• Reproductive and Developmental Biology
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• 제34권4호
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• pp.283-288
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• 2010

During normal early embryonic development in mammals, the global pattern of genomic DNA methylation undergoes marked. changes. The level of methylation is high in male and female gametes. Thus, we cloned the cDNA of the porcine DNA methyltransferase 1 (Dnmt1) gene to promote the efficiency of the generation of porcine clones. In this study, porcine Dnmt1 cDNA was sequenced, and Dnmt1 mRNA expression was detected by reverse transcription-polymerase reaction (RT-PCR) in porcine tissues during embryonic development. The porcine Dnmt1 cDNA sequence showed more homology with that of bovine than human, mouse, and rat. The complete sequence of porcine Dnmt1 cDNA was 4,774-bp long and consisted of an open reading frame encoding a protein of 1611 amino acids. The amino acid sequence of porcine DNMT1 showed significant homology with those of bovine (91%), human (88%), rat (76%), and mouse (75%) Dnmt1. The expression of porcine Dnmt1 mRNA was detected during porcine embryogenesis. The mRNA was detected at stages of porcine preimplantation development (1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages). It was also abundantly expressed in tissues (lung, ovary, kidney and somatic cells). Further investigations are necessary to understand the complex links between methyltransferase 1 and the transcriptional activity in cloned porcine tissues.

• BMB Reports
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• 제40권4호
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• pp.554-561
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• 2007

Shen and colleagues (Lin et al., 2004) have recently shown that overexpression of the Drosophila DNA methyltransferase 2 isoform C, dDnmt2c, extended life span of fruit flies, probably due to increased expression of small heat shock proteins such as Hsp22 or Hsp26. Here, I demonstrate with immunoprecipitations that overexpressed dDnmt2c interacts with endogenous Hsp70 protein in vivo in S2 cells. However, its C-terminal half, dDnmt2c(178-345) forms approximately 10-fold more Hsp70-containing protein complexe than wild-type dDnmt2c. Overexpressed dDnmt2c(178-345) but not the full length dDnmt2c is able to increase endogenous mRNA levels of the small heat shock proteins, Hsp26 and Hsp22. I provide evidence that dDnmt2c(178-345) increases Hsp26 promoter activity via two heat shock elements, HSE6 and HSE7. Simultaneously overexpressed Hsp40 or a dominant negative form of heat shock factor abrogates the dDnmt2c(178-345)-dependent increase in Hsp26 transcription. The data support a model in which the activation of heat shock factor normally found as an inactive monomer bound to chaperones is linked to the overexpressed C-terminus of dDnmt2c. Despite the differences observed in flies and S2 cells, these findings provide a possible explanation for the extended lifespan in dDnmt2c-overexpressing flies with increased levels of small heat shock proteins.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.84-84
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• 2003

DNA methylation is involved in epigenetic processes such as X-chromosome inactivation, imprinting and silencing of transposons. DNA methylation is a highly plastic and critical component of mammalian development The DNA methyltransferases (Dnmts) are responsible for the generation of genomic methylation patterns, which lead to transcriptional silencing. The maintenance DNA methyltransferase enzyme, Dnmt 1, and the de novo methyltransferase, Dnmt3a and Dnmt3b, are indispensable for development because mice homozygous for the targeted disruption of any of these genes are not viable. The occurrence of DNA methylation is not random, and it can result in gene silencing The mechanisms underlying these processes are poorly understood. It is well established that DNA methylation and histone deacetylation operate along a common mechanistic pathway to repress transcription through the action of methyl-binding domain proteins (MBDs), which are components of, or recruit, histone deacetylase (HDAC) complexes to methylated DNA. As a basis for future studies on the role of the DNA-methyl-transferase in porcine development, we have isolated and characterized a partial cDNA coding for the porcine Dnmt1. Total RNA of testis, lung and ovary was isolated with TRlzol according to the manufacture's specifications. 5 ug of total RNA was reverse transcribed with Super Script II in the presence of porcine Dnmt 1 specific primers. Standard PCRs were performed in a total volume of 50 ul with cDNA as template. Two DNA fragmenets in different position were produced about 700bp, 1500bp and were cloned into pCR II-TOPO according to the manufacture's specification. Assembly of all sequences resulted in a cDNA from 158bp of 5'to 4861bp of 3'compare with the known human maintenance methyltransferase. Now, we are cloning the unknown Dnmt 1 region by 5'-RACE method and expression of Dnmt 1 in tissues from adult porcine animals.

• Clinical and Experimental Reproductive Medicine
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• 제45권1호
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• pp.17-24
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• 2018

Objective: To investigate sperm chromatin/DNA integrity, global DNA methylation, and DNMT mRNA transcription in men with oligoasthenoteratozoospermia (OAT) compared with normozoospermic men. Methods: Semen samples from 32 OAT patients who comprised the case group and 32 normozoospermic men who comprised the control group were isolated and purified using a standard gradient isolation procedure according to World Health Organization criteria. DNMT1, DNMT3A, and DNMT3B transcripts were then compared between groups using real-time quantitative reverse-transcription polymerase chain reaction. Global DNA methylation in sperm was determined by an enzyme-linked immunosorbent assay. Protamine deficiency and the proportion of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3), aniline blue (AB), and toluidine blue (TB) staining, as well as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The p-values < 0.05 were considered to indicate statistical significance. Results: Significantly higher proportions of AB+, TB+, CMA3+, and TUNEL+ spermatozoa, as well as DNMT3A and DNMT3B transcription, were found in the OAT group. Positive correlations were detected between sperm parameters, DNA/chromatin damage, and DNMT3A and DNMT3B transcripts. Global DNA methylation was significantly higher in the OAT patients and had a significant correlation with abnormal results of all sperm chromatin integrity tests, but was not associated with DNMT1, DNMT3A, or DNMT3B expression. Conclusion: Oligoasthenoteratozoospermic men showed abnormal sperm parameters, abnormal chromatin/DNA integrity, and a higher global DNA methylation rate, as well as overexpression of DNMT mRNA.

• 대한임상검사과학회지
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• 제52권1호
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• pp.69-77
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• 2020

종양 억제 유전자 p53은 인간 간암세포 Hep3B에서는 불활성화되어 있다. 베르베린(berberine)은 암세포의 증식을 억제하는 것으로 보고되어 있다. 우리는 베르베린을 처리한 Hep3B 세포에서 세포사멸이 유도되는지를 조사하였고 이 세포사멸이 p53과 DNA methyltransferase의 발현과 연관되어 있는지를 관찰하였다. MTT 분석을 통하여 세포 생존력을 측정하였다. 세포사멸은 각각 Annexin V flow 세포 분석을 사용하여 측정하였다. 베르베린이 처리된 세포에서 DNMT 효소 활성, mRNA 발현, 단백질 발현 정도가 검사되었으며, p53 단백질 발현은 웨스턴 블롯 분석에 의해 검사되었다. 베르베린 처리는 시간 및 용량 의존적으로 Hep3B세포의 세포사멸을 증가시켰다. 베르베린 처리 시 DNMT3b의 활성 정도, mRNA 발현 그리고 단백질 발현 정도가 모두 감소되었다. 이와는 대조적으로, Hep3B에서는 비활성인 p53 단백질의 발현은 DNMT3b의 감소와 동시에 증가했다. ERK의 발현은 변화가 없었으나, P-ERK의 발현은 농도 의존적으로 감소하는 것으로 나타났다. 이러한 결과는 Hep3B 세포에 베르베린의 처리는 DNMT3b의 발현을 감소시켜서 종양 억제 유전자인 p53의 증가를 유도할 수 있고, 이를 통해서 세포사멸을 증가 시킬 수 있다는 것을 나타낸다. 이는 베르베린이 간암 세포의 증식 억제에 효과적으로 작용할 수 있음을 보여준다.

• BMB Reports
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• 제42권12호
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• pp.834-839
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• 2009

DNA methyltransferase (DNMT) 1 is the key enzyme responsible for DNA methylation, which often occurs in CpG islands located near the regulatory regions of genes and affects transcription of specific genes. In this study, we examined the possible association of DNMT1 polymorphisms with HBV clearance and the risk of hepatocellular carcinoma (HCC). Seven common polymorphic sites were selected by considering their allele frequencies, haplotype-tagging status and LDs for genotyping in larger-scale subjects (n = 1,100). Statistical analysis demonstrated that two intron polymorphisms of DNMT1, +34542G > C and +38565G > T, showed significant association with HBV clearance in a co-dominant model (OR = 1.30, $P^{corr}$ = 0.03) and co- dominant/recessive model (OR = 1.34-1.74, $P^{corr}$ = 0.01-0.03), respectively. These results suggest that two intron polymorphisms of DNMT1, +34542G > C and +38565G > T, might affect HBV clearance.

• Reproductive and Developmental Biology
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• 제36권1호
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• pp.7-12
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• 2012

DNA methyltransferase 1 (Dnmt1) gene contains three different isoform transcripts, Dnmt1s, Dnmt1o, and Dnmt1p, are produced by alternative usage of multiple first exons. Dnmt1o is specific to oocytes and preimplantation embryos, whereas Dnmt1s is expressed in somatic cells. Here we determined that porcine Dnmt1o gene had differentially methylated regions (DMRs) in 5'-flanking region, while those were not found in the Dnmt1s promoter region. The methylation patterns of the porcine Dnmt1o/Dnmt1s DMRs were investigated using bisulfite sequencing and pyrosequencing analysis through all preimplantation stages from one cell to blastocyst stage in in vivo or somatic cell nuclear transfer (SCNT). The Dnmt1o DMRs contained 8 CpG sites, which located in -640 bp to -30 bp upstream region from transcription start site of the Dnmt1o gene. The methylation status of 5 CpGs within the Dnmt1o DMRs were distinctively different at each stage from one-cell to blastocyst stage in the $in$ $vivo$ or SCNT, respectively. 55.62% methylation degree of the Dnmt1o DMRs in the $in$ $vivo$ was increased up to 84.38% in the SCNT embryo, moreover, $de$ $novo$ methylation and demethylation occurred during development of porcine embryos from the one-cell stage to the blastocyst stage. However, the DNA methylation states at CpG sites in the Dnmt1s promoter regions were hypomethylated, and dramatically not changed through one-cell to blastocyst stage in the $in$ $vivo$ or SCNT embryos. In the present study, we demonstrated that the DMRs in the promoter region of the porcine Dnmt1o was well conserved, contributing to establishment and maintenance of genome-wide patterns of DNA methylation in early embryonic development.

• Asian Pacific Journal of Cancer Prevention
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• 제13권5호
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• pp.2207-2212
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• 2012

Overexpression of DNA methyltransferase 1 (DNMT1) has been detected in many cancers. Tobacco exposure is known to induce genetic and epigenetic changes in the pathogenesis of malignancy. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important carcinogen present in tobacco smoke; however the detailed molecular mechanism of how NNK induces esophageal carcinogenesis is still unclear. We found that DNMT1 was overexpressed in ESCC tissues compared with paired non-cancerous tissues, the overexpression being correlated with smoking status and low expression of $RAR{\beta}$. The latter could be upregulated by NNK treatment in Het-1A cells, and the increased DNMT1 expression level reflected promoter hypermethylation and downregulation of retinoic acid receptor ${\beta}$($RAR{\beta}$). RNA interference mediated knockdown of DNMT1 resulted in promoter demethylation and upregulation of $RAR{\beta}$ in KYSE30 and TE-1 cells. 3-(4,5-Dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometric analysis demonstrated that NNK treatment in Het-1A cells could enhance cell proliferation and inhibit cell apoptosis in a dose-dependent manner. In conclusion, DNMT1 overexpression is correlated with smoking status and low expression of $RAR{\beta}$ in esophageal SCC patients. NNK could induce $RAR{\beta}$ promoter hypermethylation through upregulation of DNMT1 in esophageal squamous epithelial cells, finally leading to enhancement of cell proliferation and inhibition of apoptosis.

• Asian Pacific Journal of Cancer Prevention
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• 제14권10호
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• pp.5713-5718
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• 2013

Background: DNA methyltransferase-3a (DNMT3a) plays significant roles in embryogenesis and the generation of aberrant methylation in carcinogenesis. This study aimed to investigate associations between single nucleotide polymorphisms (SNPs) of the DNMT3a gene and risk of Helicobacter pylori infection, gastric atrophy and gastric cancer. Methods: The subjects comprised 447 patients with gastric cancer; 111 individuals with gastric atrophy and 961 healthy controls. Two SNPs (rs1550117 and rs13420827) of the DNMT3a gene were genotyped by Taqman assay. DNMT3a expression was analyzed in cancer tissues from 89 patients by tissue microarray technique. Odds ratio (ORs) and 95% confidence intervals were calculated by multivariate logistic regression. Results: Among healthy controls, risk of H.pylori infection was significantly higher in subjects with the rs1550117 AA genotype, compared to those with GG/AG genotypes of DNMT3a [OR=2.08, (95%CI: 1.02-4.32)]. However, no significant correlation was found between the two SNPs and risk of developing gastric atrophy or gastric cancer. In addition, no increase in DNMT3a expression was observed in the gastric cancer with H.pylori infection. Conclusions: This study revealed that DNMT3a rs1550117 polymorphism is significantly associated with an increased risk of H. pylori infection, but did not support any evidence for contributions of DNMT3a rs1550117 and rs13420827 to either gastric atrophy or gastric cancer. The biological roles of DNMT3a polymorphisms require further investigation.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.39-39
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• 2003

Methylation of DNA 5-cytosine in mammalian early embryo affects great deal in nuclear reprogramming and chromatin remodeling of developing embryo. Current efforts to clone and produce cloned animals including transgenic animals face various problems including low birth rate, irregular development, and so on. In this report, cDNA for the one of house keeping methyltransfcrase, Dnmt1 was cloned from bovine somatic tissues and was analyzed for its nucleotide sequences to investigate the structure and function of the gene in bovine early development. Nucleotide sequence of bovine Dnmt1 homologue showed 76.8% identity with that of human Dnmtl and 66.4% with mouse Dnmt1. Translated amino acid sequence showed 88.4% homology with human homologue and 75.8% homology with mouse counterpart. Three types of Dnmt1 are reported in mouse and human, and are likely present in bovine tissues. Understanding of role of Dnmt1 in bovine development may shed a light in the field of animal, especially bovine cloning.