Identification of the Establishment of Differential Methylation at the Mouse Gene gtl2

Posted May 26th, 2010 at 2:34 pm.

Elizabeth Powell and Geneva Stein
Mentor: Dr. Tamara Davis

Our work focuses on genomic imprinting, which only occurs at some mammalian genes. Genomic imprinting is relatively rare, occurring in about 70 genes. While most genes are biallelically expressed, imprinting results in the preferential transcription of one parental allele over the other. Most imprinting is believed to be controlled by the epigenetic mechanism of differential methylation of cytosine residues in CpG pairs. In some cases, methylation may serve to silence a particular parental allele. In these instances, the methylation may prevent the transcription and subsequent expression of the gene.

Since expression of imprinted genes is parent-specific, the imprint must be passed from parent to offspring, and the methylation must also be changed in the germline in order to reflect the sex of the offspring. The offspring can then pass their sex-specific imprint on to the next generation. Germline methylation is erased at a particular stage of embryonic development, around 11.5 days post coitum (d.p.c.) and replaced at a later stage. Our particular project focuses on the establishment of methylation in the male mouse germline of the imprinted gene gtl2 .

We are interested in exploring the extent of methylation on the gtl2 mouse gene as well as the timeline during which methylation occurs. Gtl2, located on mouse chromosome 14, is maternally expressed and paternally silent. Our particular regions of interest include four CpG islands in and upstream of the gtl2 promoter and the Dlk1-gtl2 reciprocally expressed region. The region in and around the gtl2 promoter will allow us to study a genomic imprinting mark while the Dlk1-gtl2 domain will allow us to study the acquisition of methylation at a region that may play a role in expression control.

We will study the acquisition of methylation in the gtl2 promoter region and the Dlk1-gtl2 domain in germ cells and during embryonic development. In order to study the methylation of both parental alleles, we will study Mus musculus domesticus (BL/6) X Mus musculus castaneus (CAST) offspring. We must first identify polymorphisms in order to determine the parental origin of each allele in these F1 hybrid animals. After polymorphisms have been identified, we will purify germ cells and early embryos at particular stages of embryonic development, mutagenize the DNA, amplify the DNA, and sequence the nucleotides in order to determine the time and extent of methylation. Bisulfite mutagenesis will allow us to distinguish between methylated and unmethylated cytosine residues. After the analysis of each parental allele, we should be able to determine when and to what extent each allele is methylated in the male germline.

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