Location: Children's Nutrition Research Center
2020 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 DNA) 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 the risk of disease throughout life (an area called 'developmental programming'). Research under Objective 1 focuses on understanding the developmental programming of obesity through evaluating a part of the brain called the hypothalamus using mouse models of epigenetic development. We are ahead of schedule, having completed Sub-objective 1A. To explore the effects of cell-type specific DNA methylation changes on risk of obesity, we generated mice lacking DNA methyltransferase 3a specifically in one neuronal cell type (Agrp neurons) in the hypothalamus. As reported last year, the most prominent effect we identified in the knockout mice is that they have reduced levels of voluntary exercise; given free access to a running wheel in their cage, adult knockout males run only half as much as wild types. We isolated the two major cell types from one region of the hypothalamus (the arcuate nucleus of the hypothalamus, or ARH) and performed whole-genome analysis of DNA methylation and gene expression. We developed a new approach for analyzing the whole-genome DNA methylation data, allowing us to identify DNA methylation changes occurring specifically in Agrp neurons. Research under Objective 2 focuses on identifying human metastable epialleles and assessing their associations with obesity. Metastable epialleles are regions of the genome that show epigenetic variation among individuals but not between different tissues of the same individual. We also made excellent progress by completing Sub-objective 2A. We performed target capture bisulfite sequencing for ~4,000 candidate metastable epialleles, in 811 samples representing multiple tissues from 188 GTEx donors. The target capture worked very effectively, delivering us excellent sequencing depth across ~80% of the targets. Also, our quality control analysis shows clear clustering of the data by donor. Research under Objective 3 focuses on developing a mouse model by which to understand the role of p16 hypermethylation (epimutation) on intestinal carcinogenesis. For Sub-objective 3A, our overall goal is to determine the functional impact of dietary folate supplementation in a novel epigenetically engineered mouse model of p16 epimutation. To achieve this goal, we established a large cohort of mice (n=29) that replicates two common events observed in human colon cancer; Apc mutation and p16 epimutation. We found that the mice had a significantly shortened overall survival as compared to control Apc mutation alone mice (the average age of survival was 18 weeks (wk) vs. 25 wk respectively). We also found that a methyl-donor diet can increase colon cancer risk. Briefly, we used the amino acid-defined NIH-31 diet supplemented with extra methyl-donor nutrients including choline, betaine, folic acid, and vitamin B12. We observed that the dietary supplementation promotes intestinal tumorigenesis based on our analyses of overall survival, tumor numbers, and tumor sizes. We have collected the samples for detailed histological characterization, but unfortunately our experiments have been on hold due to the COVID-19 pandemic and all core facilities including the histology core labs are closed as a result. In parallel, we established an ex vivo organoid culture system to directly study the nutritional epigenetic effects on intestinal epithelial cells in which cancer cells arise. Our preliminary data suggest that the methyl-donor diet increases p16 promoter methylation in the intestinal organoids. However, in the beginning of March, we had to freeze down all the organoids due to the COVID-19. As soon as the lab re-opens, we will thaw our stocks of frozen organoids, perform quality controls and validations, and reestablish our systems for follow-up studies. For SubObjective 3B, the overall goal is to determine the underlying mechanisms by which p16 epimutation promotes intestinal tumorigenesis. This year we focused on a novel sequence-specific transcription factor CCAAT/enhancer binding protein (CEBPZ) that binds to methylated p16 promoter. Using chromatin immunoprecipitation and real-time Polymerase Chain Reaction (PCR) (ChIP-qPCR), we confirmed that both the endogenous CEBPZ and the FLAG-tagged CEBPZ bind specific to the methylated p16 promoter in both human cancer cell lines and spontaneously immortalized p16 methylated mouse embryonic fibroblasts. Together, our data strongly suggest that CEBPZ acts as a methyl-cytosine reader and is involved in cancer-related epigenetic silencing.
Accomplishments
