Research Description:

Effects of Methionine and Cysteine Depletion on DNA Methylation at Imprinted Genes in Cultured Mouse Embryonic Fibroblasts

Recently, there have been a number of studies that suggest nutrient deprivation during pregnancy causes detrimental life-long changes to the health of offspring, which may explain chronic problems such as cardiovascular and respiratory diseases.  Because DNA methylation patterns are established early in embryonic development and sustained throughout adulthood, malnutrition may prevent the establishment of correct imprints, leading to chronic health problems.  Most of the studies conducted in this area concentrate on the role of folate in DNA methylation reactions, which has resulted in the addition of folic acid to fortify many common food products.  However, there is little research on methionine deprivation.  Methionine is another substrate involved in DNA methylation reactions, and it is my hypothesis that methionine deprivation causes changes to DNA methylation patterns in differentially methylated regions (DMRs).

This experiment will look at the effect of methionine deprivation on DNA methylation of DMRs at imprinted genes, where one allele is hypermethylated and the other is hypomethylated, in mouse embryonic fibroblasts.  In order to observe any changes in an allele-specific manner, female C57BL/6 (Mus musculus musculus) mice will be mated with congenic male B6.CastC7 mice.  This congenic strain contains Mus musculus castaneus sequences within the 7C region, including the imprinted genes Mkrn3 and Snrpn, on a Mus musculus musculus genomic background.  At day 13.5 of pregnancy, the embryonic fibroblast cells are harvested and cultured in medium containing 100%, 50%, 35%, 25%, and 20% normal methionine/cytosine level.  After the fibroblasts are cultured for 96 hours, the genomic DNA is extracted and processed using sodium bisulphite conversion.  The resulting DNA will be amplified by polymerase chain reaction (PCR) and sequenced as specific differentially methylated regions.  Single nucleotide polymorphisms (SNPs) in the Snrpn and Mkrn3 genes of the 7C locus will be used to determine the parent of origin of the specific alleles and comparison of DMRs between the five cell lines will demonstrate the effect of methionine deprivation on imprinted genes.