PARENT SESSION

(M421) IDENTIFICATION OF GENES WITH SPERM-SPECIFIC UNMETHYLATED REGIONS.

Sato, Shun ¹, Suzuki, Masako¹, Abe, Tetsuya¹, Hattori, Naka¹, Tanaka, Satoshi¹, Shiota, Kunio¹, ¹ The University of Tokyo, Tokyo, Japan

ABSTRACT- During germ cell differentiation, the genome undergoes epigenetic modifications and ultimately forms gamete-specific epigenetic modification patterns. DNA methylation is one of main epigenetic modifications, and is involved in gene silencing. We previously showed that there are numerous tissue-dependently, differentially methylated regions (T-DMRs) in mouse genome, by comparing restriction landmark genomic scanning (RLGS) profiles of several cell lines and tissues. We demonstrated that there are some CpG islands with T-DMRs, which control the expression of genes in vicinity region. Thus, formation of cell-specific DNA methylation profile underlies differentiation of cells. In this study, we found 30 T-DMRs specifically hypomethylated in the sperm. In order to elucidate the role of such sperm-specific T-DMRs during germ cell development, we attempted to identify them. First, we chose candidate genomic sites by simulating RLGS in silico using a mouse whole genome sequence. Then we evaluated DNA methylation statuses of the candidate sites in the genome of sperm, kidney, brain and EG cells by methylation-sensitive quantitative PCR, and identified 9 sperm-specifically hypomethylated T-DMRs, seven of them located around non-imprinted gene coding regions. Lower methylation status of these sites, exclusively in the sperm and testis, was further confirmed by pyrosequencing method using genome of sperm and testis as well as 3 and 13 different cell lines and tissues, respectively. To examine when the sperm-specific methylation pattern of the identified T-DMRs is established, we purified each stage of spermatogenic cells (spermatids, spermatocytes and spermatogonia) from adult testis sections by laser- microdissection technique and analyzed the methylation status of the T-DMRs by bisulfite sequencing method. The results indicated that the T-DMRs are demethylated in all stages of spermatogenic cells in adult testis. These findings support the possibility that the T-DMRs control the expression of neighbor genes, which are involved in germ cell differentiation.

KEY WORDS: DNA methylation, spermatogenesis, tissue-dependently, differentially methylated regions, mouse