Location: Children's Nutrition Research Center
2019 Annual Report
Objectives
Objective 1: Use transgenic mouse models, microdissection, nuclear sorting, next-generation sequencing and innovative computational approaches to alter DNA methylation in specific subpopulations of hypothalamic neurons and evaluate lifelong effects on energy metabolism, food intake, and physical activity; isolate specific neuronal (and potentially non-neuronal) hypothalamic cell types to evaluate cell type-specific alterations in DNA methylation in established models of nutritional programming.
Subobjective 1A: In a mouse model with DNA methyltransferase 3a (Dnmt3a) knocked out specifically in Agrp neurons in the arcuate nucleus of the hypothalamus, characterize the effects of this cell type-specific epigenetic perturbation on energy balance and metabolism.
Subobjective 1B: Use a mouse model with Agrp/Npy neurons genetically tagged with green fluorescence protein (GFP) to assess effects of early postnatal overnutrition on DNA methylation and gene expression specifically in Agrp/Npy neurons.
Objective 2: Advance understanding of the causes of interindividual epigenetic variation and consequences for human energy balance by conducting target-capture bisulfite sequencing in multiple tissues from an existing cohort of molecularly-phenotyped individuals to determine associations between genetic variation, epigenetic variation, and gene expression at human metastable epialleles; identify human metastable epialleles that predict risk of obesity by exploiting existing longitudinal cohorts of metabolically-phenotyped individuals; assess how DNA methylation at obesity-associated metastable epialleles is affected by maternal periconceptional nutrition.
Subobjective 2A: In multiple tissues representing hundreds of donors in the NIH Gene-Tissue Expression (GTEx) program, use target-capture bisulfite sequencing to assess DNA methylation at candidate metastable epiallele regions.
Subobjective 2B: Integrate these DNA methylation data with existing GTEx genotyping and RNA-seq data on these individuals to assess 1) genetic influences on individual variation on DNA methylation and 2) correlations between tissue-specific DNA methylation and expression.
Subobjective 2C: Exploit an existing cohort with longitudinal data on metabolically phenotyped adults to determine whether individual variation in DNA methylation at metastable epialleles predicts risk of adult weight gain.
Objective 3: Determine the functional impact of folic acid supplementation and establish the functional role of age-related p16 epimutation in genetically and epigenetically engineered mouse models of colon cancer and in intestinal carcinogenesis.
Subobjective 3A: Determine the functional impact of dietary folate supplementation in an epigenetically engineered mouse model of p16 epimutation.
Subobjective 3B: Determine the underlying mechanisms by which p16 epimutation promotes intestinal tumorigenesis.
Approach
Developmental programming occurs when nutrition and other environmental exposures affect prenatal or early postnatal development, causing structural or functional changes that persist to influence health throughout life. Researchers are working to understand epigenetic mechanisms of developmental programming. Epigenetic mechanisms regulate cell-type specific gene expression, are established during development, and persist for life. Importantly, nutrition during prenatal and early postnatal development can induce epigenetic changes that persist to adulthood. We focus on DNA methylation because this is the most stable epigenetic mechanism. The inherent cell-type specificity of epigenetic regulation motivates development of techniques to isolate and study specific cell types of relevance to obesity and digestive diseases. These projects integrate both detailed studies of animal models and characterization of epigenetic mechanisms in humans. We will use mouse models of developmental epigenetics in the hypothalamus to understand cell type-specific epigenetic mechanisms mediating developmental programming of body weight regulation. Mouse models will also be used to investigate how folic acid intake affects epigenetic mechanisms regulating intestinal epithelial stem cell (IESC) development and characterize the involvement of these mechanisms in metabolic programming related to obesity, inflammation, and gastrointestinal cancer. In human studies, we will identify human genomic loci at which interindividual variation in DNA methylation is both sensitive to maternal nutrition in early pregnancy and associated with risk of later weight gain. An improved understanding of how nutrition affects developmental epigenetics should eventually lead to the creation of early-life nutritional interventions to improve human health.
Progress Report
Our research project is related to understanding epigenetic mechanisms, which are the fundamental molecular mechanisms that enable our different cell types (each of which contains the same genome) to develop and stably maintain very different structures and functions. In particular, this project focuses on DNA methylation, which is the most stable epigenetic mark and therefore likely relevant to our over-arching goal of understanding how nutrition before conception and during embryonic, fetal, and early postnatal development has persistent influences on risk of disease throughout life (an area called 'developmental programming').
Research under Objective 1 focuses on mouse models of epigenetic development in the hypothalamus, to understand developmental programming of energy balance and obesity. To explore effects of cell-type specific DNA methylation changes on energy balance, we generated mice lacking the DNA methyltransferase 3a specifically in one neuronal cell type in the hypothalamus: Agrp neurons. We found that, as adults, the Agrp-specific Dnmt3a-/- knockout (KO) mice become slightly fatter than control mice. Surprisingly, although Agrp neurons are famous for their role in food intake regulation, extensive metabolic cage studies showed no change in food intake in KO mice. Rather, their most obvious phenotype is reduced physical activity, in particular a 50% reduction in voluntary exercise (wheel running). Hence, our metabolic phenotyping studies on these mice have identified an extremely interesting and robust phenotype, laying the groundwork for our current focus: detailed DNA methylation and gene expression analysis in hypothalamic neurons of these mice. Objective 2 focuses on identification and characterization of human metastable epialleles and their association with obesity. We have completed the design of an Agilent SureSelect capture reagent to target our human candidate metastable epiallele regions, and have begun to generate data on target capture bisulfite sequencing on the 811 genomic DNA samples from multiple tissues from genotype-tissue expression donors.
Objective 3 focuses on effects of folic acid supplementation on age-related p16 epimutation in genetically and epigenetically engineered mouse models of colon cancer and in intestinal carcinogenesis. We have expanded our mouse cohorts for the proposed studies of folic acid supplementation and colon cancer risk. We are also collecting and sending the samples for methylation-dependent proteomic analysis.
Accomplishments
