• Journal of Ginseng Research
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• 제44권5호
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• pp.747-755
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• 2020

Background: Ginsenosides accumulation responses to temperature are critical to quality formation in cold-dependent American ginseng. However, the studies on cold requirement mechanism relevant to ginsenosides have been limited in this species. Methods: Two experiments were carried out: one was a multivariate linear regression analysis between the ginsenosides accumulation and the environmental conditions of American ginseng from different sites of China and the other was a synchronous determination of ginsenosides accumulation, overall DNA methylation, and relative gene expression in different tissues during different developmental stages of American ginseng after experiencing different cold exposure duration treatments. Results: Results showed that the variation of the contents as well as the yields of total and individual ginsenosides Rg1, Re, and Rb1 in the roots were closely associated with environmental temperature conditions which implied that the cold environment plays a decisive role in the ginsenoside accumulation of American ginseng. Further results showed that there is a cyclically reversible dynamism between methylation and demethylation of DNA in the perennial American ginseng in response to temperature seasonality. And sufficient cold exposure duration in winter caused sufficient DNA demethylation in tender leaves in early spring and then accompanied the high expression of flowering gene PqFT in flowering stages and ginsenosides biosynthesis gene PqDDS in green berry stages successively, and finally, maximum ginsenosides accumulation occurred in the roots of American ginseng. Conclusion: We, therefore, hypothesized that cold-induced DNA methylation changes might regulate relative gene expression involving both plant development and plant secondary metabolites in such cold-dependent perennial plant species.

• Plant Biotechnology Reports
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• 제3권2호
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• pp.139-145
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• 2009

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we isolated NtMET1 from Nicotiana tabacum cv. Havana (SR1) and obtain transgenic plants that reduced MET1 expression level with the double-strand RNA (dsRNA) MET1 gene. Transgenic tobacco plants showed dwarf and abnormal flower development when compared with the wild type. Using methylation-sensitive amplified polymorphism (MSAP) analysis, the patterns of cytosine methylation in transformed plants and the wild type were compared. MseI/HpaII selection primers showed an interesting polymorphism, and 153 DNA bands of interest were detected. Among these, 30 selective fragments were sequenced and analyzed with a BLAST search by successful MSAP modifications. The homology search showed that the transposons and tandem repeated sequences were related to the phenotypes. These results suggested that the decreased degree of methylation by dsRNA strategy caused abnormal growth and development in N. tabacum.

• Clinical and Experimental Pediatrics
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• 제48권6호
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• pp.634-639
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• 2005

목 적 : 2세 미만의 소아에서는 임상적으로 확실한 증상이 있거나 알레르기 가족력이 있는 것을 제외하고는 실제적으로 아토피 유무를 판별하는 것은 성인에 비해 제한점이 많다. 본 연구에서는 영유아기에 알레르기 질환과 관련된 사이토카인이 유전자 배열에서 발현하는 지를 말초혈액의 Th2 림프구에서 interleukin-4 유전자의 DNA 탈메틸화를 통하여 알아보고자 하였다. 방 법 : 대상은 한양대학교 구리병원 소아알레르기클리닉을 내원한 3세 미만 소아에 대해 소아의 병력과 가족력을 조사하여 family allergy score와 개인력을 기준으로 알레르기질환에 대한 고위험군 7명, 저위험군 8명, 정상 대조군 7명이었다. 이들 모두에서 총 IgE치와 Der f II 특이 IgE를 측정하였고 이들의 말초혈액에서 T세포를 Der f II로 자극하여 배양한 후 DNA 추출을 하여 IL-4 유전자 두 번째 인트론의 CpG 섬에 대한 메틸화 분석을 실시하였다. 결 과 : 정상군에 비해 알레르기 환아군에서 IL4 유전자의 두번째 인트론에 위치한 CpG 섬의 두 부위 모두에서 메틸화 특이 PCR 신호강도가 약하게 나타나는 반면 정상아에서는 118 bp PCR 산물과 205 bp PCR 산물이 모두 강하게 나타나서 메틸화된 것으로 보이며, 저위험군 알레르기 환아에서 최소한 한 부위에서 탈메틸화가 일어났으며 고위험군 알레르기 환아에서는 두 부위 모두에서 탈메틸화가 일어났다. 결 론 : 정상 대조군의 T 세포에서는 IL-4 유전자의 메틸화가 일어나고 알레르기 환아에서 고위험군과 저위험군 모두 정도의 차이는 있으나 메틸화의 소실이 관찰되었다. 이를 이용하여 향후 영유아기에서 아토피 유무를 판별할 수 있는 가능성을 제시하였으나 더 많은 연구가 필요할 것으로 사료된다.

• 미생물학회지
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• 제55권2호
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• pp.103-111
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• 2019

DNA 메틸화는 유전체의 무결성의 유지 및 유전자 발현 조절과 같은 박테리아의 다양한 과정에 관여한다. Alphaproteobacteria 종에서 보존된 DNA 메틸 전이 효소인 CcrM은 S-아데노실 메티오닌을 공동 기질로 사용하여 $N^6$-아데닌 또는 $N^4$-시토신의 메틸 전이 효소 활성을 갖는다. Celeribacter marinus IMCC 12053는 해양 환경에서 분리된 알파프로테오박테리아로서 GpC 시토신의 외향고리 아민의 메틸기를 대체하여 $N^4$-메틸 시토신을 생산한다. 단일 분자 실시간 서열 분석법(SMRT)을 사용하여, C. marinus IMCC12053의 메틸화 패턴을 Gibbs Motif Sampler 프로그램을 사용하여 확인하였다. 5'-GANTC-3'의 $N^6$-메틸 아데노신과 5'-GpC-3'의 $N^4$-메틸 시토신을 확인하였다. 발현된 DNA 메틸전이 효소는 계통 발생 분석법을 사용하여 선택하여 pQE30 벡터에 클로닝 후 $dam^-/dcm^-$ 대장균을 사용하여 클로닝된 DNA 메틸라아제의 메틸화 활성을 확인하였다. 메틸화 효소를 코딩하는 게놈 DNA 및 플라스미드를 추출하고 메틸화에 민감한 제한 효소로 절단하여 메틸화 활성을 확인하였다. 염색체와 메틸라아제를 코드하는 플라스미드를 메틸화시켰을 때에 제한 효소 사이트가 보호되는 것으로 관찰되었다. 본 연구에서는 분자 생물학 및 후성유전학을 위한 새로운 유형의 GpC 메틸화 효소의 잠재적 활용을 위한 외향고리 DNA 메틸라제의 특성을 확인하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권6호
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• pp.2209-2213
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• 2015

DNA methylation of tumor suppressor gene promoters is the most frequent phenomenon leading to inactivation of function, consequently driving malignant cell transformation. Cyclin D2 is implicated in tumor suppression. In our study, we carried out the MSP assay to evaluation the methylation status at CpG islands in the cyclin D2 promoter in breast cancer cases from the Vietnamese population. The results showed that the frequency of methylation reached 62.1% (59 of 95 breast cancer tumors), but was low in non-cancer specimens at 10% (2 of 20 non-cancer specimens). Additionally, with an RR (relative risk) and OR (odd ratios) of 6.21 and 14.8, DNA hypermethylation of cyclin D2 increased the possibility of malignant transformation. Our results confirmed the cyclin D2 hypermethylation could be used as the potential biomarker which could be applied in prognosis and early diagnosis of Vietnamese breast cancer patients.

• Asian-Australasian Journal of Animal Sciences
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• 제21권11호
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• pp.1572-1575
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• 2008

The xenotransplantation of pig organs and cells can be related with a risk of transmission of infectious diseases to human. Previous findings indicate that the regulatory region of PERV for retroviral transcription, replication and integration into the cellular DNA is located on the 5' Long Terminal Repeat (LTR). The objective of this study is the investigation of methylation and deletion status of the PERV 5' LTR region which can be used for regulating PERV expression. We compared the sequences of genomic DNA and bisulfite-treated genomic DNA from PK-15 cells expressing PERV to observe the methylation status of the 5' LTR. Our results showed that the CpG sites of U3 were methylated and methylation was inconsistent in the R and U5 regions. Also, variable numbers of 18 bp repeats and 21 bp repeats were detected on 5' LTR by sequencing analysis. The consistent U3 methylation might be indicative of host suppression of expression of the retroviruses.

• Molecules and Cells
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• 제44권10호
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• pp.746-757
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• 2021

Plant somatic cells can be reprogrammed into a pluripotent cell mass, called callus, which can be subsequently used for de novo shoot regeneration through a two-step in vitro tissue culture method. MET1-dependent CG methylation has been implicated in plant regeneration in Arabidopsis, because the met1-3 mutant exhibits increased shoot regeneration compared with the wild-type. To understand the role of MET1 in de novo shoot regeneration, we compared the genome-wide DNA methylomes and transcriptomes of wildtype and met1-3 callus and leaf. The CG methylation patterns were largely unchanged during leaf-to-callus transition, suggesting that the altered regeneration phenotype of met1-3 was caused by the constitutively hypomethylated genes, independent of the tissue type. In particular, MET1-dependent CG methylation was observed at the blue light receptor genes, CRYPTOCHROME 1 (CRY1) and CRY2, which reduced their expression. Coexpression network analysis revealed that the CRY1 gene was closely linked to cytokinin signaling genes. Consistently, functional enrichment analysis of differentially expressed genes in met1-3 showed that gene ontology terms related to light and hormone signaling were overrepresented. Overall, our findings indicate that MET1-dependent repression of light and cytokinin signaling influences plant regeneration capacity and shoot identity establishment.

• Genomics & Informatics
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• 제20권4호
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• pp.47.1-47.13
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• 2022

Klebsiella pneumoniae is a gram-negative bacterium that is known for causing infection in nosocomial settings. As reported by the World Health Organization, carbapenem-resistant Enterobacteriaceae, a category that includes K. pneumoniae, are classified as an urgent threat, and the greatest concern is that these bacterial pathogens may acquire genetic traits that make them resistant towards antibiotics. The last class of antibiotics, carbapenems, are not able to combat these bacterial pathogens, allowing them to clonally expand antibiotic-resistant strains. Most antibiotics target essential pathways of bacterial cells; however, these targets are no longer susceptible to antibiotics. Hence, in our study, we focused on a hypothetical protein in K. pneumoniae that contains a DNA methylation protein domain, suggesting a new potential site as a drug target. DNA methylation regulates the attenuation of bacterial virulence. We integrated computational-aided drug design by using a bioinformatics approach to perform subtractive genomics, virtual screening, and fingerprint similarity search. We identified a new potential drug, koenimbine, which could be a novel antibiotic.

• Molecules and Cells
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• 제44권3호
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• pp.146-159
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• 2021

DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.

• 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.