小鼠早期胚胎中DNA羟甲基化的动态变化及Tet蛋白的功能

发布时间：2018-03-13 14:08

  本文选题：小鼠　切入点：胚胎　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：DNA去甲基化参与哺乳动物的诸多生命过程,在早期胚胎发育和原始生殖细胞生成过程中发挥重要作用。在过去几十年的研究中有关DNA主动去甲基化的机制引发了很多讨论和关注,但很多观点仍存在争议。Tet蛋白以及它的氧化产物5hmC的发现,为DNA主动去甲基化机制的研究开辟了全新的视野。5hmC是重要的表观遗传标记,它主要作为一种中间产物,参与DNA去甲基化过程。本论文研究了Tet蛋白在小鼠早期胚胎DNA去甲基化过程中的作用,为深入了解Tet蛋白的功能提供了理论基础,并且可进一步了解小鼠早期胚胎发育过程中DNA主动去甲基化所依赖的机制。本研究首先系统验证了小鼠早期胚胎发育过程中5mC和5hmC在体内受精胚胎和孤雌激活胚胎中的定位及动态变化趋势,发现：受精卵中5mC和5hmC呈不对称分布,从PN1到PN5时期雌原核5mC水平缓和下降,而5hmC水平缓和上升,雄原核5mC水平剧烈下降,而5hmC水平剧烈上升。孤雌激活胚胎在原核期发育阶段仅有少量的DNA 5hmC发生,但在2-细胞期出现剧烈的DNA 5hmC重编程,说明父本基因组大量的5hmC生成主要发生于1-细胞时期,而母本基因组大量的5hmC生成主要发生于2-细胞时期。进一步检测发现2-细胞时期Stella含量会显著下降,这可能减弱了其对母本基因组的保护作用从而有利于Tet蛋白发挥其羟甲基化的作用。从2-细胞期到囊胚期发育过程中,孤雌激活胚胎和体内受精胚胎在5mC、5hmC以及Tet家族基因表达上呈现类似的动态变化模式,但孤雌激活胚胎的5hmC水平和Tet基因表达水平普遍低于体内受精胚胎,显示出二者在DNA甲基化重编程过程中的差异。通过进一步的研究发现,无论是孤雌激活胚胎还是体内受精胚胎,2一细胞晚期的5hmC水平显著高于早期,说明在2-细胞胚胎发育过程中有大量的5hmC的生成。为了研究这一现象与Tet蛋白的相关性,通过RNAi实验下调Tet基因的表达发现：孤雌激活2-细胞胚胎和受精2-细胞胚胎DNA 5hmC的生成均受Tet基因的调控,在孤雌激活胚胎中Tetl的作用更强,而在体内受精胚胎中Tetl和Tet3的作用更强。说明Tet基因不仅介导1-细胞胚胎DNA 5hmC的生成,亦可介导2-细胞胚胎DNA 5hmC的生成。同时RNAi实验还发现,Tet基因的下调会显著影响胚胎发育的囊胚率,说明Tet家族在胚胎发育过程中发挥比较重要的作用。最后本实验利用体细胞核移植技术,将小鼠胎儿成纤维细胞转入小鼠2-细胞胚胎细胞中,发现小鼠胎儿成纤维细胞的5hmC水平显著提高,说明小鼠2-细胞胚胎细胞质仍然具有调控DNA5hmC重编程的能力。综上所述,父本基因组大量的5hmC生成主要发生于1-细胞时期,母本基因组大量的5hmC生成主要发生于2-细胞时期。Tet基因不仅参与1-细胞时期5hmC的生成,也参与2-细胞时期5hmC的生成。2-细胞胚胎细胞质具有调控5hmC重编程的能力。本研究结果为了解Tet蛋白家族功能及哺乳动物早期胚胎DNA去甲基化机制提供了新的信息。
[Abstract]:Many life processes DNA demethylation in mammals, play an important role in early embryonic development and primordial germ cell formation. In the past several decades of research on the mechanism of DNA initiative to methylation caused a lot of discussion and attention, but many ideas are still controversial.Tet protein and found its oxidation product 5hmC the study of DNA active demethylation mechanism opens up a new field of.5hmC are important epigenetic marks, it is mainly used as an intermediate product, participate in DNA demethylation process. This paper research the Tet protein demethylation function in the process of early mouse embryo DNA, provides a theoretical basis for the understanding the function of Tet protein, and to further understand the mechanism of DNA active demethylation on mouse early embryos. In this study, the system verifies the early mouse embryo 5mC and 5hmC during the development of embryo in vivo and parthenogenetic activation and trends in embryos found that asymmetric distribution of 5mC and 5hmC were fertilized eggs, from PN1 to PN5 during the period of the female pronucleus 5mC level decreased and 5hmC level ease, ease up, the male pronucleus 5mC level in severe decline, and the level of 5hmC rises sharply. The parthenogenetic embryos had only a small amount of DNA 5hmC in prokaryotic developmental stage, but in 2- cell cycle DNA 5hmC reprogramming dramatically, indicating that 5hmC generates a large number of paternal genome occurred mainly in the 1- cell stage and 5hmC generated a large number of maternal genome occurred mainly in the 2- cell stage further. Detection of 2- cells during the Stella content significantly decreased, which may weaken the protective effect of the maternal genome to Tet protein play its polymethylolated role. From the 2- cell stage to blastocyst development. In the process of embryo and in vivo fertilized embryos in 5mC parthenogenetic activation, expression of 5hmC and Tet genes showed similar patterns of dynamic changes, but the parthenogenetic activation level of 5hmC and Tet gene expression levels are generally lower than embryos fertilized embryos in vivo, showed two differences in DNA methylation reprogramming through further process. The study found that both embryos and parthenogenetic activation in vivo fertilized embryos, 2 cells in late 5hmC level was significantly higher than that in early generation of a large number of 5hmC in 2- cells during embryonic development. In order to study the correlation between this phenomenon and Tet protein, the down-regulation of Tet gene expression by RNAi experiments that generate 2- cell embryo and fertilization embryo 2- cells DNA 5hmC parthenogenetic activation are regulated by Tet gene, in stronger embryo Tetl parthenogenetic activation, and Tetl in the embryo in vivo and Tet3 for more That generation of Tet gene is not only mediated by 1- cell embryos DNA 5hmC, can generate 2- cells mediated by DNA 5hmC RNAi. At the same time, embryo experiments also found that down-regulation of Tet gene may influence embryo development rate of blastocyst, indicating that Tet family play a more important role in the embryonic development process. At the end of the experiment using the technology somatic cell nuclear transplantation, mouse fetal fibroblast cells into mouse 2- cells in embryonic cells, found that mice fetal fibroblast cells significantly increased the level of 5hmC, indicating that 2- cells of Mice Embryonic cytoplasm still has the regulation of DNA5hmC reprogramming. In summary, 5hmC generates a large number of paternal genome occurs mainly in 1- cells, 5hmC genome of creation a large number of mainly generated in.Tet 2- cells in 1- cell 5hmC gene is not only involved in the period of the generation of.2- cells from embryonic 5hmC 2- cells are also involved in the fine period Cytoplasm has the ability to regulate 5hmC reprogramming. The results provide new information for understanding the function of Tet protein family and the mechanism of DNA demethylation in early mammalian embryos.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q954.4
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本文编号：1606710