Location: Children's Nutrition Research Center
2021 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 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 mouse models of epigenetic development in a part of the brain called the hypothalamus, to understand developmental programming of obesity. We are using a classic model of developmental programming of energy balance (the postnatal small litter model), but are employing an innovative approach to test the hypothesis that early postnatal overnutrition induces epigenetic changes within specific subclasses of neurons in the hypothalamus. To do this, we are conducting small litter experiments in transgenic mice in which one type of hypothalamic neuron, called Agrp neurons, are fluorescently tagged (NPY-GFP mice). This allows us to isolate specifically Agrp neurons from mice who were over nourished postnatally and compare them with mice that were fed normally. We established a colony of NPY-GFP mice, conducted the cross-fostering (small litter) studies, collected hypothalamus from control and small litter offspring at postnatal age of 21 days, and performed nuclear sorting to isolate Agrp neurons from these animals. Scientists also performed the tissue collections at the postnatal age of 180 days. 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). This project is ahead of schedule, but our conceptual framework has evolved. Rather than focus on attempting to identify canonical metastable epialleles (at which individual variation in DNA methylation is largely independent of genetic variation), our studies have shown that systemic interindividual epigenetic variants in humans can have a stochastic (probabilistic) component, a genetic component, and be influenced by periconceptional nutrition. Hence, rather than limiting our focus to metastable epialleles, we introduced in 2019 a new term: Correlated Regions of Systemic Interindividual Variation in DNA methylation (CoRSIVs). In Fiscal Year (FY) 2020, we generated DNA methylation data on 4,086 CoRSIVs, in multiple tissues from each of 187 donors from the National Institutes of Health Gene-Tissue Expression (GTEx) program. In FY2021, we completed the bioinformatics analysis of these data (Sub-objective 2B), assessing both the influence of genetic variation at CoRSIVs, and correlations between CoRSIV DNA methylation and expression of associated genes. In addition to corroborating the major claims we made about CoRSIVs in 2019, this new data set allowed us to make a major discovery about genetic influences on DNA methylation in humans (which are called methylation quantitative trait loci, or mQTL). Since the first use of mQTL in 2010, more than 130 papers have been published on mQTL in humans. Nearly all of these, however, have employed standard commercial DNA methylation arrays. Unfortunately, the arrays are not appropriate for analysis of mQTL; in particular, most of the genomic regions included on the arrays do not show appreciable interindividual variation in DNA methylation. Accordingly, our approach, which focused on individual epigenetic variation from the outset, has mapped out much more mQTL than has ever been detected in humans. Specifically, we identified 70-fold more mQTL than detected in the most powerful previous study.
Regarding Objective 3, a major challenge in the field of nutritional epigenetics is to elucidate the causal pathways by which diet induces epigenetic changes, which then lead to phenotypic outcomes. The overall goal of Objective 3 is to determine the functional impact of dietary folate supplementation in a novel epigenetically engineered mouse model of epimutation at the p16 gene, an important tumor-suppressor gene (analagous to genetic mutation, an epimutation is a stable change in epigenetic regulation, that occurs in a subset of individuals or in a subset of cells). In prior work, we found that p16 epimutation, caused by promoter DNA methylation, predisposes mice to spontaneous tumor development. We also showed that p16 epimutation cooperates with mutations in the Apc gene to accelerate intestinal tumorigenesis. In FY2021, we continued the dietary methyl-donor supplementation studies in ApcMin/+; p16cis/cis mice carrying combined p16 epimutation and Apc mutation. We collected colon tumors and adjacent intestinal epithelial cells from mice under either control or supplemented diet for histology, DNA methylation, transcriptomic, and metabolomic analyses. We also re-derived the normal and tumor organoids to recover those that were lost due to work restrictions caused by the COVID-19 pandemic. In addition, we began functional studies of two identified methylation-dependent binding proteins (MDBPs); MBD2 and CEBPZ. Our main findings were: 1) Methyl-donor dietary supplementation exacerbates tumor progression in ApcMin/+; p16cis/cis mice. We found that the methyl-donor supplemented mice had a significantly shortened overall survival as compared to the control diet mice. In both small intestines and colons, we found the supplemented mice had a greater number of tumors and the tumors were significantly larger than those control mice; 2) We confirmed that the methyl-donor diet increases p16 promoter methylation in the intestinal organoids; 3) Our RNA-seq transcriptomic analysis revealed that p16 epimutation modulates tumor immune escape through the upregulation of IFN-y/Programmed Cell Death Ligand 1(PD-L1) pathway. Consistently, through our immune profiling by flow cytometry analysis, we observed substantially higher numbers of myeloid-derived immune-suppressive cells as well as reduced number of CD8+ killer T cells in the colon tumors with methyl-donor supplementation. Our finding may help explain why methyl-donor supplementation promotes p16 epimutant tumor growth and progression; and 4) We found that inhibition of MDBPs (MBD2 and CEBPZ) leads to reactivation of epigenetically silenced p16, without causing significant changes in DNA methylation of p16 promoter or at the global level. This finding suggests potential new and improved strategies for epigenetic prevention and treatment of colon cancer, as targeting MDBPs could avoid the side effects of global hypomethylation and genomic instability.
Accomplishments
