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METHYLATION OF IMPRINTED GENES IN THE GERM LINE.
Lucifero, Diana1,2,3, Mertineit, Carmen1,2,3, Clarke, Hugh2, Bestor, Timothy4, Trasler, Jacquetta1,2,3, 1 2 3 4
ABSTRACT- DNA methylation is an epigenetic modification of DNA that is initiated during gametogenesis and is essential for normal development. Methylation is postulated to be a molecular mark underlying genomic imprinting, the differential expression of a gene depending on its parental origin. In this study, our objective was to determine the methylation status of several imprinted genes in mature germ cells and to assess when genomic methylation imprints are established during oogenesis. DNA was isolated from germ cells and bisulfite sequencing was used to determine the methylation status of multiple CpG sites in the imprinted genes H19, Igf2r, Snrpn, Peg1/Mest, and Peg3. The 5′ region of H19, a paternally imprinted gene was completely methylated in sperm and unmethylated in oocytes. We identified regions in the maternally imprinted genes (Snrpn, Peg1/Mest, Peg3) that were unmethylated in sperm but 100% methylated in mature oocytes. Igf2r, which is expressed off the maternal allele, was completely methylated within intronic region 2 in oocytes and unmethylated in sperm. IAP, an endogenous retrovirus, was also amplified from bisulfite treated gamete DNA to investigate the methylation status of a non-imprinted sequence in the germ line. IAP was approximately 50% methylated in both mature gametes. A developmental methylation profile of Snrpn during postnatal oogenesis revealed that genomic methylation imprints are set down in a mosaic pattern during the oocyte growth phase. Bisulfite sequencing data on liver DNA confirmed that methylated and unmethylated alleles were amplified without bias. We have used bisulfite sequencing to identify regions containing 7 to 29 CpG residues within imprinted genes that show gamete-specific methylation patterns in mature germ cells. Furthermore, we have determined IAP to be partially methylated in mature gametes and have shown that maternal imprints on at least one imprinted gene are established during the post-natal growth phase of oogenesis. (Supported by CIHR and NIH)
KEY WORDS: DNA methylation, genomic imprinting, oogenesis, bisulfite sequencing