• BMB Reports
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• 제35권3호
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• pp.348-351
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• 2002

AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'CYCGRG3'. M.AquI is encoded by two overlapping ORFs (termed $\alpha$ and $\beta$) instead of the single ORF that is customary for Class II methyltransferase genes. The structural organization of the M.AquI protein sequence is quite similar to that of other bacterial C5-DNA methyltransferases. Ten conserved motifs are also present in the correct order, but only on two polypeptides. We separately subcloned the genes that encode the $\alpha$ and $\beta$ subunits of M.AquI into expression vectors. The overexpressed His-fusion $\alpha$ and $\beta$ subunits of the enzyme were purified to homogeneity in a single step by Nickel-chelate affinity chromatography. The purified recombinant proteins were assayed for biological activity by an in vitro DNA tritium transfer assay. The $\alpha$ and $\beta$ subunits of M.AquI alone have no DNA methyltransferase activity, but when both subunits are included in the assay, an active enzyme that catalyses the transfer of the methyl group from S-adenosyl-L-methionine to DNA is reconstituted. We also showed that the $\beta$ subunit alone contains all of the information that is required to generate recognition of specific DNA duplexes in the absence of the $\alpha$ subunit.

• Asian Pacific Journal of Cancer Prevention
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• 제17권sup3호
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• pp.219-224
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• 2016

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver making up more than 80 percent of cases. It is known to be the sixth most prevalent cancer and the third most frequent cause of cancer related death worldwide. Epigenetic regulation constitutes an important mechanism by which dietary components can selectively activate or inactivate target gene expression. The miR-34 family members including mir-34a, mir-34b and mir-34c are tumor suppressor micro RNAs, which are expressed in the majority of normal tissues. Several studies have indicated silencing of miR-34 expression via DNA methylation in multiple types of cancers. Bioactive nutrients like curcumin (Cur) have excellent anticarcinogenic activity and minimal toxic manifestations in biological systems. This compound has recently been determined to induce epigenetic changes. However, Cur is lipophilic and has a poor systemic bioavailability and poor absorption. Its bioavailability is increased through employing dendrosome nanoparticles. The aim of the current study was to investigate the effect of dendrosomal nanocurcumin (DNC) on expression of mir-34 family members in two HCC cell lines, HepG2 and Huh7. We performed the MTT assay to evaluate DNC and dendrosome effects on cell viability. The ability of DNC to alter expression of the mir-34 family and DNA methyltransferases (DNMT1, DNMT3A and 3B) was evaluated using semi-quantitative and quantitative PCR. We observed the entrance of DNC into HepG2 and Huh7 cells. Gene expression assays indicated that DNC treatment upregulated mir34a, mir34b and mir34c expression (P<0.05) as well as downregulated DNMT1, DNMT3A and DNMT3B expression (P<0.05) in both HepG2 and Huh7 cell lines. DNC also reduced viability of Huh7 and HepG2 cells through restoration of miR-34s expression. We showed that DNC could awaken the epigenetically silenced miR-34 family by downregulation of DNMTs. Our findings suggest that DNC has potential in epigenetic therapy of HCC.

• Journal of Periodontal and Implant Science
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• 제47권2호
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• pp.66-76
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• 2017

Purpose: Oral wound healing requires gingival fibroblasts to respond to local growth factors. Epigenetic silencing through DNA methylation can potentially decrease the responsiveness of gingival fibroblasts to local growth factors. In this study, our aim was to determine whether the inhibition of DNA methylation sensitized gingival fibroblasts to transforming growth factor-${\beta}1$ (TGF-${\beta}1$). Methods: Gingival fibroblasts were exposed to 5-aza-2'-deoxycytidine (5-aza), a clinically approved demethylating agent, before stimulation with TGF-${\beta}1$. Gene expression changes were evaluated using quantitative polymerase chain reaction (PCR) analysis. DNA methylation was detected by methylation-sensitive restriction enzymes and PCR amplification. Results: We found that 5-aza enhanced TGF-${\beta}1$-induced interleukin-11 (IL11) expression in gingival fibroblasts 2.37-fold (P=0.008). 5-aza had no significant effects on the expression of proteoglycan 4 (PRG4) and NADPH oxidase 4 (NOX4). Consistent with this, 5-aza caused demethylation of the IL11 gene commonly next to a guanosine (CpG) island in gingival fibroblasts. The TGF-${\beta}$ type I receptor kinase inhibitor SB431542 impeded the changes in IL11 expression, indicating that the effects of 5-aza require TGF-${\beta}$ signaling. 5-aza moderately increased the expression of TGF-${\beta}$ type II receptor (1.40-fold; P=0.009), possibly enhancing the responsiveness of fibroblasts to TGF-${\beta}1$. As part of the feedback response, 5-aza increased the expression of the DNA methyltransferases 1 (DNMT1) (P=0.005) and DNMT3B (P=0.002), which are enzymes responsible for gene methylation. Conclusions: These in vitro data suggest that the inhibition of DNA methylation by 5-aza supports TGF-${\beta}$-induced IL11 expression in gingival fibroblasts.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.93-98
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• 2009

In this study, bovine Dnmt1 cDNA was sequenced and detected Dnmt1 mRNA level in bovine tissues by northern blot, methylation pattern of genome by southern blot, specific localization of Dnmt1 in mouse and bovine preimplantation embryos by immunocytostaining and Dnmt1 protein level in ovary and testis by western blot. Bovine Dnmt1 cDNA sequence showed more homology with that of human than mouse and rat. The RNA level of Dnmt1 was 10 times higher expression in placenta than other tissues. This indicates that placenta was hypermethylated compared to others organs. The genomic DNA could not be cut by a specific restriction enzyme (HpaII) in placenta, lung and liver of bovine. It suggests that Dnmt1 in some somatic cells was already methylated. Dnmt1, which has the antibody epitope 1316~1616, was distributed in nucleus and cytoplasm including the stage of pronuclear stage and maturation of oocyte and gradually weaken to blastocyst stage compare to negative. In addition, Dnmt1 was strongly expressed in tetraploid embryo and cloned 8-cell than IVF 8-cell. An aberrant pattern of DNA methylation in cloned embryo may be abnormal development of fetus, embryonic lethality and placenta dysfunction. The somatic specific band (190kDa) was appeared in ovary and testis, but oocyte specific band (175kDa) was not. Further investigations are necessary to understand the complex links between the methyltransferases and the transcriptional activity of genes in the cloned bovine tissues.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제30권3호
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• pp.302-309
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• 2008

DNA 메틸화는 histone modification과 함께 DNA의 염기서열이 유지되면서 유전기능이 변화되고 자손까지 전달 될 수 있는 후생 유전의 중요한 한 부분이다. DNA 메틸화는 크로마틴의 구조를 변경시키는 과정을 통하여 유전자와 repetitive sequence의 표현을 억제시킬 수 있다. DNA 메틸화는 X-불활성화, 유전체 각인, 유전자 발현조절, 암 생성 등에 중요한 역할을 하는 것으로 밝혀졌고, DNA 메틸화 표지자 (DNA methylation marker)들은 종양의 진단과 치료에 대한 반응을 예측하는 지표로 활용되고 있다. 지금까지 많은 연구 성과에도 불구하고 DNA메틸화, 메틸화에 의한 gene silencing, DNA 메틸화의 표적부위 등에 대한 명확한 기전이 아직도 밝혀지지 않고 있어 향후 더 많은 기초적 연구가 필요할 것이다. 최근에는 후생 유전적 변화는 가역적이기 때문에 종양억제유전자를 억압하는 후생 유전적 변화를 제거한다면 그 종양억제유전자를 다시 활성화시킬 수 있다는 개념의 후생유전 치료법 연구로 DNA 메틸화 억제제와 histone deacetyaltion에 관여하는 HDAC의 억제제들이 항암제로서 개발되어 사용되고 있는데 향후 더 많은 약제 개발과 임상적 연구가 진행되어야 할 것이다.

• 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.

• 미생물학회지
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• 제37권4호
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• pp.245-252
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• 2001

MLS (macrolide-lincosamide-streptogramin B) 항생제 내성인자 단백질인 Erm 단백질들은 아미노산 서열 중 그 동일성과 유사성이 높아 구조적으로도 동등한 단백질의 한 집단을 형성한다. 최근 X-ray crystallography에 의해 구조가 결정된 ErmC\` 및 ErmAM 단백질의 구조에 근거하여 ErmSF 단백질도 catalytic domain과 substrate binding domain으로 구분하였고 N-terminal end에 존재하는 catalytic domain의 대량생산을 다양한 pET 발현 vector를 사용하여 시도하였다. 그리고 catalytic domain을 coding하는 DNA 절편은 세 종류를 사용하였다: DNA 절편 1은 Met 1부터 Glu 186까지를 coding하고 DNA 절편 2는 Arg 60부터 Glu 186까지의 정보를 가진 DNA이고 DNA 절편 3은 Arg 60부터 Arg 240까지를 encoding하는 DNA이다. 사용된 다양한 발현 vector중에서 pET19b는 DNA 절편 3, pET23b는 DNA 절편 1과 2를 성공적으로 대량생산하였다. 그러나 대량생산된 catalytic domain들은 불용성 단백질 집합체인 inclusion body를 형성하였다. ErmSF catalytic domain들의 용해성 단백질의 생산을 위하여 chaperone GroESL과 Thioredoxin의 동시 발현 및 배양온도를 $22^{\circ}C$로 낮추어 시도했으나 대량 발현된 단백질의 용해에는 도움을 얻지 못하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권1호
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• pp.9-21
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• 2015

Cancer, a serious public health problem in worldwide, results from an excessive and uncontrolled proliferation of the body cells without obvious physiological demands of organs. The gastrointestinal tract, including the esophagus, stomach and intestine, is a unique organ system. It has the highest cancer incidence and cancer-related mortality in the body and is influenceed by both genetic and environmental factors. Among the various chemical elements recognized in the nature, some of them including zinc, iron, cobalt, and copper have essential roles in the various biochemical and physiological processes, but only at low levels and others such as cadmium, lead, mercury, arsenic, and nickel are considered as threats for human health especially with chronic exposure at high levels. Cadmium, an environment contaminant, cannot be destroyed in nature. Through impairment of vitamin D metabolism in the kidney it causes nephrotoxicity and subsequently bone metabolism impairment and fragility. The major mechanisms involved in cadmium carcinogenesis could be related to the suppression of gene expression, inhibition of DNA damage repair, inhibition of apoptosis, and induction of oxidative stress. In addition, cadmium may act through aberrant DNA methylation. Cadmium affects multiple cellular processes, including signal transduction pathways, cell proliferation, differentiation, and apoptosis. Down-regulation of methyltransferases enzymes and reduction of DNA methylation have been stated as epigenetic effects of cadmium. Furthermore, increasing intracellular free calcium ion levels induces neuronal apoptosis in addition to other deleterious influence on the stability of the genome.

• Asian Pacific Journal of Cancer Prevention
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• 제17권7호
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• pp.3213-3222
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• 2016

Background: Methyl donor status influences DNA stability and DNA methylation although little is known about effects on DNA methyltransferases. The aim of this study was to determine whether methyl-donor status influences DNA methyltransferase (Dnmt) gene expression in cervical cancer cells, and if so, whether there are associated effects on global DNA methylation. Materials and Methods: The human cervical cancer cell line, C4-II, was grown in complete medium and medium depleted of folate (F-M+) and folate and methionine (F-M-). Growth rate, intracellular folate, intracellular methionine and homocysteine in the extracellular medium were measured to validate the cancer cell model of methyl donor depletion. Dnmt expression was measured by qRT-PCR using relative quantification and global DNA methylation was measured using a flow cytometric method. Results: Intracellular folate and methionine concentrations were significantly reduced after growth in depleted media. Growth rate was also reduced in response to methyl donor depletion. Extracellular homocysteine was raised compared with controls, indicating disturbance to the methyl cycle. Combined folate and methionine depletion led to a significant down-regulation of Dnmt3a and Dnmt3b; this was associated with an 18% reduction in global DNA methylation compared with controls. Effects of folate and methionine depletion on Dnmt3a and 3b expression were reversed by transferring depleted cells to complete medium. Conclusions: Methyl donor status can evidently influence expression of Dnmts in cervical cancer cells, which is associated with DNA global hypomethylation. Effects on Dnmt expression are reversible, suggesting reversible modulating effects of dietary methyl donor intake on gene expression, which may be relevant for cancer progression.

• Journal of Animal Science and Technology
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• 제53권3호
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• pp.269-274
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• 2011

DNA methyltransferases (DNMTs) are closely associated with the epigenetic change and the gene silencing through the regulation of methylation status in animal genome. But, the role of DNMTs in transgene silencing has remained unclear. So, we examined whether the knockdown of DNMT influences the reactivation of transgene expression in the transgenic quails. In this study, we investigated the expression of DNMT3a, and DNMT3b in blastoderm, quail embryonic fibroblasts (QEFs) and limited embryonic tissues such as gonad, kidney, heart and liver of E6 transgenic quails (TQ2) by RT-PCR. We further analyzed the expression of DNMT3a at different stages of whole embryos during early embryonic development by qRT-PCR. DNMT3a expression was detected in all test samples; however, it showed the highest expression in E6 whole embryo. Embryonic fibroblasts collected from TQ2 quails were treated with two DNMT3a-targeted siRNAs (siDNMT3a-51 and siDNMT3a-88) for RNA interference assay, and changes in expression were then analyzed by qRT-PCR. The siDNMT3a-51 and siDNMT3a-88 reduced 53.34% and 64.64% of DNMT3a expression in TQ2 QEFs, respectively. Subsequently the treatment of each siRNA reactivated enhanced green fluorescent protein (EGFP) expression in TQ2 (224% and 114%). Our results might provide a clue for understanding the DNA methylation mechanism responsible for transgenic animal production and stable transgene expression.