Project Number: 6250-51000-049-02
Project Type: Specific Cooperative Agreement

Start Date: Oct 01, 2005
End Date: Sep 30, 2010

Objective:
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:
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.