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Studies on expression of DNA methyltransferases during mouse germ cell development
https://ir.soken.ac.jp/records/971
https://ir.soken.ac.jp/records/971e8fe5afe-8f88-4ffc-96f0-0fe4c72ba8a4
| 名前 / ファイル | ライセンス | アクション |
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要旨・審査要旨 / Abstract, Screening Result (315.7 kB)
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本文 (2.7 MB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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| 公開日 | 2010-02-22 | |||||
| タイトル | ||||||
| タイトル | Studies on expression of DNA methyltransferases during mouse germ cell development | |||||
| タイトル | ||||||
| タイトル | Studies on expression of DNA methyltransferases during mouse germ cell development | |||||
| 言語 | en | |||||
| 言語 | ||||||
| 言語 | eng | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_46ec | |||||
| 資源タイプ | thesis | |||||
| 著者名 |
辻本, 直美
× 辻本, 直美 |
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| フリガナ |
ツジモト, ナオミ
× ツジモト, ナオミ |
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| 著者 |
TSUJIMOTO, Naomi
× TSUJIMOTO, Naomi |
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| 学位授与機関 | ||||||
| 学位授与機関名 | 総合研究大学院大学 | |||||
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| 学位名 | 博士（理学） | |||||
| 学位記番号 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | 総研大甲第601号 | |||||
| 研究科 | ||||||
| 値 | 生命科学研究科 | |||||
| 専攻 | ||||||
| 値 | 18 遺伝学専攻 | |||||
| 学位授与年月日 | ||||||
| 学位授与年月日 | 2002-03-22 | |||||
| 学位授与年度 | ||||||
| 値 | 2001 | |||||
| 要旨 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | Methylation at the 5th position of the cytosine residue is the unique physiological modification found in genomic DNA in mammals, which almost exclusively occurs in a CpG dinucleotide-sequence. This DNA methylation is catalyzed by the enzyme activity known as DNA (cytosine-5) methyltransferase (Dnmt). Two distinct families of Dnmts, Dnmt1 and Dnmt3 have been identified in mammals, both of which contain motifs evolutionarily conserved among all known cytosine methyltransferases. Dnmt1 prefers hemimathylated CpG sites as substrates rather than unmethylated ones, which implies that its major role is to maintain the methylation patterns of the genome shortly after DNA replication. The Dnmt3 family, on the other hand, contains two closely related proteins, termed Dnmt3a and Dnmt3b, which are encoded by distinct genes. Dnmt3a and Dnmt3b methylate both hemimethylated and unmethylated sites with similar kinetics. Based on studies with knockout mice and transgenic flies, it is suggested that Dnmt3a and Dnmt3b are de novo methylatransferases. <br /> In mammals, DNA methylation is essential for normal embryonic development, and plays important roles in developmental gene expression, chromosome stability, silencing of endogenous retroviruses, X chromosome inactivation, and genomic imprinting. Genomic imprinting is a process by which a subset of genes in mammals is differentially marked in the parental germlines so as to be expressed in a parent-of-origin-specific manner in the embryo and adult. Most of the imprinted genes examined so far show differences in DNA methylation between the parental alleles, suggesting a crucial role for differential methylation in imprinting. <br />The 5' flank of the mouse imprinted gene, H19, is more methylated on the paternal chromosome than on the maternal chromosome in somatic tissues, suggesting that this differential methylation may be the gametic imprints that differentiate parental H19 alleles. It has been reported that the paternal methylation imprint of H19 is, for the most part, acquired in gonocyte or prospermatogonia, which are premeiotic germ cells mitotically arrested in the fetal testis. In contrast, the maternal methylation imprints of Igf2r and some transgenes seem to be established during oocyte growth, corresponding to the diplotene or dictyotene stage of meiotic prophase I, which is consistent with the improved development of diploid parthenogenetic embryos with one genome from a nongrowing newborn oocyte and the other from a fully-grown oocyte. <br /> To ask which methyltransferase is responsible for the de novo methylation at imprinted loci in the male and female germline, I carried out immunohistochemistry on the developing testes and ovaries with polyclonal antibodies specific to Dnmt1 , Dnmt3a, and dnmt3b. In the fetal testes, a moderate level of Dnmt1 was observed in the gonocyte nuclei at embryonic day 14.5 (E14.5), which eventually declined to an undetectable level at E18.5. Dnmt3a was detected in somatic cells but not in gonocytes between E14.5 and E18.5. In contrast, Dnmt3b was localized in the nuclei of gonocytes at E16.5 and E18.5. <br /> There are, at least, three different isoforms for Dnmt3b, which are produced by alternative splicing. Dnmt3b1 and Dnmt3b2 are enzymatically active isoforms, whereas Dnmt3b3 seems to be inactive because it lacks a part of the indispensable catalytic domain. RT-PCR confirmed expression of the active isoforms of Dnmt3b in gonocytes at the above stages, implying the presence of a functional enzyme(s) when the sperm-specific methylation imprints were acquired. In addition, RT-PCR also revealed that a novel isoform of Dnmt3b was transcribed in spermatogonia at postnatal day 1 (P1), although this isoform seemed to be enzymatically inactive. <br /> In female gonads, on the other hand, Dnmt1 and Dnmt3b were present in the nucleus of growing oocytes at P7 when the oocyte-specific methylation imprints were established. It has been shown that mice deficient for Dnmt1o, and oocyte-specific isoform of Dnmt1, produce oocytes with appropriate maternal methylation imprints. This excludes Dnmt1 playing a major role in the de novo methylation at imprinted loci in oogenesis. RT-PCR confirmed that Dnmt3b present in oocytes were the active isoforms. Taken together, immunostaining with these polyclonal antibodies implied that Dnmt3b is involved in the establishment of the germline-specific methylation imprints in both sexes.<br /> However, additional study using a monoclonal antibody specific to Dnmt3a unexpectedly demonstrated the presence of dnmt3a in the nuclei of both gonocytes and growing oocytes at the stages critical for methylation imprinting. The apparent discrepancy of immunostaining obtained by the polyclonal and the monoclonal antibodies may be explained by the fact that the former had been raised against the N-terminus of the protein, whereas the latter had been raised against the entire protein. Although the epitope for the monoclonal antibody has not been mapped, it is possible that there is an unidentified isoform(s) of Dnmt3a, which could not be recognized by the polyclonal antibodies. These results suggest that not only Dnmt3b but also a novel isoform(s) of Dnmt3a may be the key methylatransferases that are responsible for the de novo methylation at the imprinted loci in the male and female germline. It also appears that these enzymes are also responsible for the global methylation occurring at the gonocyte stage in the male germline. | |||||
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| 値 | 有 | |||||
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| 内容記述タイプ | Other | |||||
| 内容記述 | application/pdf | |||||
