We observed that variations in exposure to choline and folic acid during fetal brain development were associated with profound differences in proliferation and apoptosis of neural progenitor cells in hippocampus and septum. In this proposal we seek to identify a mechanistic link between diet and these effects. Since choline and folate have similar effects we are focusing on the metabolic pathway that they share - methylation - and hypothesize that gene expression is modified by methylation in response to dietary variation of these two nutrients. In cultured progenitor cells from fetal mouse hippocampus (in vitro) we will identify the effects of exposure to choline on cell proliferation, apoptosis and differentiation and on likely candidate genes that regulate these processes during brain development. We will determine whether specific gene promoter regions are methylated or hypomethylated after varying choline. We will determine whether pharmacologic inhibition of methyltransferases or absence of methyltransferase prevents choline responsiveness, and determine if methylation status of promoter regions alter binding of MeCP or known transcription factors for the gene. We will determine if siRNA inhibition of selected target genes prevents changes in cell proliferation or apoptosis. Using fetal mouse hippocampus after in vivo exposure, we will determine whether the choline effects identified in cells can be replicated in whole brain regions or in cells from the neuroepithelial layer of hippocampus. We will determine whether histone methylation and acetylation in regions of specific genes mediates the observed choline-mediated changes using cell culture and using fetal mouse hippocampus. In similar experiments, we will determine whether the choline effects can be replicated by varying folate content of diet rather than choline. Finally, we will determine whether the choline and folate effects can be replicated by using knockout mice with defects in choline or folate metabolism.
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