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首页> 外文期刊>Cell cycle >Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification
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Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification

机译：在人类多能干细胞谱系规范过程中复制灶的时空重组与复制域合并

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Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification.
机译：小鼠和人类多能干细胞（PSC）的谱系规范都伴随着染色体结构域的空间整合和其复制时机的暂时整合。复制时序和染色质组织都在G1阶段的时序决策点（TDP）上建立。在这里，我们已经开发了活细胞成像工具来追踪分化过程中的时空复制域整合。首先，我们证明了荧光泛素化细胞周期指示剂（Fucci）系统无法划分G1 / S或G2 / M细胞周期转换。取而代之的是，我们采用荧光PCNA来监视S期进程，通过胞质分裂来区分有丝分裂，并使用荧光核苷酸来标记早期和晚期复制灶，并在所有细胞周期转换过程中跟踪其3D组织进入亚核染色质区室的组合。我们发现，随着人类PSC的分化，致力于空间簇状复制灶复制的S期长度增加，这与结构域向染色质的时间簇状块的整体区室化相吻合。重要的是，结构域的重新定位和锚定在S期开始之前就已经完成，即使在缩写为PSC G1期的情况下也是如此。该方法也可用于研究单个PSC中的细胞命运转变，可以看出它优先与G1期区分开。总之，我们的结果建立了实时的活细胞成像方法，用于跟踪人PSC分化过程中的细胞周期转变，该方法可用于研究染色体结构域整合和谱系规格的其他方面。

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