methylation-based and H3K27me3-based regulation of imprinted expression, some putative IREs also overlapped with differentially methylated or histone-marked regions. Finally, we identified a progenitor-specific putatively imprinted gene overlapping with copy number variation that is associated with uniparental disomy-like phenotypes. Our results can therefore be useful in interpreting the function of variants identified in future parent-of-origin association studies.

Ex vivo exposures to arsenite and its methylated trivalent metabolites alter gene transcription in mouse sperm cells.

We have previously reported that preconception exposure to iAs may contribute to the development of diabetes in mouse offspring by altering gene expressions in paternal sperm. However, the individual contributions of iAs and its methylated metabolites, monomethylated arsenic (MAs) and dimethylated arsenic (DMAs), to changes in the sperm transcriptome could not be determined because all three As species are present in sperm after in vivo iAs exposure. The goal of the present study was to assess As species-specific effects using an ex vivo model. We exposed freshly isolated mouse sperm to either 0.1 or 1 µM arsenite (iAsIII) or the methylated trivalent arsenicals, MAsIII and DMAsIII, and used RNA-sequencing to identify differentially expressed genes, enriched pathways, and associated protein networks. For all arsenicals tested, the exposures to 0.1 µM concentrations had greater effects on gene expression than 1 µM exposures. Transcription factor AP-1 and B cell receptor complexes were the most significantly enriched pathways in sperm exposed to 0.1 µM iAsIII. The Mre11 complex and Antigen processing were top pathways targeted by exposure to 0.1 µM MAsIII and DMAsIII, respectively. While there was no overlap between gene transcripts altered by ex vivo exposures in the present study and those altered by in vivo exposure in our prior work, several pathways were shared, including PI3K-Akt signaling, Focal adhesion, and Extracellular matrix receptor interaction pathways. Notably, the protein networks associated with these pathways included those with known roles in diabetes. This study is the first to assess the As species-specific effects on sperm transcriptome, linking these effects to the diabetogenic effects of iAs exposure.

Gestational diabetes mellitus placentas exhibit epimutations at placental development genes.

Gestational diabetes mellitus (GDM) is a maternal metabolic disorder that perturbs placental development and increases the risk of offspring short- and long-term metabolic disorders. The mechanisms by which GDM impairs placental development remain poorly understood. Here, we defined the DNA methylome of GDM placentas and determined whether GDM perturbs methylation at genes important for placental development. We conducted an epigenome-wide association study of 42 placentas from pregnancies in the South African Soweto First 1000 days cohort (S1000). Using genome-wide bisulfite sequencing, we compared non-GDM placentas to GDM placentas with similar proportions from obese and non-obese mothers. Compared to non-GDM, GDM placentas exhibited a distinct methylation profile consisting of 12,210 differentially methylated CpGs (DMCs) that mapped to 3,875 genes. Epigenetically altered genes were enriched in Wnt and cadherin signalling pathways, both critical in placentation and embryogenesis. We also defined regional DNA methylation perturbation in GDM placentas at 11 placental development genes. These findings reveal extensive changes to the placental epigenome of GDM pregnancies and highlight perturbation enriched at important placental development genes. These molecular changes represent potential mechanisms for GDM-induced placental effects that may serve as candidate biomarkers for placental, maternal, and foetal health. Using a study design that used similar proportions of obese and non-obese mothers in our case and control pregnancies, we minimized the detection of changes due to obesity alone. Further work will be necessary to investigate the extent of the influence of obesity on these GDM-related placental epigenetic changes.

Sex-dependent effects of preconception exposure to arsenite on gene transcription in parental germ cells and on transcriptomic profiles and diabetic phenotype of offspring.

Chronic exposure to inorganic arsenic (iAs) has been linked to diabetic phenotypes in both humans and mice. However, diabetogenic effects of iAs exposure during specific developmental windows have never been systematically studied. We have previously shown that in mice, combined preconception and in utero exposures to iAs resulted in impaired glucose homeostasis in male offspring. The goal of the present study was to determine if preconception exposure alone can contribute to this outcome. We have examined metabolic phenotypes in male and female offspring from dams and sires that were exposed to iAs in drinking water (0 or 200 µg As/L) for 10 weeks prior to mating. The effects of iAs exposure on gene expression profiles in parental germ cells, and pancreatic islets and livers from offspring were assessed using RNA sequencing. We found that iAs exposure significantly altered transcript levels of genes, including diabetes-related genes, in the sperm of sires. Notably, some of the same gene transcripts and the associated pathways were also altered in the liver of the offspring. The exposure had a more subtle effect on gene expression in maternal oocytes and in pancreatic islets of the offspring. In female offspring, the preconception exposure was associated with increased adiposity, but lower blood glucose after fasting and after glucose challenge. HOMA-IR, the indicator of insulin resistance, was also lower. In contrast, the preconception exposure had no effects on blood glucose measures in male offspring. However, males from parents exposed to iAs had higher plasma insulin after glucose challenge and higher insulinogenic index than control offspring, indicating a greater requirement for insulin to maintain glucose homeostasis. Our results suggest that preconception exposure may contribute to the development of diabetic phenotype in male offspring, possibly mediated through germ cell-associated inheritance. Future research can investigate role of epigenetics in this phenomenon. The paradoxical outcomes in female offspring, suggesting a protective effect of the preconception exposure, warrant further investigation.

Maternal Microdeletion at the H19/Igf2 ICR in Mice Increases Offspring Susceptibility to In Utero Environmental Perturbation.

Deficiency of methyl donor nutrients folate, choline, and methionine (methyl deficiency) during gestation can impair fetal development and perturb DNA methylation. Here, we assessed genetic susceptibility to methyl deficiency by comparing effects in wildtype C57BL/6J (B6) mice to mutant mice carrying a 1.3 kb deletion at the H19/Igf2 Imprinting Control Region (ICR) (H19 ICRΔ2,3). The H19 ICRΔ2,3 mutation mimics microdeletions observed in Beckwith-Wiedemann syndrome (BWS) patients, who exhibit epimutations in cis that cause loss of imprinting and fetal overgrowth. Dams were treated during pregnancy with 1 of 4 methyl sufficient (MS) or methyl deficient (MD) diets, with or without the antibiotic commonly used to deplete folate producing gut microbes. As expected, after ~9 weeks of treatment, dams in MD and MD + antibiotic groups exhibited substantially reduced plasma folate concentrations. H19 ICRΔ2,3 mutant lines were more susceptible to adverse pregnancy outcomes caused by methyl deficiency (reduced birth rate and increased pup lethality) and antibiotic (decreased litter size and litter survival). Surprisingly, pup growth/development was only minimally affected by methyl deficiency, while antibiotic treatment caused inverse effects on B6 and H19 ICRΔ2,3 lines. B6 pups treated with antibiotic exhibited increased neonatal and weanling bodyweight, while both wildtype and mutant pups of heterozygous H19 ICRΔ2,3/+ dams exhibited decreased neonatal bodyweight that persisted into adulthood. Interestingly, only antibiotic-treated pups carrying the H19 ICRΔ2,3 mutation exhibited altered DNA methylation at the H19/Igf2 ICR, suggesting ICR epimutation was not sufficient to explain the altered phenotypes. These findings demonstrate that genetic mutation of the H19/Igf2 ICR increases offspring susceptibility to developmental perturbation in the methyl deficiency model, maternal and pup genotype play an essential role, and antibiotic treatment in the model also plays a key independent role.

Content and Performance of the MiniMUGA Genotyping Array: A New Tool To Improve Rigor and Reproducibility in Mouse Research.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.

Baseline and innate immune response characterization of a Zfp30 knockout mouse strain.

Airway neutrophilia is correlated with disease severity in a number of chronic and acute pulmonary diseases, and dysregulation of neutrophil chemotaxis can lead to host tissue damage. The gene Zfp30 was previously identified as a candidate regulator of neutrophil recruitment to the lungs and secretion of CXCL1, a potent neutrophil chemokine, in a genome-wide mapping study using the Collaborative Cross. ZFP30 is a putative transcriptional repressor with a KRAB domain capable of inducing heterochromatin formation. Using a CRISPR-mediated knockout mouse model, we investigated the role that Zfp30 plays in recruitment of neutrophils to the lung using models of allergic airway disease and acute lung injury. We found that the Zfp30 null allele did not affect CXCL1 secretion or neutrophil recruitment to the lungs in response to various innate immune stimuli. Intriguingly, despite the lack of neutrophil phenotype, we found there was a significant reduction in the proportion of live Zfp30 homozygous female mutant mice produced from heterozygous matings. This deviation from the expected Mendelian ratios implicates Zfp30 in fertility or embryonic development. Overall, our results indicate that Zfp30 is an essential gene but does not influence neutrophilic inflammation in this particular knockout model.

Maternal Liver Metabolic Response to Chronic Vitamin D Deficiency Is Determined by Mouse Strain Genetic Background.

BACKGROUND: Liver metabolite concentrations have the potential to be key biomarkers of systemic metabolic dysfunction and overall health. However, for most conditions we do not know the extent to which genetic differences regulate susceptibility to metabolic responses. This limits our ability to detect and diagnose effects in heterogeneous populations. OBJECTIVES: Here, we investigated the extent to which naturally occurring genetic differences regulate maternal liver metabolic response to vitamin D deficiency (VDD), particularly during perinatal periods when such changes can adversely affect maternal and fetal health. METHODS: We used a panel of 8 inbred Collaborative Cross (CC) mouse strains, each with a different genetic background (72 dams, 3-6/treatment group, per strain). We identified robust maternal liver metabolic responses to vitamin D depletion before and during gestation and lactation using a vitamin-D-deficient (VDD; 0 IU vitamin D3/kg) or -sufficient diet (1000 IU vitamin D3/kg). We then identified VDD-induced metabolite changes influenced by strain genetic background. RESULTS: We detected a significant VDD effect by orthogonal partial least squares discriminant analysis (Q2 = 0.266, pQ2 = 0.002): primarily, altered concentrations of 78 metabolites involved in lipid, amino acid, and nucleotide metabolism (variable importance to projection score ≥1.5). Metabolites in unsaturated fatty acid and glycerophospholipid metabolism pathways were significantly enriched [False Discovery Rate (FDR) <0.05]. VDD also significantly altered concentrations of putative markers of uremic toxemia, acylglycerols, and dipeptides. The extent of the metabolic response to VDD was strongly dependent on genetic strain, ranging from robustly responsive to nonresponsive. Two strains (CC017/Unc and CC032/GeniUnc) were particularly sensitive to VDD; however, each strain altered different pathways. CONCLUSIONS: These novel findings demonstrate that maternal VDD induces different liver metabolic effects in different genetic backgrounds. Strains with differing susceptibility and metabolic response to VDD represent unique tools to identify causal susceptibility factors and further elucidate the role of VDD-induced metabolic changes in maternal and/or fetal health for ultimately translating findings to human populations.

Assessment of placental metal levels in a South African cohort.

The placenta plays an important role in mediating the effect of maternal metal exposure on fetal development, acting as both barrier and transporter. Term-placenta metal levels serve as an informative snapshot of maternal/fetal exposure during pregnancy and could be used to predict offspring short- and long-term health outcomes. Here, we measured term-placenta metal levels of 11 metals in 42 placentas from the Soweto First 1000 days cohort (S1000, Soweto-Johannesburg, SA). We compared these placental metal concentrations with previously reported global cohort measurements to determine whether this cohort is at increased risk of exposure. Placental metals were tested for correlations to understand potential interactions between metals. Since these samples are from a birth cohort study, we also performed exploratory analyses to determine whether metal levels were associated with placenta and birth outcomes. Most S1000 placental metal levels were similar to other cohorts; however, cadmium (Cd) levels up to 50-fold lower, and essential elements nickel (Ni) and chromium (Cr) level up to 6- and 16-fold lower, respectively. Cd, Se, and Ni were associated with placenta and birth outcomes. Studies are ongoing to examine underlying mechanisms and how these developmental differences affect long-term health.

Maternal vitamin D deficiency and developmental origins of health and disease (DOHaD).

Vitamin D is an essential nutrient that is metabolized in the body to generate an active metabolite (1,25(OH)2D) with hormone-like activity and highly diverse roles in cellular function. Vitamin D deficiency (VDD) is a prevalent but easily preventable nutritional disturbance. Emerging evidence demonstrates the importance of sufficient vitamin D concentrations during fetal life with deficiencies leading to long-term effects into adulthood. Here, we provide a detailed review and perspective of evidence for the role of maternal VDD in offspring long term health, particularly as it relates to Developmental Origins of Health and Disease (DOHaD). We focus on roles in neurobehavioral and cardiometabolic disorders in humans and highlight recent findings from zebrafish and rodent models that probe potential mechanisms linking early life VDD to later life health outcomes. Moreover, we explore evidence implicating epigenetic mechanisms as a mediator of this link. Gaps in our current understanding of how maternal VDD might result in deleterious offspring outcomes later in life are also addressed.

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

Suboptimal environmental conditions during development can substantially alter the epigenome. Stable environmentally-induced changes to the germline epigenome, in particular, have important implications for the health of the next generation. We showed previously that developmental vitamin D depletion (DVD) resulted in loss of DNA methylation at several imprinted loci over two generations. Here, we assessed the impact of DVD on genome-wide methylation in mouse sperm in order to characterize the number, extent and distribution of methylation changes in response to DVD and to find genes that may be susceptible to this prevalent environmental perturbation. We detected 15,827 loci that were differentially methylated in DVD mouse sperm vs. controls. Most epimutations (69%) were loss of methylation, and the extent of methylation change and number of CpGs affected in a region were associated with genic location and baseline methylation state. Methylation response to DVD at validated loci was only detected in offspring that exhibited a phenotypic response to DVD (increased body and testes weight) suggesting the two types of responses are linked, though a causal relationship is unclear. Epimutations localized to regions enriched for developmental and metabolic genes and pathway analyses showed enrichment for Cadherin, Wnt, PDGF and Integrin signaling pathways. These findings show for the first time that vitamin D status during development leads to substantial DNA methylation changes across the sperm genome and that locus susceptibility is linked to genomic and epigenomic context.

Intergenerational response to the endocrine disruptor vinclozolin is influenced by maternal genotype and crossing scheme.

In utero exposure to vinclozolin (VIN), an antiandrogenic fungicide, is linked to multigenerational phenotypic and epigenetic effects. Mechanisms remain unclear. We assessed the role of antiandrogenic activity and DNA sequence context by comparing effects of VIN vs. M2 (metabolite with greater antiandrogenic activity) and wild-type C57BL/6 (B6) mice vs. mice carrying mutations at the previously reported VIN-responsive H19/Igf2 locus. First generation offspring from VIN-treated 8nrCG mutant dams exhibited increased body weight and decreased sperm ICR methylation. Second generation pups sired by affected males exhibited decreased neonatal body weight but only when dam was unexposed. Offspring from M2 treatments, B6 dams, 8nrCG sires or additional mutant lines were not similarly affected. Therefore, pup response to VIN over two generations detected here was an 8nrCG-specific maternal effect, independent of antiandrogenic activity. These findings demonstrate that maternal effects and crossing scheme play a major role in multigenerational response to in utero exposures.

Dietary Modulation of the Epigenome.

Epigenetics is the study of heritable mechanisms that can modify gene activity and phenotype without modifying the genetic code. The basis for the concept of epigenetics originated more than 2,000 yr ago as a theory to explain organismal development. However, the definition of epigenetics continues to evolve as we identify more of the components that make up the epigenome and dissect the complex manner by which they regulate and are regulated by cellular functions. A substantial and growing body of research shows that nutrition plays a significant role in regulating the epigenome. Here, we critically assess this diverse body of evidence elucidating the role of nutrition in modulating the epigenome and summarize the impact such changes have on molecular and physiological outcomes with regards to human health.

Tissue-specific and mosaic imprinting defects underlie opposite congenital growth disorders in mice.

Differential DNA methylation defects of H19/IGF2 are associated with congenital growth disorders characterized by opposite clinical pictures. Due to structural differences between human and mouse, the mechanisms by which mutations of the H19/IGF2 Imprinting Control region (IC1) result in these diseases are undefined. To address this issue, we previously generated a mouse line carrying a humanized IC1 (hIC1) and now replaced the wildtype with a mutant IC1 identified in the overgrowth-associated Beckwith-Wiedemann syndrome. The new humanized mouse line shows pre/post-natal overgrowth on maternal transmission and pre/post-natal undergrowth on paternal transmission of the mutation. The mutant hIC1 acquires abnormal methylation during development causing opposite H19/Igf2 imprinting defects on maternal and paternal chromosomes. Differential and possibly mosaic Igf2 expression and imprinting is associated with asymmetric growth of bilateral organs. Furthermore, tissue-specific imprinting defects result in deficient liver- and placenta-derived Igf2 on paternal transmission and excessive Igf2 in peripheral tissues on maternal transmission, providing a possible molecular explanation for imprinting-associated and phenotypically contrasting growth disorders.

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

BACKGROUND: Environmental 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. METHODS: We 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. RESULTS: Maternal 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. CONCLUSIONS: These 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.

Humanized H19/Igf2 locus reveals diverged imprinting mechanism between mouse and human and reflects Silver-Russell syndrome phenotypes.

Genomic imprinting affects a subset of genes in mammals, such that they are expressed in a monoallelic, parent-of-origin-specific manner. These genes are regulated by imprinting control regions (ICRs), cis-regulatory elements that exhibit allele-specific differential DNA methylation. Although genomic imprinting is conserved in mammals, ICRs are genetically divergent across species. This raises the fundamental question of whether the ICR plays a species-specific role in regulating imprinting at a given locus. We addressed this question at the H19/insulin-like growth factor 2 (Igf2) imprinted locus, the misregulation of which is associated with the human imprinting disorders Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). We generated a knock-in mouse in which the endogenous H19/Igf2 ICR (mIC1) is replaced by the orthologous human ICR (hIC1) sequence, designated H19(hIC1) We show that hIC1 can functionally replace mIC1 on the maternal allele. In contrast, paternally transmitted hIC1 leads to growth restriction, abnormal hIC1 methylation, and loss of H19 and Igf2 imprinted expression. Imprint establishment at hIC1 is impaired in the male germ line, which is associated with an abnormal composition of histone posttranslational modifications compared with mIC1. Overall, this study reveals evolutionarily divergent paternal imprinting at IC1 between mice and humans. The conserved maternal imprinting mechanism and function at IC1 demonstrates the possibility of modeling maternal transmission of hIC1 mutations associated with BWS in mice. In addition, we propose that further analyses in the paternal knock-in H19(+/hIC1) mice will elucidate the molecular mechanisms that may underlie SRS.

Nutrigenomics, the Microbiome, and Gene-Environment Interactions: New Directions in Cardiovascular Disease Research, Prevention, and Treatment: A Scientific Statement From the American Heart Association.

Cardiometabolic diseases are the leading cause of death worldwide and are strongly linked to both genetic and nutritional factors. The field of nutrigenomics encompasses multiple approaches aimed at understanding the effects of diet on health or disease development, including nutrigenetic studies investigating the relationship between genetic variants and diet in modulating cardiometabolic risk, as well as the effects of dietary components on multiple "omic" measures, including transcriptomics, metabolomics, proteomics, lipidomics, epigenetic modifications, and the microbiome. Here, we describe the current state of the field of nutrigenomics with respect to cardiometabolic disease research and outline a direction for the integration of multiple omics techniques in future nutrigenomic studies aimed at understanding mechanisms and developing new therapeutic options for cardiometabolic disease treatment and prevention.