Project Number: 6250-51000-049-00
Project Type: Appropriated

Start Date: May 21, 2004
End Date: Apr 30, 2009

Objective:
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.

Approach:
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.