Progress 10/01/05 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) The objective of this cooperative research is to characterize the components and dynamics of the GATA transcription factor complexes; characterize the adipocyte role of PPARs in mediation of omega-3 fatty acid-regulation and conjugated linoleic acid regulation of decreased fat deposition; investigate maternal dietary methyl donor supplementation; 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; and determine the role of dietary elements such as cholesterol and retinoic acid on regulation of early embryonic patterning via the Hedgehog developmental pathway. 1) The Role of Cholesterol in Regulation of the Hedgehog Developmental Pathway - Aims to provide a greater understanding of the role that cholesterol plays in regulating the activity of the Hedgehog (Hh) receptor, which is critical for early embryonic patterning of nearly every organ system of the developing fetus, and this will shed light on the mechanisms regulating other developmentally important signaling molecules. 2) Role of GATA Protein Complexes in Adipocyte Differentiation - Purify and characterize the components of the GATA protein complexes in adipocyte differentiation and to determine the dynamics and function of the GATA protein complexes in the adipogenesis process. 3) Nutritional Influences on Epigenetic Gene Regulation During Development - Determine if dietary levels of methyl donors during pregnancy, such as folic acid, affect DNA methylation in the offspring resulting in lifelong changes in gene expression. 4) Nutritional Influences on Mammalian Developmental Epigenetics - Determine if nutrition during prenatal and early postnatal development has permanent effects on epigenetic gene regulation in humans. Approach (from AD-416) 1) The Role of Cholesterol in Regulation of the Hedgehog Developmental Pathway - Investigate the effects of cholesterol inhibitors and caveolin- 1 mutants by utilizing transient transfection of Hedgehog pathway components, confocal imaging, Western blot and membrane fractionation. Transcriptional regulatory elements and tissue-specific RNA splicing mechanisms will be identified by electric mobility shift assays (gel shifts and supershifts), luciferase reporter assays, in situ hybridization and quantitative RT-PCR. 2) Role of GATA Protein Complexes in Adipocyte Differentiation - Purify the GATA protein complexes and identify their constituents. 3) Nutritional Influences on Epigenetic Gene Regulation During Development - Study in animal models how nutritional supply of methyl donors during development can alter the epigenetic state of the genome and to discover the altered genes. 4) Nutritional Influences on Mammalian Developmental Epigenetics - studying mouse models, two gene classes: genes adjacent to transposon elements, and genomically imprinted genes, to determine if maternal dietary methyl donor supplementation before conception and during pregnancy alters DNA methylation at specific genomic regions in the early embryo. Significant Activities that Support Special Target Populations We have generated adipocytes with PU.1 RNAi knockdown to suppress PU.1 expression. These cells, together with adipocytes with constitutive PU.1 expression generated before, were used for the study of the effects of PU. 1 on adipocyte differentiation, glucose uptake, lipolysis and insulin signaling. (Project 2) We previously reported a slight modification from the original aims and extension of our analysis to muscle and other tissues of mice exposed to maternal high- and low-protein diets in utero and neonatally. We have nearly finished the glucose, tissue, and body weight analysis on these mice and have performed Affymetrix mouse Gene ST expression arrays for gene expression profiling on Soleus muscle and liver at 1 year of age. Data from 3 arrays in each group (total N = 12) are being analyzed and qPCR validation is in progress. We previously reported added studies on placental imprinting, which is directly relevant to the role of placenta in nutrition. We continued investigation of the role of the NLRP7 gene in the establishment or maintenance of imprinting in the placenta. Mutations in this gene cause hydatidiform moles: abnormal pregnancies with imprinting abnormalities in the placenta. We further analyzed potential interaction with CTCF and YY1 and performed a yeast two-hybrid screen to find other NLRP7 binding partners. Results of these are being verified, but support a role for NLRP7 in control of imprinting. We have now also performed methylation profiling with the Illumina Humanmethylation27 BeadChip on tissues from 6 androgenetic hydatidiform moles to find new placental imprinted genes; data analysis and verification are in progress. (Project 3) We performed supplementation studies with several potential pro-methylation diets and, importantly, confirmed that the original 'methyl supplemented' diet still has the same effect on coat color in our current Avy mouse population. Additionally, our first objective focused on using a bioinformatics approach to identify additional mouse metastable epialleles. We have instead used our methylation-specific amplification microarray (MSAM) approach to screen directly for murine metastable epialleles. The microarray studies are completed, and we are currently validating some of the microarray hits by bisulfite pyrosequencing. (Project 4)
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) The objective of this cooperative research is to characterize the components and dynamics of the GATA transcription factor complexes; characterize the adipocyte role of PPARs in mediation of omega-3 fatty acid-regulation and conjugated linoleic acid regulation of decreased fat deposition; investigate maternal dietary methyl donor supplementation; 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; and determine the role of dietary elements such as cholesterol and retinoic acid on regulation of early embryonic patterning via the Hedgehog developmental pathway. 1) The Role of Cholesterol in Regulation of the Hedgehog Developmental Pathway - Aims to provide a greater understanding of the role that cholesterol plays in regulating the activity of the Hedgehog (Hh) receptor, which is critical for early embryonic patterning of nearly every organ system of the developing fetus, and this will shed light on the mechanisms regulating other developmentally important signaling molecules. 2) Role of GATA Protein Complexes in Adipocyte Differentiation - Purify and characterize the components of the GATA protein complexes in adipocyte differentiation and to determine the dynamics and function of the GATA protein complexes in the adipogenesis process. 3) Nutritional Influences on Epigenetic Gene Regulation During Development - Determine if dietary levels of methyl donors during pregnancy, such as folic acid, affect DNA methylation in the offspring resulting in lifelong changes in gene expression. 4) Nutritional Influences on Mammalian Developmental Epigenetics - Determine if nutrition during prenatal and early postnatal development has permanent effects on epigenetic gene regulation in humans. Approach (from AD-416) 1) The Role of Cholesterol in Regulation of the Hedgehog Developmental Pathway - Investigate the effects of cholesterol inhibitors and caveolin- 1 mutants by utilizing transient transfection of Hedgehog pathway components, confocal imaging, Western blot and membrane fractionation. Transcriptional regulatory elements and tissue-specific RNA splicing mechanisms will be identified by electric mobility shift assays (gel shifts and supershifts), luciferase reporter assays, in situ hybridization and quantitative RT-PCR. 2) Role of GATA Protein Complexes in Adipocyte Differentiation - Purify the GATA protein complexes and identify their constituents. 3) Nutritional Influences on Epigenetic Gene Regulation During Development - Study in animal models how nutritional supply of methyl donors during development can alter the epigenetic state of the genome and to discover the altered genes. 4) Nutritional Influences on Mammalian Developmental Epigenetics - studying mouse models, two gene classes: genes adjacent to transposon elements, and genomically imprinted genes, to determine if maternal dietary methyl donor supplementation before conception and during pregnancy alters DNA methylation at specific genomic regions in the early embryo. Significant Activities that Support Special Target Populations The ADODR monitors activities for the project through communication with the Center Director, consulation with the On-Site Representative, review of cooperator reports, conference calls, and site visits. 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)
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