인간의 MBD2 유전자는 in vitro에서 demethylating activity를 가진다고 알려져 있다. 생쥐 Mbd2유전자의 가장 짧은 형태인 testis form을 cloning하여 demethylation assay를 수행하였다. N-말단의 GR rich domain과 C-말단의 coiled-coil domain이 없이도 demethylating activity가 존재하였다. 또, 생쥐 발생 초기에 일어나는 genome-wide de...
인간의 MBD2 유전자는 in vitro에서 demethylating activity를 가진다고 알려져 있다. 생쥐 Mbd2유전자의 가장 짧은 형태인 testis form을 cloning하여 demethylation assay를 수행하였다. N-말단의 GR rich domain과 C-말단의 coiled-coil domain이 없이도 demethylating activity가 존재하였다. 또, 생쥐 발생 초기에 일어나는 genome-wide demethylation에 Mbd2가 관여하는 지의 단서를 알아내기 위하여 발생 초기 여러 단계의 embryo에서 발현 여부를 조사하였다. 수정란에서부터 gastrula까지 모두 Mbd2 mRNA가 발견되었지만 미수정란에서는 발견되지 않았다. 처녀 생식에서도 조사한 결과, Mbd2 transcript가 발견되지 않았다. 그렇지만, Mbd2 단백질과 mRNA 모두가 sperm에서 Western blot과 Northern hybridization 분석으로 발견되었다. 이 결과는 Mbd2가 paternal factor 중의 하나이고, 초기 embryo에서의 Mbd2 발현에는 paternal messages를 필요로 하다는 것으로 생각할 수 있다. 이는 수정란에서 paternal 염색체들만이 먼저 genome-wide-demethylation되는 사실과 부합한다. 종합하면, Mbd2가 생쥐 발생 초기에 활성화되는 genome-wide demethylase일 것으로 생각된다.
인간의 MBD2 유전자는 in vitro에서 demethylating activity를 가진다고 알려져 있다. 생쥐 Mbd2유전자의 가장 짧은 형태인 testis form을 cloning하여 demethylation assay를 수행하였다. N-말단의 GR rich domain과 C-말단의 coiled-coil domain이 없이도 demethylating activity가 존재하였다. 또, 생쥐 발생 초기에 일어나는 genome-wide demethylation에 Mbd2가 관여하는 지의 단서를 알아내기 위하여 발생 초기 여러 단계의 embryo에서 발현 여부를 조사하였다. 수정란에서부터 gastrula까지 모두 Mbd2 mRNA가 발견되었지만 미수정란에서는 발견되지 않았다. 처녀 생식에서도 조사한 결과, Mbd2 transcript가 발견되지 않았다. 그렇지만, Mbd2 단백질과 mRNA 모두가 sperm에서 Western blot과 Northern hybridization 분석으로 발견되었다. 이 결과는 Mbd2가 paternal factor 중의 하나이고, 초기 embryo에서의 Mbd2 발현에는 paternal messages를 필요로 하다는 것으로 생각할 수 있다. 이는 수정란에서 paternal 염색체들만이 먼저 genome-wide-demethylation되는 사실과 부합한다. 종합하면, Mbd2가 생쥐 발생 초기에 활성화되는 genome-wide demethylase일 것으로 생각된다.
Human MBD2 (methyl-CpG-binding domain containing protein 2) is known to have a demethylation activity in vitro. MBD2 has been found as a member of methyl-CpG binding domain proteins in human. Two translational initiation sites produce MBD2a (longer form) and MBD2b (shorter form). MBD2a contain N-ter...
Human MBD2 (methyl-CpG-binding domain containing protein 2) is known to have a demethylation activity in vitro. MBD2 has been found as a member of methyl-CpG binding domain proteins in human. Two translational initiation sites produce MBD2a (longer form) and MBD2b (shorter form). MBD2a contain N-terminal GR rich domain, methyl CpG binding domain (MBD), and C-terminal coiled-coil domain. MBD2b form lacks GR rich domain. Testis form of MBD2a (MBD2aT) is preterminated before C-terminal coiled-coil domain. MBD2b protein possesses a demethylation activity that can processively transfer the methyl group from methyl-cytosine of fully- or hemi-methylated CpG dinucleotides to water molecule in vitro. It has been proposed that the mechanisms of genome-wide demethylation of paternal and maternal chromosomes are different. The paternal chromosomes are actively demethylated immediately after fertilization, while the maternal chromosomes are passively demethylated by DNA replication during early embryonic development. A plausible explanation is that the demethylase activity is delivered from sperm to oocyte during fertilization or activated by paternal factors during fertilization. However, any demethylase directly responsible for the genome-wide demethylation during early embryonic development has not been identified yet. The testis form of mouse Mbd2b (Mbd2bT) was cloned into GFP-fusion vector and transfected into NIH3T3. The GFP-Mbd2bT protein was highly localized in the nucleus of NIH3T3. The nucleus localization signal was contained in Mbd2bT form. Demethylation assay with Mbd2bT protein expressed in Esherichia coli confirmed that Mbd2bT had also a limited demethylating activity. The coiled-coil domain was not required for the demethylating activity. Expression pattern of Mbd2 in mouse embryos at various stages of early development was investigated to obtain clues whether Mbd2 is involved in the genome-wide demethylation during embryonic development. We showed that Mbd2 mRNA was detected in normal embryos ranging from fertilized eggs to gastrulae, but not in unfertilized oocytes. Mbd2 was transcriptionally inert in parthenogenetic embryos. Both Mbd2 protein and mRNA were also detected in sperm by Western and Northern hybridization analyses. The results suggest that Mbd2 is one of the paternal factors and that its expression requires the contribution of paternal messages. These imply that Mbd2 is a candidate for a genome-wide demethylase.
Human MBD2 (methyl-CpG-binding domain containing protein 2) is known to have a demethylation activity in vitro. MBD2 has been found as a member of methyl-CpG binding domain proteins in human. Two translational initiation sites produce MBD2a (longer form) and MBD2b (shorter form). MBD2a contain N-terminal GR rich domain, methyl CpG binding domain (MBD), and C-terminal coiled-coil domain. MBD2b form lacks GR rich domain. Testis form of MBD2a (MBD2aT) is preterminated before C-terminal coiled-coil domain. MBD2b protein possesses a demethylation activity that can processively transfer the methyl group from methyl-cytosine of fully- or hemi-methylated CpG dinucleotides to water molecule in vitro. It has been proposed that the mechanisms of genome-wide demethylation of paternal and maternal chromosomes are different. The paternal chromosomes are actively demethylated immediately after fertilization, while the maternal chromosomes are passively demethylated by DNA replication during early embryonic development. A plausible explanation is that the demethylase activity is delivered from sperm to oocyte during fertilization or activated by paternal factors during fertilization. However, any demethylase directly responsible for the genome-wide demethylation during early embryonic development has not been identified yet. The testis form of mouse Mbd2b (Mbd2bT) was cloned into GFP-fusion vector and transfected into NIH3T3. The GFP-Mbd2bT protein was highly localized in the nucleus of NIH3T3. The nucleus localization signal was contained in Mbd2bT form. Demethylation assay with Mbd2bT protein expressed in Esherichia coli confirmed that Mbd2bT had also a limited demethylating activity. The coiled-coil domain was not required for the demethylating activity. Expression pattern of Mbd2 in mouse embryos at various stages of early development was investigated to obtain clues whether Mbd2 is involved in the genome-wide demethylation during embryonic development. We showed that Mbd2 mRNA was detected in normal embryos ranging from fertilized eggs to gastrulae, but not in unfertilized oocytes. Mbd2 was transcriptionally inert in parthenogenetic embryos. Both Mbd2 protein and mRNA were also detected in sperm by Western and Northern hybridization analyses. The results suggest that Mbd2 is one of the paternal factors and that its expression requires the contribution of paternal messages. These imply that Mbd2 is a candidate for a genome-wide demethylase.
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