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配偶子形成とゲノムインプリントの分子メカニズムを,DNAメチル化を中心に話した.Dnmt3Lノックアウトマウスを用いて,インプリント機構が個体の発生維持だけでなく,胎盤形成にも重要な役割を果たしていることを説明した.また,RLGS法を用いて,新規インプリント遺伝子,「HYMAI/ZAC」を同定し,このDMR,アレル特異的メチル化領域のメチル化の獲得が卵成長過程で起こり,それと同時に,インプリントが確立することを証明した.最後に,HYMAI/ZACのインプリントの異常が,新生児一過性糖尿病や,卵巣癌と関連を示しているということも説明した.
A subset of genes on the mammalian genomes are differentially expressed depending on whether they are inherited from the mother or the father. These genes are imprinted during gametogenesis so that they remember, in the offspring, the sex of the parent from which they are derived. The expression of imprinted genes were reported accompanied with imprinted patterns of DNA methylation on the locus. To identify endogenously imprinted genes, we screened CpG island using RLGS method. We represented novel imprinted genes, HYMAI and ZAC and both expressed only from the paternal allele. Another object was to identify cis-regulatory elements responsible for controlling imprinting in this region. A high homology CpG island was detected between human and mouse sequence. The CpG island was examined for differentially methylated region (DMR). We performed bisulphite-modified genomic sequencing of the conserved region in the mouse DMR. 23 CpG sites were predominantly unmethylated in sperm DNA. In contrast, in E3.5 blastcysts and E13.5 embryos, the percentage of methylation showed that nearly 50% were methylated. The methylation of the DMR were examined in detail in several stage oocytes. We found their methylation were established in the oocytes from 5-day-old mice, which is maturating phase in oocyte. Therefore, the maternal-specific methylation of the DMR that is established in female germ cells, was maintained during pre-implantation development and persists thereafter in advanced embryos and adults. These results suggest that the conserved region represents a methylation imprint mark. To determine whether the DMR has the capacity to function as regulatory element, we employed a transient transfection assay in Hela cells in which a reporter Luciferase gene was expressed from an SV40 promoter. Fragments from the human DMR were assayed for their effect on transcription of the reporter when either unmethylated or methylated. DMR fragment was shown to act as a strong transcriptional silencer, but only when methylated. Therefore, the methylation of DMR on the maternall allele may result in transcriptional regulation of the imprinted genes in this region. Mutations which affect the epigenetic status of imprinted loci underlie a number of disease in cluding cancer. Transient neomatal diabetes mellitus (TNDM) patients mainly exhibit paternal uniparental disomy (UPD) of HYMAI/ZAC imprinted domain. We showed that in the majority of TNDM patients with a normal karyotype, there is a loss of methylation within DMR. In human ovarian cancer, LOH has been reported at high frequency includeing the region where the putative tumor suppressor gene. We analyzed ZAC expression level in eleven cell lines and tumor tissues. The expression level was reduced or absent as compared with normal ovarian tissues. Furthermore the methylation status of DMR was predominantly hypermethylated. These results suggest that DMA methylation may be involved in the ovarian cancer development. Finally, DNA methyltransferase enzyme (Dnmt3L) was shown to be essential to the maternall methylation imprinting. The maternall allele deficent mouse was demonstrated the placental abnormalities. We propose genomic imprinting is one of the most important mechanisms in the both development of embryo and placenta in mammals.
