ANALYSIS OF DNA METHYLATION AT THE UDMR LOCUS OF THE IMPRINTED RASGRF1 IN MOUSE EMBRYONIC DEVELOPMENT

 

Rachel Shields and Dr. Tamara Davis

Department of Biology, Bryn Mawr College, 101 N. Merion Ave., Bryn Mawr, PA, 19010

 

Genomic imprinting refers to the differential expression of the parental alleles, which is achieved through epigenetic modifications to the chromatin structure that distinguish between the maternally and paternally derived alleles, allowing expression to be regulated. DNA methylation and histone modification are two mechanisms through which the parent of origin-specific allele can be differentiated. Rasgrf1 is a paternally-methylated and paternally-expressed imprinted gene located on mouse chromosome 9. This imprinted gene’s differentially methylated region (DMR) is believed to be more expansive than originally proposed. The methylation patterns of this extended, paternally-methylated locus upstream of the differentially methylated region (uDMR) of Rasgrf1, which contains five known potential methylation sites, are still being investigated and are not as established as those of the DMR.

As Rasgrf1 is a paternally-methylated imprinted gene, it was established that differential DNA methylation is inherited through the gametes.  Both the maternal and paternal alleles of the Rasgrf1 uDMR in sperm cells are favorably methylated at the five known sites, while oocytes exhibit very low levels of DNA methylation on both parental alleles.  These epigenetic modifications in gametic DNA have been compared to the methylation patterns of the uDMR in 12.5 dpc embryos, which depict different levels of allele-specific DNA methylation.  It has been observed that differential DNA methylation in imprinted genes contracts and re-expands during early embryonic development, however it is not known at what embryonic developmental stage this occurs.

This study will examine how the DNA methylation pattern in the Rasgrf1 uDMR is maintained during mouse embryonic development by utilizing a chemical process, bisulfite mutagenesis, which allows differentiation between methylated and unmethylated cytosines by converting unmethylated cytosines to uracils.  Subsequent gel analysis will be employed to identify the sites of the non-mutagenized, methylated cytosines after sequencing the purified plasmid DNA containing clones of the uDMR.  This analysis will be done utilizing DNA extracted from blastocysts as well as 6.5 dpc, 9.5 dpc, and 12.5 dpc embryonic tissues in order to determine when the DNA methylation contraction and re-expansion occur in the Rasgrf1 uDMR.  It is hypothesized that this rearrangement and reorganization of the DNA methylation occurs at some point during the first 12.5 days of embryonic development.  The aim of this research study is to determine when the DNA methylation pattern observed in neonatal tissue is acquired.