Dynamic changes in the epigenomic state and nuclear organization of differentiating mouse embryonic stem cells
Dynamic changes in the epigenomic state and nuclear organization of differentiating mouse embryonic stem cells
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DOI：
10.1111/j.1365-2443.2007.01063.x
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发表时间：
2007-04-01
期刊：
影响因子：
2.1
通讯作者：
Abe, Kuniya
中科院分区：
文献类型：
--
作者：
Kobayakawa, Satoru;Miike, Koichiro;Abe, Kuniya
细胞核组织的变化和基因组的表观遗传状态是发育基因表达的重要驱动力。然而，一个战略，允许同时可视化的动态的表观基因组状态和核结构一直缺乏日期。我们建立了一个实验系统来观察活体小鼠胚胎干细胞（ES）的DNA甲基化。将编码人甲基CpG结合域蛋白1（MBD 1）的甲基化DNA结合域（MBD）和核定位信号（nls）序列与增强型绿色荧光蛋白（EGFP）报告基因融合，建立了携带该构建体的ES细胞系（EGFP-MBD-nls）。EGFP-MBD-nls蛋白用于在生理条件下原位跟踪DNA甲基化。我们还使用EGFP-MBD-nls监测甲基化异染色质的形成和重排。多能小鼠ES细胞显示出独特的核组织，甲基化的着丝粒异染色质聚结在核仁周围形成大簇。分化后，这些异染色质簇的组织发生了显着变化。延时显微镜成功地捕捉到了在两个分化阶段之间的过渡期间染色体定位的戏剧性变化。因此，该实验系统应有助于研究细胞核组织，表观遗传状态和细胞分化之间的关系。
Changes in nuclear organization and the epigenetic state of the genome are important driving forces for developmental gene expression. However, a strategy that allows simultaneous visualization of the dynamics of the epigenomic state and nuclear structure has been lacking to date. We established an experimental system to observe global DNA methylation in living mouse embryonic stem (ES) cells. The methylated DNA binding domain (MBD) and the nuclear localization signal (nls) sequence coding for human methyl CpG-binding domain protein 1 (MBD1) were fused to the enhanced green fluorescent protein (EGFP) reporter gene, and ES cell lines carrying the construct (EGFP-MBD-nls) were established. The EGFP-MBD-nls protein was used to follow DNA methylation in situ under physiological conditions. We also monitored the formation and rearrangement of methylated heterochromatin using EGFP-MBD-nls. Pluripotent mouse ES cells showed unique nuclear organization in that methylated centromeric heterochromatin coalesced to form large clusters around the nucleoli. Upon differentiation, the organization of these heterochromatin clusters changed dramatically. Time-lapse microscopy successfully captured a moment of dramatic change in chromosome positioning during the transition between two differentiation stages. Thus, this experimental system should facilitate studies focusing on relationships between nuclear organization, epigenetic status and cell differentiation.