Partial Sequencing and Characterization of Porcine DNA Methyltransferase I cDNA

Lee Y.Y.,Kim M.S.,Park J.J.,H.Y. Kang,Y.M. Chang,Yoon J.T.,K.S. Min 한국발생생물학회 2003 한국발생생물학회 학술발표대회 Vol.2003 No.1

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.

Cytoplasmic localization of oocyte-specific variant of porcine DNA methyltransferase-1 during early development

Jeong, Young Sun,Oh, Keon Bong,Park, Jung Sun,Kim, Ji-Su,Kang, Yong-Kook Wiley-Liss, Inc. 2009 Developmental dynamics Vol.238 No.7

<P>DNA methyltransferase-1 (Dnmt1) is involved in the maintenance of genomic methylation patterns. Rather than full-length Dnmt1, mouse oocytes have a truncated variant called Dnmt1o. Immunofluorescence data showed that Dnmt1o localized to the cytoplasm, but this has not been confirmed using more direct methods. The cytoplasmic localization of Dnmt1o has been assigned to the main cause of global DNA demethylation in early mouse embryos. We studied localization of Dnmt1o in mouse and pig embryos. We identified pig Dnmt1o protein and its transcript with unique 5′-end sequence. Physically separating mouse and pig 2-cell embryos into their nuclear and cytoplasmic components demonstrated that Dnmt1o of both species localized to the cytoplasm. Cloned pig embryos had Dnmt1o as the main form, with no indication of somatic Dnmt1. These findings indicate that Dnmt1o is cytoplasmic during early development; its presence in both pig and mouse embryos further suggests that Dnmt1o is conserved in mammals. Developmental Dynamics 238:1666–1673, 2009. © 2009 Wiley-Liss, Inc.</P>

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Induces Retinoic Acid Receptor β Hypermethylation through DNA Methyltransferase 1 Accumulation in Esophageal Squamous Epithelial Cells

Wang, Jing,Zhao, Shu-Lei,Li, Yan,Meng, Mei,Qin, Cheng-Yong Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.5

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.

DNA Methylation Change of Dnmt1o 5’-terminal Region in Cloned Pig during Preimplantation Development

Yeoung-Gyu Ko,Sung-woo Kim,Sang-Rae Cho,Hyun Kim,Boh-SukYang 한국동물생명공학회(구 한국동물번식학회) 2011 발생공학 국제심포지엄 및 학술대회 Vol.2011 No.1

There are replete numbers of reports which have apparently shown that established patterns of methylation are critical for normal mammalian development. DNA methyltransferase 1 (Dnmt1) gene contains three different isoform transcripts, Dnmt1s, Dnmt- 1o, 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.

Estrogen이 전신홍반루푸스 환자 B세포의 DNA Methylation에 미치는 영향

박미경 ( Mi Kyung Park ),박성환 ( Sung Hwan Park ),곽승기 ( Seung Ki Kwok ),조미라 ( Mi La Cho ),김호연 ( Ho Youn Kim ) 대한류마티스학회 2007 대한류마티스학회지 Vol.14 No.1

Objective: Epigenetics is an important, alternative mechanism of gene regulation that is independent of the nucleotide sequences of DNA. We investigated mRNA levels for DNA methyltransferase-1 (DNMT-1), and the effect of estrogen on the expression of DNMT-1 level in T cells and B cells from patients with systemic lupus erythematosus (SLE) and healthy subjects, and assessed the possible etiological role of DNA methylation in the pathogenesis of SLE. Methods: mRNA levels for DNMT-1 in CD4+ T cells and CD19+ B cells from 37 patients with SLE and 12 healthy controls were examined using RT-PCR. We used specific primer for DNMT-1 and β actin, The effect of estrogen on the DNA methylation was measured by the mRNA level of DNMT-1 CD4+ T cells and CD19+ B cells treated with 100 nM of 17 β-estradiol for 72 hour. Results: The levels of DNMT-1 mRNA were significantly lower in CD4+ T cells and CD19+ B cells from SLE patients compared with healthy controls. We observed the suppression of the levels of DNMT-1 mRNA by stimulated with estrogen in patients with SLE patients, especially in CD19+B cells. DNA hypomethylation of B cells was tend to be correlated with the level of anti-ds DNA antibody without statistical significance (r=-0.43, p=0.3). Conclusion: Our observations suggest that suppression of DNMT-1 by estrogen in B cells from patients with SLE might be related to the pathogenesis of SLE. Epigenetic studies may provide clues for developing new treatment strategies of SLE.

MicroRNA-148a Can Regulate Runt-Related Transcription Factor 3 Gene Expression via Modulation of DNA Methyltransferase 1 in Gastric Cancer

Junbo Zuo,Hong Zhou,Jiazeng Xia,Feng Ju,Jiang Yan,Akao Zhu,Shimao Jin,Ting Shan 한국분자세포생물학회 2013 Molecules and cells Vol.35 No.4

Underexpression of the gene runt-related transcription fac-tor 3 (RUNX3), an important tumor suppressor, is known to contribute to gastric cancer progression. However, the mechanism underlying aberrant RUNX3 expression has not been fully elucidated. We investigated the role of microRNA-148a (miR-148a) and DNA methyltransferases (DNMTs) in RUNX3 promoter methylation and gene expression. RUNX3 mRNA, RUNX3 protein, and methylation levels were assayed in human gastric cancer tissues and matched normal tissues, and AGS and BGC-823 cells by real-time reverse transcription PCR, Western blot, and methylation-specific PCR, respectively. A correlation between RUNX3 mRNA levels and that of miR-148a was also investigated in gastric cancer tissues. We found that RUNX3 mRNA levels were significantly downregulated in gastric cancer tissues compared with their matched normal tissues, and were closely associated with miR-148a expression. After treatment of human gastric cancer AGS and BGC-823 cells with the DNA methylation inhibitor 5-aza-2’-deoxycytidine, a significant increase in RUNX3 mRNA, RUNX3 protein, and the non-methylated form of the RUNX3 promoter were observed relative to untreated cells. Enforced expression of miR-148a, which can modulate DNMT1 and DNMT3B, also increased the expression of RUNX3 in gastric cancer cells. Knockdown of DNMT1 was associated with increased levels of RUNX3 mRNA and RUNX3 protein, while knockdown of DNMT3B did not have any effect on these in BGC-823 cells. Our results show that miR-148a may regulate RUNX3 expression through modulation of DNMT1-dependent DNA methylation in gastric cancer and highlight a miRNA-epigenetics regulation mechanism of gene expression.

Elevated expression of DNMT1 is associated with increased expansion and proliferation of hematopoietic stem cells co-cultured with human MSCs

Moharram Ahmadnejad,Naser Amirizadeh,Roya Mehrasa,Ahmad Karkhah,Mahin Nikougoftar,Arezoo Oodi 대한혈액학회 2017 Blood Research Vol.52 No.1

Background: Mesenchymal stem cells (MSCs) play an important role in hematopoietic stem cell (HSC) maintenance, proliferation, and apoptosis. DNA methyltransferase 1 (DNMT1) is consid-ered an essential factor in the maintenance of HSCs in mammalian cells. Therefore, this study was conducted to evaluate the mRNA expression level of DNMT1 during cord blood (CB)-HSC ex vivo expansion with MSCs. Methods: Ex vivo cultures of CB-HSCs were performed in three culture conditions for 7 days: cyto-kines, cytokines with MSCs, and only MSCs. Total and viable cell numbers were counted after 5 and 7 days using trypan blue stain, and the stem cell percentage was then evaluated by flow cytometry. Moreover, in vitro colony-forming unit assay was carried out to detect clonogenic potential of HSCs at days 0 and 7 using MethoCult H4434. Finally, DNMT1 mRNA expression level was evaluated by real-time polymerase chain reaction. Results: Maximum CB-CD34+ cell expansion was observed on day 7 in all the three cultures. After 7 days, ex vivo expansion of CB-CD34+ cells indicated a significant decrease in DNMT1 expression in the cytokine cultures, whereas in the two co-culture conditions DNMT1 ex-pression was increased. A significant difference between the number of CD34+ and CD34−cells in the cytokine co-culture system was observed. Conclusion: These data indicated that an elevated expression of DNMT1 is associated with increased expansion and proliferation of HSCs co-cultured with human MSCs. Hence, DNMT1 may be a potential factor in the maintenance of expanded HSCs co-cultured with human MSCs.

Malignant Glioma with Neuronal Marker Expression : A Clinicopathological Study of 18 Cases

Kim, Hong Rye,Lee, Jae Jun,Lee, Jung-Il,Nam, Do Hyun,Suh, Yeon-Lim,Seol, Ho Jun The Korean Neurosurgical Society 2016 Journal of Korean neurosurgical society Vol.59 No.1

Objective : Malignant gliomas with neuronal marker expression (MGwNM) are rare and poorly characterized. Increasingly diverse types of MGwNM have been described and these reported cases underscore the dilemmas in the classification and diagnosis of those tumors. The aim of this study is to provide additional insights into MGwNM and present the clinicopathological features of 18 patients. Methods : We reviewed the medical records of 18 patients diagnosed as MGwNM at our institute between January 2006 and December 2012. Macroscopic total resection was performed in 11 patients (61%). We evaluated the methylation status of $O^6$-methylguanine-DNA methyltransferase (MGMT) and expression of isocitrate dehydrogenase 1 (IDH-1) in all cases, and deletions of 1p and 19q in available cases. Results : The estimated median overall survival was 21.2 months. The median progression-free survival was 6.3 months. Six patients (33%) had MGMT methylation but IDH1 mutation was found in only one patient (6%). Gene analysis for 1p19q performed in nine patients revealed no deletion in six, 19q deletion only in two, and 1p deletion only in one. The extent of resection was significantly correlated with progression free survival on both univariate analysis and multivariate analysis (p=0.002 and p=0.013, respectively). Conclusion : In this study, the overall survival of MGwNM was not superior to glioblastoma. The extent of resection has a significant prognostic impact on progression-free survival. Further studies of the prognostic factors related to chemo-radio therapy, similar to studies with glioblastoma, are mandatory to improve survival.

Dynamic methylation pattern of the methyltransferase1o (Dnmt1o) 5′‐flanking region during mouse oogenesis and spermatogenesis

Ko, Yeoung‐,Gyu,Yun, Jisoo,Park, Hyoung Joon,Tanaka, Satoshi,Shiota, Kunio,Cho, Jae‐,Hyeon Wiley Subscription Services, Inc., A Wiley Company 2013 Molecular reproduction and development Vol.80 No.3

<P><B>Abstract</B></P><P>DNA methyltransferase1o (<I>Dnmt1o</I>), which is specific to oocyte and preimplantation embryo, plays a role in maintaining DNA methylation in mammalian cells. Here, we investigated the methylation status of CpGs sites in the <I>Dnmt1o</I> 5′‐flanking region in germ cells at different stages of oogenesis or spermatogenesis. The methylation levels of the CpG sites at the 5′‐flanking regions were hypermethylated in growing oocytes of all follicular stages, while the oocytes in meiotic metaphase II (MII) were demethylated. The methylation pattern within the CpGs sites in the 5′‐flanking region, however, was dramatically changed during spermatogenesis. We observed that there was significant non‐CpG methylation both in MII oocytes and spermatocytes. Although a low methylation level in non‐CpG sites was observed in primary and secondary oocytes, the CpA site of position 25 and CpT site of position 29 within the no‐CpG region in the 5′‐flanking region of <I>Dnmt1o</I> was highly methylated in MII oocytes. During spermatogenesis, the low degree of methylation at CpG sites in spermatocytes increased to a higher degree in sperm, while the high ratio of methylation in non‐CpG sites in spermatocytes decreased. Together, germ cells showed inverted methylation patterns between CpG and non‐CpG sites in the <I>Dnmt1o</I> 5′‐upstream region, and the methylation pattern during oogenesis did not drastically change, remaining generally hypomethylated at the MII stage. Mol. Reprod. Dev. 80: 212–222, 2013. © 2013 Wiley Periodicals, Inc.</P>

돼지 체세포 복제란 초기발달 과정 중 Dnmt1o상류 영역의 다이내믹한 DNA 메틸화 변화

고응규,김성우,조상래,도윤정,김재환,김상우,김현,박재홍,박수봉 한국동물번식학회 2012 Reproductive & developmental biology Vol.36 No.1