Mentor: Dr. Tamara Davis
Genomic imprinting is an unusual form of gene regulation. While most genes are expressed from both inherited parental copies, imprinting results in the preferential transcription of one parental allele over the other. Most imprinting is believed to be controlled by DNA methylation. Methylation occurs when a CH3 group is bonded to the cytosine residue of a CpG dinucleotide in the DNA. Only one parental allele of a specific imprinted gene is methylated allowing alleles to be distinguished. Differential methylation at some imprinted genes is inherited directly from the sperm or egg at fertilization. In contrast, other imprinted genes acquire differential methylation on a particular parental allele after fertilization. While we may know if a specific allele acquires methylation before or after fertilization, we do not yet know how the acquisition of methylation occurs on a specific parental allele in an embryo, or, for most genes, at what time the differential methylation is acquired. It is these questions that our research will attempt to address.
Our research focuses on understanding the acquisition of methylation on the paternal allele of the mouse gene Gtl2. Gtl2 is a maternally expressed, paternally methylated gene that codes for an untranslated mRNA. We are looking specifically at a CpG-dense region around the Gtl2 promoter. We will study the methylation of these differentially methylated regions (DMR) in order to discover at what time differential methylation is acquired.
We are focusing on the Gtl2 promoter because this region has been shown to contain a DMR. We will analyze DNA from hybrid offspring which will allow us to distinguish between the copy inherited from the mother vs. the father based on differences in the DNA sequences. A technique called bisulfite mutagenesis followed by DNA sequencing will allow us to determine if the DNA is methylated or unmethylated. Our goal is to determine when during embryonic development methylation is acquired. To accomplish this we will look at embryos at various stages of development. We have shown that all of the potentially methylated sites are methylated solely on the paternal allele in adult tissues. We are also examining sperm DNA in order to verify that methylation of this region is not inherited from sperm. We will then examine the methylation patterns of maternal and paternal alleles in 7.5, 8.5, and 9.5 day embryos to investigate if methylation is acquired during this period of early embryonic development. We are using 7.5,8.5 and 9.5 dpc DNA because another imprinted gene has recently been shown to acquire methylation within that time frame. By comparing the timing of methylation acquisition at Gtl2 with other paternally methylated genes, we hope to better understand the mechanisms leading to the monallelic expression of imprinted genes.