• Reproductive and Developmental Biology
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• 제35권1호
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• pp.93-97
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• 2011

Previously, we reported that the osmolarity conditions in the satellite region were affected CpG DNA methylation status while Pre-1 sequence was not affected CpG DNA methylation in pNT blastocyst stage. This study was conducted to investigate the DNA methylation status of repeat sequences in pig nuclear transfer (pNT) embryos produced under different osmolarity culture conditions. Control group of pNT embryos was cultured in PZM-3 for six days. Other two treatment groups of pNT embryos were cultured in modified PZM-3 with 138 mM NaCl or 0.05 M sucrose (mPZM-3, 320 mOsmol) for two days, and then cultured in PZM-3 (270 mOsmol) for four days. The DNA methylation status of the Pre-1 sequences in blastocysts was characterized using a bisulfite-sequencing method. Intriguingly, in the present study, we found the unique DNA methylation at several non-CpG sequences at the Pre-1 sequences in all groups. The non-CpG methylation was hypermethylated in all three groups, including in vivo group (86.90% of PZM-3; 83.87% of NaCl; 84.82% of sucrose; 90.94% of in vivo embryos). To determine whether certain non-CpG methylated sites were preferentially methylated, we also investigated the methylation degree of CpA, CpT and CpC. Excepting in vivo group, preference of methylation was CpT>CpC>CpA in all three groups investigated. These results indicate that DNA methylation of Pre-1 sequences was hypermethylated in CpG as well as non-CpG site, regardless modification of osmolarity in a culture media.

• Interdisciplinary Bio Central
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• 제1권1호
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• pp.4.1-4.7
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• 2009

Introduction: Histone modifications and DNA methylation are the major factors in epigenetic gene regulation. Especially, revealing how histone modifications are related to DNA methylation is one of the challenging problems in this field. In this paper, we address this issue and propose several plausible mechanisms for precise controlling of DNA methylation status at CpG islands. Materials and Methods: To establish the regulatory relationships, we used 38 histone modification types including H2A.Z and CTCF, and DNA methylation status at CpG islands across chromosome 6, 20, and 22 of human CD4+ T cell. We utilized Bayesian network to construct regulatory network. Results and Discussion: We found several meaningful relationships supported by previous studies. In addition, our results show that histone modifications can be clustered into several groups with different regulatory properties. Based on those findings we predicted the status of methylation level at CpG islands with high accuracy, and suggested core-regulatory network to control DNA methylation status.

• Asian-Australasian Journal of Animal Sciences
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• 제31권8호
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• pp.1141-1149
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• 2018

Objective: Cluster of differentiation 4 protein (CD4) gene is an important immune related gene which plays a significant role in T cell development and host resistance during viral infection. Methods: In order to unravel the relationship of CpG island methylation level of CD4 gene with its gene expression and T lymphocyte subpopulation traits, we used one typical Chinese indigenous breed (Dapulian, DP) and one commercial breed (Landrace), then predicted the CpG island of CD4 gene, determined the methylation status of CpG sites by bisulfite sequencing polymerase chain reaction (BSP), and carried out the correlation analyses of methylation frequencies of CpG sites with mRNA expression and T lymphocyte subpopulation traits. Results: There was one CpG island predicted in the upstream -2 kb region and exon one of porcine CD4 gene, which located 333 bp upstream from the start site of gene and contained nine CpG sites. The correlation analysis results indicated that the methylation frequency of CpG_2 significantly correlated with CD4 mRNA expression in the DP and Landrace combined population, though it did not reach significance level in DP and Landrace separately. Additionally, 15 potential binding transcription factors (TFs) were predicted within the CpG island, and one of them (Jumonji) contained CpG_2 site, suggesting that it may influence the CD4 gene expression through the potential binding TFs. We also found methylation frequency of CpG_2 negatively correlated with T lymphocyte subpopulation traits CD4+CD8-CD3-, CD4-CD8+CD3- and CD4+/CD8+, and positively correlated with CD4-CD8+CD3+ and CD4+CD8+CD3+ (for all correlation, p<0.01) in DP and Landrace combined population. Thus, the CpG_2 was a critical methylation site for porcine CD4 gene expression and T lymphocyte subpopulation traits. Conclusion: We speculated that increased methylation frequency of CpG_2 may lead to the decreased expression of CD4, which may have some kind of influence on T lymphocyte subpopulation traits and the immunity of DP population.

• Asian Pacific Journal of Cancer Prevention
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• 제15권6호
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• pp.2485-2489
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• 2014

In the present study, we studied the hypermethylation of the human riboflavin transporter 2 (hRFT2) gene and regulation of protein expression in biopsies from resected tissues from Uighur cervical squamous cell carcinoma (CSCC) patients and their neighboring normal tissues. hRFT2 gene promoter region methylation sequences were mapped in cervical cancer cell line SiHa by bisulfite-sequencing PCR and quantitative detection of methylated DNA from 30 pairs of Uighur's CSCCs and adjacent normal tissues by MassARRAY (Sequenom, San Diego, CA, USA) and hRFT2 protein expression was analyzed by immunohistochemistry. In SiHa, we identified 2 CG sites methylated from all of 12CpG sites of the hRFT2 gene. Analysis of the data from quantitative analysis of single CpG site methylation by Sequenom MassARRAY platform showed that the methylation level between two CpG sites (CpG 2 and CpG 3) from CpG 1~12 showed significant differences between CSCC and neighboring normal tissues. However, the methylation level of whole target CpG fragments demonstrated no significant variation between CSCC ($0.476{\pm}0.020$) and neighboring normal tissues ($0.401{\pm}0.019$, p>0.05). There was a tendency for translocation the hRFT2 proteins from cytoplasm/membrane to nucleus in CSCC with increase in methylation of CpG 2 and CpG 3 in hRFT2gene promoter regions, which may relate to the genesis of CSCC. Our results suggested that epigenetic modifications are responsible for aberrant expression of the hRFT2 gene, and may help to understand mechanisms of cervical carcinogenesis.

• 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.

• 생명과학회지
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• 제15권5호
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• pp.802-808
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• 2005

사람의 Disabled-2 (Dab2)는 정상세포에서 c-Fos의 발현을 억제하여 세포 성장을 조절하는 암억제 유전자로 추정되어 지고 있다. 많은 암세포에서 Dab2는 유방과 난소의 정상세포에서는 발현이 되지만, 약 $85\%$의 유방과 난소의 종양세포에서는 발현이 줄어들거나, 발현이 억제되는 것으로 알려져 있다. 본 연구에서는 bisulfite 반응에 의한 염기서열 분석법과 MSP방법 등을 이용하여 유방암 세포주인 MDA MB-231세포에서 Dab2 유전자의 promoter상에 존재하는 Cpg island의 methylation된 상태를 분석하였다. 그 결과, 사람의 정상 자궁내막세포에서는 Dab2 promoter 부위가 완전하게 methylation되어 있지 않았다. 그러나 MDA MB-231세포에서는 TATA box 근처 의 CpG dinucleotide에서 비정상적으로 methylation되어 있었다. 이런 비정상적인 CpG dinucleotide의 methylation은 MDA MB-231세포를 5-azacytidine으로 처리하였을 때 methylation이 풀리고, Dab2의 발현이 회복되는 것으로 나타났다. 따라서 인간 유방암 세포주인 MBA MB-231세포에서 Dab2의 발현억제는 Dab2 유전자의 promoter부위의 CpG island의 비정상적인 methylation과 관련이 있는 것으로 여겨진다.

• Parasites, Hosts and Diseases
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• 제55권2호
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• pp.115-120
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• 2017

Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba. To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1-3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba. In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.

• 대한임상검사과학회지
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• 제41권4호
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• pp.180-188
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• 2009

Plakoglobin (PKG) is a protein linking cadherin adhesion receptors to the actin cytoskeleton and its overexpression has been known to suppress cell proliferation and tumorigenesis in thyroid cancer. We investigated the effect of 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, on the methylation status of the promoter and the expression of the plakoglobin gene in a thyroid carcinoma cell line (ARO) and papillary thyroid carceinoma. In cultures of ARO cell line incubated without 5-Aza-2'-deoxycytidine (5-Aza-CdR), five of the fifteen CpG sites in the promoter spanning -225 and -54 were methylated at 4.2 - 12.5%. When the cells were treated with 5-Aza-CdR, all the methylated CpG sites were induced to be demethylated except one. In addition, a new methylation at one CpG site, CpG4, was identified at level of 12.0%. The expression level of PKG decreased approximately 10-fold in the 5-Aza-CdR treated cells compared to untreated cells. Different pattern of promoter methylation and expression of PKG was also observed in the tissue samples. CpG10 and CpG12 sites were methylated at 9.0-27.0% in normal tissues. However, in cancer tissues, CpG5 and CpG10 sites were methylated at 10.0-22.0%. Three of ten normal thyroid tissue samples and one of thirteen papillary carcinoma tumor samples showed increased PKG mRNA expression level. PKG protein expression analyzed by the immunohistochemical staining showed higher expression in the tumor compared with normal.

• Clinical and Experimental Reproductive Medicine
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• 제37권1호
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• pp.25-31
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• 2010

목 적: X 염색체 불활성화는 여성과 남성 사이에 X 염색체의 유전자 발현 유지를 위해 여성의 X 염색체 중 하나가 불활성화 되는 현상이다. 이러한 X 염색체 불활성화는 해독되지 않는 XIST 유전자에 의해 조절된다. XIST 유전자는 오직 불활성화된 X 염색체 에서만 발현되고, 활성화된 X 염색체 에서는 발현되지 않는다. 따라서 체세포에서 활성화된 X 염색체의 XIST 유전자는 promoter 부분이 메틸화 되어있고, 불활성화된 X 염색체에서는 메틸화가 거의 되어 있지 않다. 연구방법: 본 연구에서는 정상 여성과 정상 남성의 XIST 유전자의 promoter와 5'-end 지역의 메틸화 차이를 측정하기 위해 정상여성과 남성의 혈액에서 DNA를 추출하여 파이로시퀀싱 (Pyrosequencing) 방법을 통해 XIST 유전자의 총 8부분의 CpG 영역 (-1696, -1679, -1475, -1473, -1469, +947, +956, +971)을 분석하였다. 결 과: 총 8부분의 CpG 영역을 분석한 결과, promoter 부분인 CpG 1-5 영역 (-1696, -1679, -1475, -1473, -1469)에서는 여성과 남성의 메틸화 정도에 차이가 없었다. 그러나 5'-end 부분인 CpG6-8 영역 (+947, +956, +971)에서는 여성이 45.2% 49.9% 44.2%, 남성이 90.6%, 96.7%, 87.8%으로 메틸화 정도가 차이를 나타냈다. 결 론: 따라서 본 연구에 사용한 방법은 XIST 유전자의 메틸화 패턴의 차이를 기존의 방법보다 신속하고 정확하게 분석할 수 있다는 장점이 있기 때문에 유용하게 사용될 수 있을 것이다.

• Tuberculosis and Respiratory Diseases
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• 제82권2호
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• pp.126-132
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• 2019

Background: The development of lung cancer results from the interaction between genetic mutations and dynamic epigenetic alterations, although the exact mechanisms are not completely understood. Changes in DNA methylation may be a promising biomarker for early detection and prognosis of lung cancer. We evaluated the serial changes in genome-wide DNA methylation patterns in blood samples of lung cancer patients. Methods: Blood samples were obtained for three consecutive years from three patients (2 years before, 1 year before, and after lung cancer detection) and from three control subjects (without lung cancer). We used the MethylationEPIC BeadChip method, which covers the 850,000 bp cytosine-phosphate-guanine (CpG) site, to conduct an epigenome-wide analysis. Significant differentially methylated regions (DMRs) were identified using p-values <0.05 in a correlation test identifying serial methylation changes and serial increase or decrease in ${\beta}$ value above 0.1 for three consecutive years. Results: We found three significant CpG sites with differentially methylated ${\beta}$ values and 7,105 CpG sites with significant correlation from control patients without lung cancer. However, there were no significant DMRs. In contrast, we found 11 significant CpG sites with differentially methylated ${\beta}$ values and 10,562 CpG sites with significant correlation from patients with lung cancer. There were two significant DMRs: cg21126229 (RNF212) and cg27098574 (BCAR1). Conclusion: This study revealed DNA methylation changes that might be implicated in lung cancer development. The DNA methylation changes may be the possible candidate target regions for the early detection and prevention of lung cancer.