Systemic interindividual epigenetic variation in humans is associated with transposable elements and under strong genetic control.

BACKGROUND: Genetic variants can modulate phenotypic outcomes via epigenetic intermediates, for example at methylation quantitative trait loci (mQTL). We present the first large-scale assessment of mQTL at human genomic regions selected for interindividual variation in CpG methylation, which we call correlated regions of systemic interindividual variation (CoRSIVs). These can be assayed in blood DNA and do not reflect interindividual variation in cellular composition. RESULTS: We use target-capture bisulfite sequencing to assess DNA methylation at 4086 CoRSIVs in multiple tissues from each of 188 donors in the NIH Gene-Tissue Expression (GTEx) program. At CoRSIVs, DNA methylation in peripheral blood correlates with methylation and gene expression in internal organs. We also discover unprecedented mQTL at these regions. Genetic influences on CoRSIV methylation are extremely strong (median R2=0.76), cumulatively comprising over 70-fold more human mQTL than detected in the most powerful previous study. Moreover, mQTL beta coefficients at CoRSIVs are highly skewed (i.e., the major allele predicts higher methylation). Both surprising findings are independently validated in a cohort of 47 non-GTEx individuals. Genomic regions flanking CoRSIVs show long-range enrichments for LINE-1 and LTR transposable elements; the skewed beta coefficients may therefore reflect evolutionary selection of genetic variants that promote their methylation and silencing. Analyses of GWAS summary statistics show that mQTL polymorphisms at CoRSIVs are associated with metabolic and other classes of disease. CONCLUSIONS: A focus on systemic interindividual epigenetic variants, clearly enhanced in mQTL content, should likewise benefit studies attempting to link human epigenetic variation to the risk of disease.

Sex-specific epigenetic development in the mouse hypothalamic arcuate nucleus pinpoints human genomic regions associated with body mass index.

Recent genome-wide association studies corroborate classical research on developmental programming indicating that obesity is primarily a neurodevelopmental disease strongly influenced by nutrition during critical ontogenic windows. Epigenetic mechanisms regulate neurodevelopment; however, little is known about their role in establishing and maintaining the brain's energy balance circuitry. We generated neuron and glia methylomes and transcriptomes from male and female mouse hypothalamic arcuate nucleus, a key site for energy balance regulation, at time points spanning the closure of an established critical window for developmental programming of obesity risk. We find that postnatal epigenetic maturation is markedly cell type and sex specific and occurs in genomic regions enriched for heritability of body mass index in humans. Our results offer a potential explanation for both the limited ontogenic windows for and sex differences in sensitivity to developmental programming of obesity and provide a rich resource for epigenetic analyses of developmental programming of energy balance.

A machine learning case-control classifier for schizophrenia based on DNA methylation in blood.

Epigenetic dysregulation is thought to contribute to the etiology of schizophrenia (SZ), but the cell type-specificity of DNA methylation makes population-based epigenetic studies of SZ challenging. To train an SZ case-control classifier based on DNA methylation in blood, therefore, we focused on human genomic regions of systemic interindividual epigenetic variation (CoRSIVs), a subset of which are represented on the Illumina Human Methylation 450K (HM450) array. HM450 DNA methylation data on whole blood of 414 SZ cases and 433 non-psychiatric controls were used as training data for a classification algorithm with built-in feature selection, sparse partial least squares discriminate analysis (SPLS-DA); application of SPLS-DA to HM450 data has not been previously reported. Using the first two SPLS-DA dimensions we calculated a "risk distance" to identify individuals with the highest probability of SZ. The model was then evaluated on an independent HM450 data set on 353 SZ cases and 322 non-psychiatric controls. Our CoRSIV-based model classified 303 individuals as cases with a positive predictive value (PPV) of 80%, far surpassing the performance of a model based on polygenic risk score (PRS). Importantly, risk distance (based on CoRSIV methylation) was not associated with medication use, arguing against reverse causality. Risk distance and PRS were positively correlated (Pearson r = 0.28, P = 1.28 × 10-12), and mediational analysis suggested that genetic effects on SZ are partially mediated by altered methylation at CoRSIVs. Our results indicate two innate dimensions of SZ risk: one based on genetic, and the other on systemic epigenetic variants.

Contribution of growth hormone secretagogue receptor (GHSR) signaling in the ventral tegmental area (VTA) to the regulation of social motivation in male mice.

Most psychiatric disorders are characterized by deficits in the ability to interact socially with others. Ghrelin, a hormone normally associated with the regulation of glucose utilization and appetite, is also implicated in the modulation of motivated behaviors including those associated with food and sex rewards. Here we hypothesized that deficits in ghrelin receptor (growth hormone secretagogue receptor; GHSR) signaling are also associated with deficits in social motivation in male mice. To test this hypothesis, we compared social motivation in male mice lacking GHSR or mice treated with the GHSR antagonist JMV2959 with that of WT or vehicle-treated mice. GHSR signaling in dopamine cells of the ventral tegmental area (VTA) has been implicated in the control of sexual behavior, thus we further hypothesized that GHSR signaling in the VTA is important for social motivation. Thus, we conducted studies where we delivered JMV2959 to block GHSR in the VTA of mice, and studies where we rescued the expression of GHSR in the VTA of GHSR knockout (KO) mice. Mice lacking GHSR or injected with JMV2959 peripherally for 3 consecutive days displayed lower social motivation as reflected by a longer latency to approach a novel conspecific and shorter interaction time compared to WT or vehicle-treated controls. Furthermore, intra-VTA infusion of JMV2959 resulted in longer latencies to approach a novel conspecific, whereas GHSR KO mice with partial rescue of the GHSR showed decreased latencies to begin a novel social interaction. Together, these data suggest that GHSR in the VTA facilitate social approach in male mice, and GHSR-signaling deficits within the VTA result in reduced motivation to interact socially.

Identification of cell type-specific methylation signals in bulk whole genome bisulfite sequencing data.

BACKGROUND: The traditional approach to studying the epigenetic mechanism CpG methylation in tissue samples is to identify regions of concordant differential methylation spanning multiple CpG sites (differentially methylated regions). Variation limited to single or small numbers of CpGs has been assumed to reflect stochastic processes. To test this, we developed software, Cluster-Based analysis of CpG methylation (CluBCpG), and explored variation in read-level CpG methylation patterns in whole genome bisulfite sequencing data. RESULTS: Analysis of both human and mouse whole genome bisulfite sequencing datasets reveals read-level signatures associated with cell type and cell type-specific biological processes. These signatures, which are mostly orthogonal to classical differentially methylated regions, are enriched at cell type-specific enhancers and allow estimation of proportional cell composition in synthetic mixtures and improved prediction of gene expression. In tandem, we developed a machine learning algorithm, Precise Read-Level Imputation of Methylation (PReLIM), to increase coverage of existing whole genome bisulfite sequencing datasets by imputing CpG methylation states on individual sequencing reads. PReLIM both improves CluBCpG coverage and performance and enables identification of novel differentially methylated regions, which we independently validate. CONCLUSIONS: Our data indicate that, rather than stochastic variation, read-level CpG methylation patterns in tissue whole genome bisulfite sequencing libraries reflect cell type. Accordingly, these new computational tools should lead to an improved understanding of epigenetic regulation by DNA methylation.

DNA methylation in AgRP neurons regulates voluntary exercise behavior in mice.

DNA methylation regulates cell type-specific gene expression. Here, in a transgenic mouse model, we show that deletion of the gene encoding DNA methyltransferase Dnmt3a in hypothalamic AgRP neurons causes a sedentary phenotype characterized by reduced voluntary exercise and increased adiposity. Whole-genome bisulfite sequencing (WGBS) and transcriptional profiling in neuronal nuclei from the arcuate nucleus of the hypothalamus (ARH) reveal differentially methylated genomic regions and reduced expression of AgRP neuron-associated genes in knockout mice. We use read-level analysis of WGBS data to infer putative ARH neural cell types affected by the knockout, and to localize promoter hypomethylation and increased expression of the growth factor Bmp7 to AgRP neurons, suggesting a role for aberrant TGF-ß signaling in the development of this phenotype. Together, these data demonstrate that DNA methylation in AgRP neurons is required for their normal epigenetic development and neuron-specific gene expression profiles, and regulates voluntary exercise behavior.

S100a4-Cre-mediated deletion of Patched1 causes hypogonadotropic hypogonadism: role of pituitary hematopoietic cells in endocrine regulation.

Hormones produced by the anterior pituitary gland regulate an array of important physiological functions, but pituitary hormone disorders are not fully understood. Herein we report that genetically-engineered mice with deletion of the hedgehog signaling receptor Patched1 by S100a4 promoter-driven Cre recombinase (S100a4-Cre;Ptch1fl/fl mutants) exhibit adult-onset hypogonadotropic hypogonadism and multiple pituitary hormone disorders. During the transition from puberty to adult, S100a4-Cre;Ptch1fl/fl mice of both sexes develop hypogonadism coupled with reduced gonadotropin levels. Their pituitary glands also display severe structural and functional abnormalities, as revealed by transmission electron microscopy and expression of key genes regulating pituitary endocrine functions. S100a4-Cre activity in the anterior pituitary gland is restricted to CD45+ cells of hematopoietic origin, including folliculo-stellate cells and other immune cell types, causing sex-specific changes in the expression of genes regulating the local microenvironment of the anterior pituitary. These findings provide in vivo evidence for the importance of pituitary hematopoietic cells in regulating fertility and endocrine function, in particular during sexual maturation and likely through sexually dimorphic mechanisms. These findings support a previously unrecognized role of hematopoietic cells in causing hypogonadotropic hypogonadism and provide inroads into the molecular and cellular basis for pituitary hormone disorders in humans.

A genomic atlas of systemic interindividual epigenetic variation in humans.

BACKGROUND: DNA methylation is thought to be an important determinant of human phenotypic variation, but its inherent cell type specificity has impeded progress on this question. At exceptional genomic regions, interindividual variation in DNA methylation occurs systemically. Like genetic variants, systemic interindividual epigenetic variants are stable, can influence phenotype, and can be assessed in any easily biopsiable DNA sample. We describe an unbiased screen for human genomic regions at which interindividual variation in DNA methylation is not tissue-specific. RESULTS: For each of 10 donors from the NIH Genotype-Tissue Expression (GTEx) program, CpG methylation is measured by deep whole-genome bisulfite sequencing of genomic DNA from tissues representing the three germ layer lineages: thyroid (endoderm), heart (mesoderm), and brain (ectoderm). We develop a computational algorithm to identify genomic regions at which interindividual variation in DNA methylation is consistent across all three lineages. This approach identifies 9926 correlated regions of systemic interindividual variation (CoRSIVs). These regions, comprising just 0.1% of the human genome, are inter-correlated over long genomic distances, associated with transposable elements and subtelomeric regions, conserved across diverse human ethnic groups, sensitive to periconceptional environment, and associated with genes implicated in a broad range of human disorders and phenotypes. CoRSIV methylation in one tissue can predict expression of associated genes in other tissues. CONCLUSIONS: In addition to charting a previously unexplored molecular level of human individuality, this atlas of human CoRSIVs provides a resource for future population-based investigations into how interindividual epigenetic variation modulates risk of disease.

A plurality of molecular targets: The receptor ecosystem for bisphenol-A (BPA).

Bisphenol-A (BPA) is a well-known endocrine disrupting compound (EDC), capable of affecting the normal function and development of the reproductive system, brain, adipose tissue, and more. In spite of these diverse and well characterized effects, there is often comparatively little known about the molecular mechanisms which bring them about. BPA has traditionally been regarded as a primarily estrogenic EDC, and this perspective is often what guides research into the effects of BPA. However, emerging data from in-vitro and in-silico models show that BPA binds with a significant number of hormone receptors, including a number of nuclear and membrane-bound estrogen receptors, androgen receptors, as well as the thyroid hormone receptor, glucocorticoid receptor, and PPARγ. With this increased diversity of receptor targets, it may be possible to explain some of the more puzzling aspects of BPA pharmacology, including its non-monotonic dose-response curve, as well as experimental results which disagree with estrogenic positive controls. This paper reviews the receptors for which BPA has a known interaction, and discusses the implications of taking these receptors into account when studying the disruptive effects of BPA on growth and development.

Measuring the "transfer of meaning" through members of equivalence classes merged via a class-specific reinforcement procedure.

Seven participants received conditional discrimination training that established the 12 conditional relations A1B1, A2B2, A3B3, A1C1, A2C2, A3C3, D1E1, D2E2, D3E3, D1F1, D2F2, and D3F3. The A stimuli were pictures of faces portraying emotional expressions; the others were arbitrary forms. Correct responses resulted in presentations of class-specific reinforcers, Sr1, Sr2, and Sr3. After training, tests confirmed the formation of ABC and DEF equivalence classes. Further tests then documented the merger of the classes and the emergence of SrB, SrC, SrE, and SrF relations, showing that the class-specific reinforcers were equivalence class members. Finally, participants did Semantic Differential ratings that tested whether the emotional valence of the A stimuli transferred to the arbitrary forms, B and E. The results show that participants' evaluations of the B and E stimuli were similar to evaluations of the A stimuli made by participants of a control group. This finding is considered as a demonstration that class-specific outcomes can mediate class merger phenomena and the transfer of functions through members of merged classes.

POMC and NPY mRNA expression during development is increased in rat offspring brain from mothers fed with a high fat diet.

Developmental programing is influenced by perinatal nutrition and it has long-lasting impacts on adult metabolism in the offspring. In particular, maternal high fat diet has been associated with increased risk of obesity and metabolic disorders during adulthood in the descendants. These effects may be due to the effects of the high fat diet on the development of the systems that regulate food intake and energy balance in the offspring hypothalamus. The arcuate nucleus (ARC) may be a particularly sensitive region to it as this nucleus contains the POMC and AgRP/NPY neurons that integrate the melanocortin system. Thus, the aim of this study was to investigate the effects of maternal high fat diet during pregnancy on the transcription factors that regulate hypothalamic development in the offspring as a potential mechanism that may result in altered neuronal expression of POMC, NPY and/or AgRP. To this end, pregnant females exposed to high fat diet (60% fat diet since day 0 of pregnancy) or standard rat chow were sacrificed on days 12, 14, 16 and 18 of gestation to obtain brains from their developing fetuses and examine the mRNA expression of transcription factors associated with the development of cells in the ARC. Results show that, while no changes in transcription factor expression between groups were observed, POMC and NPY mRNA expression were higher on embryonic day 18 in the high fat group. These results suggest that POMC and NPY expression are altered by in utero exposure to a high fat diet, but these changes in gene expression are not associated with changes in the expression of transcription factors known to determine the fate of ARC cells.

Perinatal Exposure to Low-Dose Bisphenol-A Disrupts the Structural and Functional Development of the Hypothalamic Feeding Circuitry.

Bisphenol-A (BPA) is a component of polycarbonate and other plastics to which humans are regularly exposed at low levels. BPA is characterized as an endocrine disruptor because of observations of its estrogenic activity in various experimental models. We have previously shown evidence of disrupted hypothalamic feeding circuitry and leptin sensitivity in adult BPA-exposed animals subjected to a high-fat diet, but because these animals were already exhibiting a diet-induced obese phenotype, we could not rule out the possibility that these observations were simply consequences of the obesity, not a preexisting phenotype produced by BPA exposure. Here, we studied leptin sensitivity and hypothalamic structure in young BPA-exposed animals before the onset of a body weight or metabolic phenotype. Pregnant and lactating CD-1 mice were exposed to either BPA or diethylstilbestrol (DES) at low, environmentally relevant doses via their diet. Studies of leptin function and neurobiology were conducted on offspring at several time points. Young adult offspring from this experiment were resistant to leptin-induced suppression of food intake, body weight loss, and hypothalamic pro-opiomelanocortin (POMC) upregulation. Both male and female BPA-exposed mice showed a reduced density of POMC projections into the paraventricular nucleus of the hypothalamus (PVN). BPA- and DES-exposed pups had respectively delayed and blunted postnatal leptin surges, and POMC projections into the PVN were rescued in female BPA-exposed animals given daily injections of supplemental leptin. Our findings suggest that BPA, a putative obesogen, may exert its effects through developmental programming of the hypothalamic melanocortin circuitry, permanently altering the neurobiology of metabolic homeostasis.

Meeting summary: the inaugural meeting of the US DOHaD society.

The US chapter of the International Developmental Origins of Health and Disease (DOHaD) Society recently held its inaugural meeting in Detroit, MI. US-based DOHaD researchers gathered both to create this new society chapter and share their latest research. The US DOHaD Society will provide a much-needed domestic forum for a broad range of DOHaD topics including nutrition, toxicology, stress, epidemiology, epigenetics, and more.

Rats with a truncated ghrelin receptor (GHSR) do not respond to ghrelin, and show reduced intake of palatable, high-calorie food.

Ghrelin, a peptide hormone produced by the stomach, is the endogenous ligand for the Growth Hormone Secretagogue Receptor (GHSR). Ghrelin acts on the GHSR to increase food intake, appetitive behaviors, and adiposity. Recently, a rat model with a null mutation to the GHSR gene (FHH-GHSR(m1/Mcwi)) was generated and used in behavioral studies, but the basic metabolic phenotype of this strain as well as that of the background strain (Fawn Hooded Hypertensive, FHH) has not been characterized in detail. Here we compared male FHH-GHSR(m1/Mcwi) rats with their wild-type littermates (FHH-WT) in a number of metabolic parameters. In the 24h of recovery following an acute overnight fast, FHH-GHSR(m1/Mcwi) rats consumed less food than FHH-WT animals, and relative to their body weights, adult FHH-GHSR(m1/Mcwi) rats consumed fewer calories when placed on a high-fat diet. Despite this, FHH-GHSR(m1/Mcwi) rats did not show a difference in diet-induced obesity or weight gain. Fasted FHH-GHSR(m1/Mcwi) rats exhibited increased Agouti-Related Peptide (AgRP) and Neuropeptide Y (NPY) expression in the Arcuate Nucleus (ARC), indicative of altered central regulation of feeding and energy balance. FHH-GHSR(m1/Mcwi) rats exhibited lower levels of home cage locomotor behavior over the entire light/dark cycle, and reduced levels of food anticipatory activity when placed on a restricted feeding schedule. Finally, FHH-GHSR(m1/Mcwi) rats consumed less of a palatable dessert (cookie dough) given after the completion of the scheduled meal. Altogether, our data show that rats lacking a functional GHSR tend to eat less than their wild-type counterparts in the face of acute fasts, chronic high-fat diet exposure, and exposure to a palatable dessert, despite not showing differences in body weight and glucose homeostasis that are characteristic of GHSR null mice. These data indicate that many, but not all responses to GHSR ablation are conserved between rats and mice. The FHH-GHSR(m1/Mcwi) rat thus represents a novel and useful model for studying GHSR function in rats.

Triticale Bran Alkylresorcinols Enhance Resistance to Oxidative Stress in Mice Fed a High-Fat Diet.

Triticale (× Triticosecale Whitm.) is a cereal grain with high levels of alkyresorcinols (AR) concentrated in the bran. These phenolic lipids have been shown to reduce or inhibit triglyceride accumulation and protect against oxidation; however, their biological effects have yet to be evaluated in vivo. The purpose of this study was to determine the effects of ARs extracted from triticale bran (TB) added to a high-fat diet on the development of obesity and oxidative stress. CF-1 mice were fed a standard low-fat (LF) diet, a 60% high-fat diet (HF) and HF diets containing either 0.5% AR extract (HF-AR), 10% TB (HF-TB), or 0.5% vitamin E (HF-VE). Energy intake, weight gain, glucose tolerance, fasting blood glucose (FBG) levels, and body composition were determined. Oxygen radical absorbance capacity (ORAC), superoxide dismutase (SOD) activity, and glutathione (GSH) assays were performed on mice liver and heart tissues. The findings suggest that ARs may serve as a preventative measure against risks of oxidative damage associated with high-fat diets and obesity through their application as functional foods and neutraceuticals. Future studies aim to identify the in vivo mechanisms of action of ARs and the individual homologs involved in their favorable biological effects.

Novel Regulator of Acylated Ghrelin, CF801, Reduces Weight Gain, Rebound Feeding after a Fast, and Adiposity in Mice.

Ghrelin is a 28 amino acid hormonal peptide that is intimately related to the regulation of food intake and body weight. Once secreted, ghrelin binds to the growth hormone secretagogue receptor-1a, the only known receptor for ghrelin and is capable of activating a number of signaling cascades, ultimately resulting in an increase in food intake and adiposity. Because ghrelin has been linked to overeating and the development of obesity, a number of pharmacological interventions have been generated in order to interfere with either the activation of ghrelin or interrupting ghrelin signaling as a means to reducing appetite and decrease weight gain. Here, we present a novel peptide, CF801, capable of reducing circulating acylated ghrelin levels and subsequent body weight gain and adiposity. To this end, we show that IP administration of CF801 is sufficient to reduce circulating plasma acylated ghrelin levels. Acutely, intraperitoneal injections of CF801 resulted in decreased rebound feeding after an overnight fast. When delivered chronically, they decreased weight gain and adiposity without affecting caloric intake. CF801, however, did cause a change in diet preference, decreasing preference for a high-fat diet and increasing preference for regular chow diet. Given the complexity of ghrelin receptor function, we propose that CF801, along with other compounds that regulate ghrelin secretion, may prove to be a beneficial tool in the study of the ghrelin system, and potential targets for ghrelin-based obesity treatments without altering the function of ghrelin receptors.

Embryonic development of the hypothalamic feeding circuitry: Transcriptional, nutritional, and hormonal influences.

BACKGROUND: Embryonic neurogenesis and differentiation in the hypothalamic feeding circuitry is under the control of a variety of diffused morphogens and intrinsic transcription factors, leading to the unique structural and functional characteristics of each nucleus. SCOPE OF REVIEW: The transcriptional regulation of the development of feeding neuroendocrine systems during the period of embryonic neurogenesis and differentiation will be reviewed here, with a special emphasis on genetic and environmental manipulations that yield an adverse metabolic phenotype. MAJOR CONCLUSIONS: Emerging data suggest that developmental mechanisms can be perturbed not only by genetic manipulation, but also by manipulations to maternal nutrition during the gestational period, leading to long-lasting behavioral, neurobiological, and metabolic consequences. Leptin is neurotrophic in the embryonic brain, and given that it varies in proportion to maternal energy balance, may mediate these effects through an interaction with the mechanisms of hypothalamic development.

Rapid Teaching of Arbitrary Matching in Individuals with Intellectual Disabilities.

This research extended to arbitrary matching-to-sample procedures a method that was successful in rapidly establishing identity matching in children with and without intellectual disabilities (Mackay et al., 2002). The method involves increasing the number of identical comparison stimuli in a choice array in order to create a homogenous background that makes the target more salient, thus likely to prompt selection. The number of comparison stimuli then is faded systematically contingent on accurate responding. This method unites cognitive research on visual search and behavior analytic research on conditional stimulus control. Two experiments examined use of the method to teach arbitrary relations between visual stimuli (numerals and colors and their printed names) and between visual and auditory stimuli (e.g., numerals and colors and their dictated names). Results demonstrated the generality of the method to symbolic matching. This finding is important for conceptual reasons and for its relevance to special education.