2007 Annual Report 1a.Objectives (from AD-416) Characterize the components and dynamics of the GATA transcription factor complexes which suppress adipogenesis. Characterize the adipocyte role of PPARs in mediation of omega-3 fatty acid-regulation and conjugated linoleic acid regulation of decreased fat deposition. Prove that maternal dietary methyl donor supplementation before conception and during pregnancy alters DNA methylation of specific gene regions in the early embryo, and identify which genes have altered methylation as a result of these prenatal diet changes. Optimize experimental dietary protocols and screen offspring of females treated with high and low methyl donor diets for metabolic and pathological effects. Elucidate mechanisms regulating normal embryonic development and diseases of cholesterol homeostasis. Determine the role of dietary elements such as cholesterol and retinoic acid on regulation of early embryonic patterning via the Hedgehog developmental pathway. Determine if nutrition during prenatal and early postnatal development has permanent effects on epigenetic gene regulation in humans. 1b.Approach (from AD-416) Establish protein expression and subcellular distribution pattern of GATA-2, GATA-3 and the other known GATA interacting proteins during the course of fat cell formation; purify GATA protein complexes from various stages of adipocyte differentiation & identify their components; characterize the roles that GATA protein complexes play in adipose tissue development & obesity. Characterize altered beta-oxidation & lipogenesis following treatment of cultured human adipocytes with DHA, CLA-10,12, specific PPARs, PPARa, or PPARy agonists, subtype-specific pharmacological antagonists, a combination of these various treatments, & knockdown studies of specific PPAR subtype mRNAs by the use of RNA interference; determine the role of PPARs & potential differential effects on lipogenesis & beta oxidation in several adipose tissue depots induced by fish oil and CLA-10,12 using in vivo rat diet studies; characterize the role of PPARs, independent of PPARa, in mediating altered beta-oxidative and lipogenic gene expression in mouse gonadal white adipose tissue induced by fish oil and CLA-10,12 through use of in vivo chronic diet studies & in vivo intraperitoneal injection acute studies using a PPARa-null mouse model. Standardize the experimental diets containing variable levels of methyl donor agents given to female mice before and during pregnancy; levels of metabolites predicted to be altered by these diets, & coat color changes in Avy/a mice will be used to select two diets with significant opposite influence on DNA methylation; perform metabolic assays & phenotypic evaluation of 129/SvEv wild-type and Mecp2R308/Y mutant mice on the most effective experimental high/low methyl donor diet; identify other transposon insertions which, like the Avy transposon, cause the epigenetic state of neighboring genomic regions to be labile to early nutritional influences; determine if maternal dietary methyl donor supplementation before and during pregnancy alters allelic expression of genomically imprinted genes in offspring; use Avy animals to evaluate the effectiveness of various 'pro-methylation' diets; and search for the specific target CpG sites with changed methylation using a method, called methylation-sensitive restriction landmark genome scanning (RLGS), that can evaluate 2000-3000 methylatable CpG's in the genome at once; determine the developmental stage at which individual levels of CpG methylation become established at the Avy locus. Characterize both the human and mouse promoter regions of the Hedgehog receptor Patched and identify transcriptional regulatory elements which modulate expression of the Patched gene especially with regard to Gli, SP-1, c/EBP and sterol and retinoid response elements; characterize Patched alternate splice form expression in various tissues during different stages of development; determine the effect of plasma membrane cholesterol content on Hedgehog receptor localization, ligand binding, and function. Through studying mouse models, two gene classes will determine if maternal dietary methyl donor supplementation before conception & during pregnancy alters DNA methylation at specific genomic regions in the early embryo. 4.Accomplishments Development of a Genome-wide Methylation Microarray: Researchers at the Children's Nutrition Research Center, in collaboration with scientists at the M.D. Anderson Cancer Center, developed an approach to assess site-specific DNA methylation on a genome-wide scale. Our originally proposed research objectives focused on candidate genes; however, rapid developments in technology in the past two years now make it possible to survey the entire genome to identify regions at which DNA methylation (chemical modification of DNA) varies among individuals and is influenced by diet during development. The custom array we have designed enables researchers to screen for methylation differences at 31,000 genomic loci in one hybridization experiment. This is important since this tool will allow researchers to make rapid progress in identifying genomic regions that are epigenetically likely to be affected by nutrition during development. [NP107, Component 6 Prevention of Obesity and Diesease: Relationship between Diet, Genetics, and Lifestyle] (CNRC Project 4) Diet-induced Hypermethylation is not Inherited Transgenerationally: Recently published findings indicate that, contrary to expectations, DNA methylation (the chemical modification of DNA), is not the transgenerationally inherited mark at agouti viable yellow (Avy), a mutant gene locus in the mouse model. Children's Nutrition Research Center scientists conducted a three-generation study in mice to determine if diet-induced hypermethylation at Avy is inherited across multiple generations. Our findings show that diet effects on Avy epigenotype are not inherited. Thus our data indicate that epigenetic modifications other than DNA methylation mediate transgenerational epigenetic inheritance at Avy. [NP107, Component 6 Prevention of Obesity and Diesease: Relationship between Diet, Genetics, and Lifestyle] (CNRC Project 4) Folic-Acid Supplementation Alone Does Not Affect Coat Color: Previous studies at the Children's Nutrition Research Center have shown that a methyl donor supplement containing folic acid, vitamin B12, betaine, and choline can induce epigenetic changes in mice offspring (changes that affect a cell, organ, or individual without directly affecting the DNA). These results raised our concern that too much folic acid in the diet of pregnant women might induce permanent epigenetic changes in her offspring. CNRC researchers have recently completed studies that show that maternal folic acid supplementation alone does not affect the coat color distribution of offspring. Likewise, our measurements of metabolic biomarkers show that supplementation with folic acid alone induces a completely different "metabolic signature" than the multicomponent (folic acid, vitamin B12, betaine, and choline) supplement. These results suggest that, at least in terms of epigenetic effects during development, fortification of the food supply with folic acid is safe. [NP107, Component 5 Health Promoting Properties of Plant and Animal Foods] (CNRC Project 4) Gene Expression of Fat Cells: Researchers at the Children's Nutrition Research Center have purified a multi-protein aggregate containing the GATA2 protein from fat cells. Individual components of the purified protein complex were separated by electrophoresis and visualized with staining. Our lab has confirmed previous reports that one of GATA-2 interacting protein is C/EBPBeta. By use of mass spectrometry we have identified one unknown GATA-2 complex protein as phosphofructo kinase-1 and the other as a porin homolog protein. These findings may shed light on the regulation of GATA2 gene expression regulator function in fat cells. For example, since phosphofructo kinase-1 is a key enzyme controlling glucose metabolism, our finding may provide a novel mechanistic link between metabolism (phosphofructo kinase-1) and gene expression regulation (GATA2). [NP107, Component 6 Prevention of Obesity and Diesease: Relationship between Diet, Genetics, and Lifestyle] (CNRC Project 2) Nkx Proteins Regulate the Expression of Patched Genes During Embryonic Development: Children's Nutrition Research Center researchers have shown that two proteins known to be important in embryonic development, called Nkx factors 3.2 and 2.5, bind to specific areas of the Patched gene which control when the Patched gene is turned on and off. The Patched gene is responsible for helping form nearly every organ in the developing embryo. Binding of these Nkx factors to the control areas causes the Patched gene to be turned on in specific tissues, at specific times in the developing embryo, leading to proper organ formation. Without tight regulation of these developmental genes, severe fetal malformations are known to occur, and these studies help explain how these important genes overlap and work together. Our results are among the first to address the complex regulation of this critical embryonic developmental pathway. This data will help shed light on the crucial relationships between nutrition, gene expression, and embryonic malformations, and help us begin to understand how regulation of these genes occurs and how disregulation causes human malformation and disease. [NP107, Component 6 Prevention of Obesity and Diesease: Relationship between Diet, Genetics, and Lifestyle] (CNRC Project 1) 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 4 Review Publications Waterland, R.A., Michels, K.B. 2007. Epigenetic epidemiology of the developmental origins hypothesis. Annual Review of Nutrition. 27:363-388. Waterland, R.A. 2006. Epigenetic mechanisms and gastrointestinal development. Journal of Pediatrics. 149(5):S137-S142. Tong, Q., Tsai, J., Tan, G., Dalgin, G., Hotamisligil, G.S. 2005. Interaction between GATA and the C/EBP family of transcription factors is critical in GATA-mediated supression of adipocyte differentiation. Molecular and Cellular Biology. 25(2):706-715. Motil, K.J. 2006. Cholecystitis. In: McMillian, J.A., Feigin, R.D., DeAngelis, C.D., Jones, M.D., editors. Oski's Pediatrics: Principles and Practice of Pediatrics. 4th edition. Philadelphia, PA: Lippincott Williams & Wilkins. p. 2042-2045. Waterland, R.A., Rached, M. 2006. Developmental establishment of epigenotype: a role for dietary fatty acids? Scandinavian Journal of Food & Nutrition. 50(S2):21-26. Waterland, R.A., Dolinoy, D.C., Lin, J.R., Smith, C.A., Shi, X., Tahiliani, K.G. 2006. Maternal methyl supplements increase offspring DNA methylation at axin fused. Genesis. 44(9):401-406. Marchard, V., Motil, K.J. 2006. Nutrition support for neurologically impared children: A clinical report of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition. Journal of Pediatric Gastroenterology and Nutrition. 43(1):123-135. Motil, K.J. 2005. Personal leadership protects research subjects. Society of Clinical Research Associates (SOCRA). 5:15-19. Tremain, W.J., Carlson, M.R., Isaaks, K.L., Motil, K.J., Robuck, P.R., Wurzelman, J.I. 2005. Ethical issues, safety, and data integrity in clinical trials. Inflammatory Bowel Diseases. 11(Supplement1):S17-S21. Dolinoy, D.C., Weidman, J.R., Waterland, R.A., Jirtle, R.L. 2006. Maternal genistein alters coat color and protects Avy mouse offspring from obesity by modifying the fetal epigenome. Environmental Health Perspectives. 114(4):567-572.