Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, have been identified in humans and mice to contribute to the methylation of unmodified DNA. We recently showed a transition of de novo DNA methyltransferase expre...Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, have been identified in humans and mice to contribute to the methylation of unmodified DNA. We recently showed a transition of de novo DNA methyltransferase expression from Dnmt3b to Dnmt3a during mouse embryogenesis and in tissue-specific stem cells, suggesting distinct functions of Dnmt3a and Dnmt3b during these processes. In this study, to characterize the functions of Dnmt3a and Dnmt3b in pluripotent stem cells, we exogenously transfected ES cells with Dnmt3a and Dnmt3b cDNAs linked to an internal ribosome entry site-green fluorescent protein gene, and then analyzed the effects of expression of these de novo DNA methyltransferases on ES cell growth and differentiation. ES cells expressing Dnmt3b showed specific downregulation of pluripotency marker genes such as Nanog and Oct 3/4. In addition, Dnmt3a-transfected ES cells showed a specific increase in mitotic index, while Dnmt3b-transfected ES cells showed a decrease in mitotic index. These results suggest that Dnmt3b has important physiological roles in the initial process of stem cell differentiation and that Dnmt3a has a function in stem cell proliferation.展开更多
BACKGROUND: Alterations in DNA methylation occur during the pathogenesis of human tumors. In this study, we investigated the influence of DNA methyltransferase 3b (DNMT3b) on fragile histidine trial (FHIT) expression ...BACKGROUND: Alterations in DNA methylation occur during the pathogenesis of human tumors. In this study, we investigated the influence of DNA methyltransferase 3b (DNMT3b) on fragile histidine trial (FHIT) expression and on DNA methylation of the FHIT promoter region in the hepatoma cell line SMMC-7721. METHODS: DNMT3b siRNA was used to down-regulate DNMT3b expression. DNMT3b and FHIT proteins were determined by Western blotting. Methylation-specific PCR was used to analyze the methylation status of the FHIT gene. RESULTS: After DNMT3b siRNA transfection, the expression of DNMT3b was inhibited in SMMC-7721 cells, and the expression of FHIT was significantly higher than that in the control group. There was no significant difference in methylation status between the DNMT3b siRNA transfected cells and control cells. CONCLUSION: DNMT3b may play an important role in regulation of FHIT expression in hepatoma SMMC-7721 cells, but not through methylation of the FHIT promoter. (Hepatobiliary Pancreat Dis Int 2009; 8: 273-277)展开更多
目的构建反义DNA甲基化酶3b(DNM T 3b)基因片断真核表达载体,为研究DNM T 3b基因功能提供工具。方法根据DNM T 3b基因cDNA序列中编码序列设计PCR引物,在上下游引物5′端分别添加X baⅠ和K pnⅠ酶切位点,RT-PCR从胆管癌细胞QBC-939中获得...目的构建反义DNA甲基化酶3b(DNM T 3b)基因片断真核表达载体,为研究DNM T 3b基因功能提供工具。方法根据DNM T 3b基因cDNA序列中编码序列设计PCR引物,在上下游引物5′端分别添加X baⅠ和K pnⅠ酶切位点,RT-PCR从胆管癌细胞QBC-939中获得485bp的DNA片断;将该片断反向插入真核表达载体pcDNA 3.1(+)的多克隆位点,构建反义DNM T 3b基因片断真核表达载体,并用PCR、酶切法和DNA测序鉴定。结果PCR鉴定得到467bp特异条带,双酶切鉴定得到471bp片断和5.4kb载体片断,DNA测序说明插入片断序列正确。结论本研究构建的反义DNM T 3b基因片断真核表达载体可为进一步研究DNM T 3b基因功能提供实验工具。展开更多
文摘Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, have been identified in humans and mice to contribute to the methylation of unmodified DNA. We recently showed a transition of de novo DNA methyltransferase expression from Dnmt3b to Dnmt3a during mouse embryogenesis and in tissue-specific stem cells, suggesting distinct functions of Dnmt3a and Dnmt3b during these processes. In this study, to characterize the functions of Dnmt3a and Dnmt3b in pluripotent stem cells, we exogenously transfected ES cells with Dnmt3a and Dnmt3b cDNAs linked to an internal ribosome entry site-green fluorescent protein gene, and then analyzed the effects of expression of these de novo DNA methyltransferases on ES cell growth and differentiation. ES cells expressing Dnmt3b showed specific downregulation of pluripotency marker genes such as Nanog and Oct 3/4. In addition, Dnmt3a-transfected ES cells showed a specific increase in mitotic index, while Dnmt3b-transfected ES cells showed a decrease in mitotic index. These results suggest that Dnmt3b has important physiological roles in the initial process of stem cell differentiation and that Dnmt3a has a function in stem cell proliferation.
基金supported by a grant from the National Natural Science Foundation of China(No.30571814)
文摘BACKGROUND: Alterations in DNA methylation occur during the pathogenesis of human tumors. In this study, we investigated the influence of DNA methyltransferase 3b (DNMT3b) on fragile histidine trial (FHIT) expression and on DNA methylation of the FHIT promoter region in the hepatoma cell line SMMC-7721. METHODS: DNMT3b siRNA was used to down-regulate DNMT3b expression. DNMT3b and FHIT proteins were determined by Western blotting. Methylation-specific PCR was used to analyze the methylation status of the FHIT gene. RESULTS: After DNMT3b siRNA transfection, the expression of DNMT3b was inhibited in SMMC-7721 cells, and the expression of FHIT was significantly higher than that in the control group. There was no significant difference in methylation status between the DNMT3b siRNA transfected cells and control cells. CONCLUSION: DNMT3b may play an important role in regulation of FHIT expression in hepatoma SMMC-7721 cells, but not through methylation of the FHIT promoter. (Hepatobiliary Pancreat Dis Int 2009; 8: 273-277)
文摘目的构建反义DNA甲基化酶3b(DNM T 3b)基因片断真核表达载体,为研究DNM T 3b基因功能提供工具。方法根据DNM T 3b基因cDNA序列中编码序列设计PCR引物,在上下游引物5′端分别添加X baⅠ和K pnⅠ酶切位点,RT-PCR从胆管癌细胞QBC-939中获得485bp的DNA片断;将该片断反向插入真核表达载体pcDNA 3.1(+)的多克隆位点,构建反义DNM T 3b基因片断真核表达载体,并用PCR、酶切法和DNA测序鉴定。结果PCR鉴定得到467bp特异条带,双酶切鉴定得到471bp片断和5.4kb载体片断,DNA测序说明插入片断序列正确。结论本研究构建的反义DNM T 3b基因片断真核表达载体可为进一步研究DNM T 3b基因功能提供实验工具。
