[학위논문]Expression of DNA Methylation-related Protein Genes During in vitro Differentiation of Mouse Embryonic Stem Cells : 생쥐 줄기세포의 시험관내 분화 동안 DNA 메틸화 관련 단백질 유전자의 발현원문보기
에피제네틱 기전을 통하여 다세포 생물의 세포들은 유전적으로 균질하나 차등적인 유전자 발현으로 인하여 구조와 기능적 이질성을 보인다. 이런 에피제네틱 변화는 발생과정과 성체에서 유전체 기능에 중요하며, 그리고 외부환경에 의해 에피제네틱 조절이 변화되므로 표현형에 장기간 효과를 미친다. 에피제네틱 조절의 중요한 도구인 DNA 메틸화는 유전자발현, 게놈 안정성, 유전자각인, X염색체불활성 등의 세포의 여러 기능을 조절하는 역할을 한다. 그리고 DNMT와 MBD를 포함하는 다양한 단백질은 에피제네틱 조절에 관여한다. ...
에피제네틱 기전을 통하여 다세포 생물의 세포들은 유전적으로 균질하나 차등적인 유전자 발현으로 인하여 구조와 기능적 이질성을 보인다. 이런 에피제네틱 변화는 발생과정과 성체에서 유전체 기능에 중요하며, 그리고 외부환경에 의해 에피제네틱 조절이 변화되므로 표현형에 장기간 효과를 미친다. 에피제네틱 조절의 중요한 도구인 DNA 메틸화는 유전자발현, 게놈 안정성, 유전자각인, X염색체불활성 등의 세포의 여러 기능을 조절하는 역할을 한다. 그리고 DNMT와 MBD를 포함하는 다양한 단백질은 에피제네틱 조절에 관여한다. 포유동물 발생과정 동안의 DNA 메틸화 양상은 게놈 전반적으로 재프로그램되지만 메틸화에 관련된 구성성분의 재현성에 대해서는 아직 많이 알려져 있지 않다. 따라서 초기 발생과정에서의 DNA 메틸화가 미치는 영향을 연구하기 위하여, 생쥐배아줄기세포의 시험관내 분화(in vitro differentiation, IVD)동안 Dnmt and Mbd 유전자의 발현양상을 시간별로 RT-PCR 방법으로 분석하였다. 또한, 5-Aza-2-deoxycytidine(5-AzadC)이 이들의 유전자 발현에 미치는 영향에 대해서도 조사하였다. RT-PCR분석 결과, IVD동안 다양한 발달표지인자의 변화를 확인할 수 있으면 이는 in vivo 상에서의 정상적인 배아발생과정에서 역시 반영될 수 있는 결과이다. 세포배양과정 4일과 5일 의 Dnmt3L, Mbd2, and Mbd4 mRNA는 감소하다가 다시 점차 증가하는 경향을 보였다. 게다가, 5-AzadC 처리 후 세포 배양과정 5일에는 Dnmt3L, Mbd2, Kaiso, Brachyury 와 Msx2 의 유전자가 발현되는 것을 알 수 있었다. 이들의 결과는 에피제네틱 사건의 하나인 DNA 메틸화 패턴 변화가 세포 초기분화 과정에서 다양한 세포 유형의 발현에 중요한 역할을 하는 것으로 사료된다.
에피제네틱 기전을 통하여 다세포 생물의 세포들은 유전적으로 균질하나 차등적인 유전자 발현으로 인하여 구조와 기능적 이질성을 보인다. 이런 에피제네틱 변화는 발생과정과 성체에서 유전체 기능에 중요하며, 그리고 외부환경에 의해 에피제네틱 조절이 변화되므로 표현형에 장기간 효과를 미친다. 에피제네틱 조절의 중요한 도구인 DNA 메틸화는 유전자발현, 게놈 안정성, 유전자각인, X염색체불활성 등의 세포의 여러 기능을 조절하는 역할을 한다. 그리고 DNMT와 MBD를 포함하는 다양한 단백질은 에피제네틱 조절에 관여한다. 포유동물 발생과정 동안의 DNA 메틸화 양상은 게놈 전반적으로 재프로그램되지만 메틸화에 관련된 구성성분의 재현성에 대해서는 아직 많이 알려져 있지 않다. 따라서 초기 발생과정에서의 DNA 메틸화가 미치는 영향을 연구하기 위하여, 생쥐배아줄기세포의 시험관내 분화(in vitro differentiation, IVD)동안 Dnmt and Mbd 유전자의 발현양상을 시간별로 RT-PCR 방법으로 분석하였다. 또한, 5-Aza-2-deoxycytidine(5-AzadC)이 이들의 유전자 발현에 미치는 영향에 대해서도 조사하였다. RT-PCR분석 결과, IVD동안 다양한 발달표지인자의 변화를 확인할 수 있으면 이는 in vivo 상에서의 정상적인 배아발생과정에서 역시 반영될 수 있는 결과이다. 세포배양과정 4일과 5일 의 Dnmt3L, Mbd2, and Mbd4 mRNA는 감소하다가 다시 점차 증가하는 경향을 보였다. 게다가, 5-AzadC 처리 후 세포 배양과정 5일에는 Dnmt3L, Mbd2, Kaiso, Brachyury 와 Msx2 의 유전자가 발현되는 것을 알 수 있었다. 이들의 결과는 에피제네틱 사건의 하나인 DNA 메틸화 패턴 변화가 세포 초기분화 과정에서 다양한 세포 유형의 발현에 중요한 역할을 하는 것으로 사료된다.
Epigenetic modification of the genome occurs during early development of embryos and DNA methylation of cytosine residues is a major cause of the modification[1-3]. In vertebrates, 60% - 90% of all CpGs are methylated, leaving a minor part of the genome methylation free. Many of the remaining non-me...
Epigenetic modification of the genome occurs during early development of embryos and DNA methylation of cytosine residues is a major cause of the modification[1-3]. In vertebrates, 60% - 90% of all CpGs are methylated, leaving a minor part of the genome methylation free. Many of the remaining non-methylated CpGs (~15% of all CpGs in human DNA) are found in CpG islands, which usually include functional promoters[4]. DNA methylation at CpG dinucleotides in mammals plays important roles in a variety of biological processes during embryogenesis and in adult tissues such as tissue specific gene expression, cell differentiation, geneomic imprinting, X-chromosome inactivation, carcinogenesis, and aging. DNA methylation also functions to set up chromatin structure during mouse development[5,6](Fig. 1). DNA methylation enhances the ability of cells to regulate and package of genetic information (Fig. 2). Four mammalian DNA cytosine methyltransferases, Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b have been isolated (Fig. 3). Dnmt1 preferentially methylates hemi-methylated substrates in vivo and is associated with replication foci[7]. Inactivation of Dnmt1 leads to a genome-wide loss of methylation, indicating Dnmt1 functions as a maintenance methylase. Unlike Dnmt1, Dnmt3a and Dnmt3b lack a preference for hemi-methylated substrates in vivo, and are required for de novo methylation in embryonic stem (ES) cells and early post implantation embryous, suggesting Dnmt3a and Dnmt3b function as de novo methylases. No direct evidence exists that Dnmt2, the only mammalian Dnmt lacking a large N-terminal domain, is a functional cytosine methyltransferase[8-11]. Another possible mechanism of gene regulation by DNA methylation is through preferential binding of proteins to methylated promoters, leading to hampering the binding of transcription factors to their target sequences[12,13]. The methyl-CpG binding protein family consists of five members, MECP2 and MBD1-4, sharing a common methyl-CpG binding domain (MBD)[14](Fig. 3). All MBDs except MBD4, relating to DNA mismatch-repair, form complexes with histone deacetylase and are involved in recruiting histone deacetylases to methyl-CpG enriched regions in the genome to repress transcription[13]. The unrelated Kaiso protein family binds to DNA sequences containing methyl-CpGs via a distinctive zinc finger motif[15]. MeCP2 is an abundant chromosomeal protein that requires only a single methylated CpG site for preferential binding to DNA and to repress transcription in vitro[16-18]. In keeping with evidence that DNA methylation is a signal for transcriptional repression, MeCP2, MBD1, MBD2 and Kaiso all repress transcription in vitro and associate with co-repressors. Genome-wide changes of DNA methylation by active and passive demethylation processes are typical features during preimplantation development. Here we provide an insight that epigenetic programming of DNA methylation is regulated in a region-specific manner, not a genome-wide fashion. To address this hypothesis, methylation states of three repetitive genomic regions were monitored at various development stages in the mouse embryos[19-23]. 5-Aza-2-deoxycytidin (5AzadC), one of the demethylating agents, that is a cytosine analog, can cause extensive demethylation of 5-methylcytosine residues and reduce DNA methyltransferase activity in the cells[24,25]. It has been used as a useful experimental tool to study the roles of DNA methylation in the cell differentiationand and gene activation mechanisms[26]. For instance 5-AzaC could induce muscle cell differentiation from fibroblast cell lines and cardiomyogenic cell differentiation from mesenchymal stem cells[27-30]. In the present study, I investigated the expression of DNMTs and MBDs gene and effect of 5-AzaC during in vitro of differentiation of mouse ES cells. I found that various genes for developmental marker were differentially regulated during in vitro differentiation, indicating in vitro differentiation may be reflect the normal embryogenesis in vivo.
Epigenetic modification of the genome occurs during early development of embryos and DNA methylation of cytosine residues is a major cause of the modification[1-3]. In vertebrates, 60% - 90% of all CpGs are methylated, leaving a minor part of the genome methylation free. Many of the remaining non-methylated CpGs (~15% of all CpGs in human DNA) are found in CpG islands, which usually include functional promoters[4]. DNA methylation at CpG dinucleotides in mammals plays important roles in a variety of biological processes during embryogenesis and in adult tissues such as tissue specific gene expression, cell differentiation, geneomic imprinting, X-chromosome inactivation, carcinogenesis, and aging. DNA methylation also functions to set up chromatin structure during mouse development[5,6](Fig. 1). DNA methylation enhances the ability of cells to regulate and package of genetic information (Fig. 2). Four mammalian DNA cytosine methyltransferases, Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b have been isolated (Fig. 3). Dnmt1 preferentially methylates hemi-methylated substrates in vivo and is associated with replication foci[7]. Inactivation of Dnmt1 leads to a genome-wide loss of methylation, indicating Dnmt1 functions as a maintenance methylase. Unlike Dnmt1, Dnmt3a and Dnmt3b lack a preference for hemi-methylated substrates in vivo, and are required for de novo methylation in embryonic stem (ES) cells and early post implantation embryous, suggesting Dnmt3a and Dnmt3b function as de novo methylases. No direct evidence exists that Dnmt2, the only mammalian Dnmt lacking a large N-terminal domain, is a functional cytosine methyltransferase[8-11]. Another possible mechanism of gene regulation by DNA methylation is through preferential binding of proteins to methylated promoters, leading to hampering the binding of transcription factors to their target sequences[12,13]. The methyl-CpG binding protein family consists of five members, MECP2 and MBD1-4, sharing a common methyl-CpG binding domain (MBD)[14](Fig. 3). All MBDs except MBD4, relating to DNA mismatch-repair, form complexes with histone deacetylase and are involved in recruiting histone deacetylases to methyl-CpG enriched regions in the genome to repress transcription[13]. The unrelated Kaiso protein family binds to DNA sequences containing methyl-CpGs via a distinctive zinc finger motif[15]. MeCP2 is an abundant chromosomeal protein that requires only a single methylated CpG site for preferential binding to DNA and to repress transcription in vitro[16-18]. In keeping with evidence that DNA methylation is a signal for transcriptional repression, MeCP2, MBD1, MBD2 and Kaiso all repress transcription in vitro and associate with co-repressors. Genome-wide changes of DNA methylation by active and passive demethylation processes are typical features during preimplantation development. Here we provide an insight that epigenetic programming of DNA methylation is regulated in a region-specific manner, not a genome-wide fashion. To address this hypothesis, methylation states of three repetitive genomic regions were monitored at various development stages in the mouse embryos[19-23]. 5-Aza-2-deoxycytidin (5AzadC), one of the demethylating agents, that is a cytosine analog, can cause extensive demethylation of 5-methylcytosine residues and reduce DNA methyltransferase activity in the cells[24,25]. It has been used as a useful experimental tool to study the roles of DNA methylation in the cell differentiationand and gene activation mechanisms[26]. For instance 5-AzaC could induce muscle cell differentiation from fibroblast cell lines and cardiomyogenic cell differentiation from mesenchymal stem cells[27-30]. In the present study, I investigated the expression of DNMTs and MBDs gene and effect of 5-AzaC during in vitro of differentiation of mouse ES cells. I found that various genes for developmental marker were differentially regulated during in vitro differentiation, indicating in vitro differentiation may be reflect the normal embryogenesis in vivo.
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