5-Aza-dC在mES细胞向生殖细胞分化中的DNA甲基化调控机制

中国生物工程杂志

2011, Vol. 31

Issue (11): 44-50

研究报告

5-Aza-dC在mES细胞向生殖细胞分化中的DNA甲基化调控机制

秦洁, 漆正宇, 张艳敏, 郭新, 崔光辉, 桂耀庭

北京大学深圳医院男性生殖和遗传广东省重点实验室深圳 518036

5-Aaz-2'Deoxycitydine-induced DNA Methylation Involved in Differentiation Mouse ES Cells into Germ Cells

QIN Jie, QI Zheng-yu, ZHANG Yan-min, GUO Xin, CUI Guang-hui, GUI Yao-ting

The Key Laboratory of Male Reproductive Medicine and Genetics of Guangdong Province, Shenzhen Hospital Peking University, Shenzhen 518036, China

全文: PDF(831 KB) HTML

摘要：

目的: 探讨小鼠胚胎干细胞(mouse embryonic stem cells, mESCs)向生殖细胞(Embryonic germ cells,EG)分化过程中5-杂氮-2'-脱氧胞苷(5-Aza-2'-deoxycytidine,5-Aza-dC) 对DNA甲基化转移酶Dnmt1和Dnmt3a及生殖细胞特征基因Mvh表达变化的DNA甲基化调控机制。方法:将mES细胞分化形成拟胚体(embryoid bodies, EBs) 作为向生殖细胞分化的启动步骤,采用不同浓度(0.05μmol/L,0.1μmol/L,0.5μmol/L,1μmol/L,3μmol/L)处理EBs,RT-PCR实时荧光定量RT-PCR和Western blot分别检测检测在5-Aza-dC处理前后Dnmt1和Dnmt3a在ES细胞和EBs中的表达,甲基化特异性PCR(MSP)检测原始生殖细胞分化特征基因Mvh启动子甲基化状态。结果: 5-Aza-dC的浓度在0.05 μmol/L~1 μmol/L之间时,EBs保持较高的存活率而EBs的形态明显发生了变化;5-Aza-dC 处理后, Dnmt1和Dnmt3a在EBs中mRNA表达量明显降低,其变化特点与WB结果相一致。MSP和测序结果显示, Mvh启动子区表现为部分甲基化,5-Aza-dC 处理后的4d EBs中Mvh CpG岛有4个CG位点发生突变,而mES细胞中未见突变。结论: EBs经5-Aza-dC处理后,Dnmt1和Dnmt3a的表达明显下调;同时,Mvh启动子发生部分甲基化,有可能启动了向生殖细胞的分化进程。

关键词： DNA甲基化; 5-杂氮-2'-脱氧胞苷; 拟胚体

Abstract:

Objective: The deoxycytidine analog 5-Aza-2'-deoxycytidine(5-Aza-dC) has been widely used as a DNA methylation inhibitor to experimentally induce gene expression and cellular differentiation. The aim was to investigate role of 5-Aza-dC-induced DNA methylation involved in differentiation mouse ES cells into germ cells. Methods: To measure the sensitivity to the toxic effect of the drug, mouse ES cells-derived embryoid bodies(EBs) were exposed to various concentrations of 5-Aza-dC at 0.05μmol/L~1μmol/L drug concentration. The expression of Dnmt3a and Dnmt1 were detected by RT-PCR, real-time PCR and Western blot, CpG island methylation status of germ cell-specific gene Mvh was evaluated by methylated specific PCR(MSP) combined with sequence analyse. Results: When exposed to 5-Aza-dC, the morphology and phenotype of EBs were changed, with increasing expression of Dnmt3a and Dnmt1 in mRNA and protein levels. While the germ cell-specific gene Mvh were demethylated in EB cells, but not in mES cells after 5-Aza-dC treatment. Conclusions: The effect of 5-Aza-dC may be directly affecting mouse embryonic stem cells-derived EBs through the covalent binding of DNA methyltransferase, the expression of Mvh gene is under the control of an epigenetic mechanism mediated by DNA methylation by trapping of Dnmt1 and Dnmt3a.

Key words: DNA methylation 5-Aza-2'-deoxycytidine(5-Aza-dC) Embryoid bodies(EBs)

收稿日期: 2011-07-20 出版日期: 2011-11-25

ZTFLH:

R321.1

基金资助:

国家自然科学基金 (81100471) 、高等学校博士学科点专项科研基金(20090001120130)、深圳市科技计划项目(20110421005)、深圳市科技研发资金基础研究计划 (JC201005260214A)、深圳市科技计划重点项目(201001014)资助项目

通讯作者: 桂耀庭 E-mail: guiyaoting2007@yahoo.com.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章

引用本文:

秦洁, 漆正宇, 张艳敏, 郭新, 崔光辉, 桂耀庭. 5-Aza-dC在mES细胞向生殖细胞分化中的DNA甲基化调控机制[J]. 中国生物工程杂志, 2011, 31(11): 44-50.

QIN Jie, QI Zheng-yu, ZHANG Yan-min, GUO Xin, CUI Guang-hui, GUI Yao-ting. 5-Aaz-2'Deoxycitydine-induced DNA Methylation Involved in Differentiation Mouse ES Cells into Germ Cells. China Biotechnology, 2011, 31(11): 44-50.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I11/44

[1] Enrique S, Hatten M E. Differentiation of ES cells into cerebellar neurons. Proc Natl Acad Sci USA, 2007, 104(8): 2997-3002.

[2] Qiong G, Yoshida T, McDonald O G, et al. Concise Review: Epigenetic Mechanisms Contribute to Pluripotency and Cell Lineage Determination of Embryonic Stem Cells. Stem Cells, 2007, 25(1): 2-9.

[3] Bird A.DNA mehtylation patterns and epigenetic memory. Genes Dev, 2002, 16(1):6-21.

[4] Takebayashi S,Nakao M,Fujita N,et al. 5-Aza-2-deoxycytidine induces histone hyperacetylation of mouse centromeric heterochromatin by a mechanism independent of DNA demethylation. Biochem Biophys Res Commun, 2001, 4 (288): 921-926.

[5] Tsuji-Takayama K, Toshiya I, Yoshihiro I, et al. Demethylating agent, 5-azacytidine, reverses differentiation of embryonic stem cells. Biochemical and Biophysical Research Communications, 2004, 323 (1): 86-90.

[6] Lim M L, Vassiliev I, Richings N M, et al. A novel, efficient method to derive bovine and mouse embryonicstemcells with in vivo differentiation potential by treatment with 5-azacytidine. Theriogenology, 2011, 76(1): 133-142.

[7] Kremenskoy M, Kremenska Y, Ohgane J, et al. Genome-wide analysis of DNA methylation status of CpG islands in embryoid bodies, teratomas, and fetuses. Biochem Biophys Res Commun, 2003, 311(4): 884-890.

[8] Shiota K, Kogo Y, Ohgane J, et al. Epigenetic marks by DNA methylation specific to stem, germ and somatic cells in mice. Genes Cells, 2002, 7(9): 961-969.

[9] Maatouk D M, Kellam L D, Mann M R W, et al. DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell lineages. Development, 2006, 133(17): 3411-3418.

[10] Lee E R, McCool K W, Murdoch F E, et al. Dynamic Changes in Histone H3 Phosphoacetylation during Early Embryonic Stem Cell Differentiation Are Directly Mediated by Mitogen-and Stress-activated Protein Kinase 1 via Activation of MAPK Pathways. J Biol Chem, 2006, 281(30): 21162-21172.

[11] Qin J, Guo X, Zhang J R, et al. Cluster characterization of mES-derived EBs in four distinct developmental stages. Biologicals, 2009, 37(4): 556-564.

[12] Jones P A, Takai D. The role of DNA methylation in mammalian epigenetics. Science, 2001, 293(5532): 1068-1070.

[13] Maatouk D M, Resnick J L. Continuing primordial germ cell differentiation in the mouse embryo is a cell-intrinsic program sensitive to DNA methylation. Dev Biol, 2003, 258 (1):201-208.

[14] Enright B P, Kubota C, Yang X, et al. Epigenetic Characteristics and Development of Embryos Cloned from Donor Cells treated by Trichostatin A or 5-Aza-29-deoxycytidine. Biol Reprod, 2003, 69(3): 869-901.

[15] Li J Y, Pu M T, Hirasawa R, et al. Function of DNA Methyltransferases Dnmt3a and Dnmt3b in the Methylation of Oct4 and Nanog. Molecular and Cellular Biology, 2007, 24(27): 8748-8759.

[16] Naka H, Abe T, Hattori N, et al. Preference of DNA Methyltransferases for CpG Islands in Mouse Embryonic Stem Cells. Genome Res, 2004, 14 (9): 1733-1740.

[17] Iwatani M, Kohta I, Yuliya K, et al. Dimethyl Sulfoxide Has an Impact on Epigenetic Profile in Mouse Embryoid Body. Stem Cells, 2007, 24 (11): 2549-2556.

[18] Hubner K, Fuhrmann G, Christenson L K, et al. Derivation of Oocytes from Mouse Embryonic Stem Cells. Science, 2003, 23, 300(5623):1251-1256.

[19] Toyooka Y, Tsunekawa N, Akasu R, et al. Embryonic stem cells can form germ cells in vitro. Proc Natl Acad Sci USA, 2003, 100(20):11457-11462.

[20] Geijsen N, Horoschak M, Kim K, et al. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature, 2004, 427(6970):148-154.

[21] Toyooka Y, Tsunekawa N, Takahashi Y, et al. Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development. Mech Dev, 2000, 93(1-2):139-149.

[22] Fujiwara Y, Komiya T, Kawabata H, et al. Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage. Proc Natl Acad Sci USA, 1994, 91(25): 12258-12262.

[23] Tanaka S S, Toyooka Y, Akasu R, et al.The mouse homolog of Drosophila Vasa is required for the development of male germ cells. Genes Dev, 2000, 14(7):841-853.

[24] Noce T, Okamoto-Ito S, Tsunekawa N. Vasa homolog genes in mammalian germ cell development. Cell Struct Funct, 2001, 26(3):131-136.

[1]	满朝来, 唐高霞, 赵丽, 李凤, 甄鑫. DNA甲基化与microRNA[J]. 中国生物工程杂志, 2014, 34(8): 81-87.
[2]	徐妍, 张海玲, 徐香玲, 张会新, 姚琳, 张必弦, 王全伟. 逆境胁迫下植物DNA甲基化变异的研究进展[J]. 中国生物工程杂志, 2014, 34(10): 87-93.
[3]	马浪浪, 江舟, 黄小波, 沈亚欧, 潘光堂. 植物DNA甲基化调控研究进展[J]. 中国生物工程杂志, 2013, 33(9): 101-110.
[4]	史玉杰, 李庆贺, 刘晓辉. DNA甲基化与基因表达调控研究进展[J]. 中国生物工程杂志, 2013, 33(7): 90-96.
[5]	毛建平王全会周颖方静崔玉芳. 桥式PCR，一种简易连接DNA标签序列的方法[J]. 中国生物工程杂志, 2009, 29(11): 66-69.
[6]	何文俊叶玲玲李世崇刘红王启伟王海涛谢靖陈昭烈. 搅拌式生物反应器培养促进拟胚体的形成及其向心肌细胞分化[J]. 中国生物工程杂志, 2009, 29(11): 1-6.
[7]	杨媛媛, 王根林. 表遗传学研究进展及其应用[J]. 中国生物工程杂志, 2005, 25(S1): 95-99.
[8]	黄庆, 府伟灵, 张华, 郭颖. DNA甲基化微阵列[J]. 中国生物工程杂志, 2005, 25(10): 54-57.
[9]	王忠华, 夏英武. DNA甲基化与植物转基因沉默研究进展[J]. 中国生物工程杂志, 2001, 21(3): 23-25.

Viewed

Full text

Abstract

Cited

Shared

Discussed