Rif1 prolongs the embryonic S phase at the Drosophila mid-blastula transition.
Rif1 prolongs the embryonic S phase at the Drosophila mid-blastula transition.
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DOI：
10.1371/journal.pbio.2005687
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发表时间：
2018-05
期刊：
影响因子：
9.8
通讯作者：
O'Farrell PH
中科院分区：
文献类型：
--
作者：
Seller CA;O'Farrell PH
为了准备原肠胚形成的戏剧性形态发生事件，快速的胚胎细胞周期在囊胚中期转变（MBT）时减慢。在果蝇胚胎中，细胞周期蛋白依赖性激酶 1 (Cdk1) 活性的下调通过延迟异染色质卫星序列的复制和延长 S 期来启动这种减慢。我们发现 Cdk1 活性抑制 Rap1 相互作用因子 1 (Rif1)（一种候选复制抑制因子）的染色质关联。此外，在 MBT 中 Cdk1 下调后，Rif1 选择性地与卫星序列结合。在下一个 S 阶段，Rif1 以有序的时间表与不同的卫星分离，预计它们的复制。缺乏潜在磷酸化位点的 Rif1 无法解离并显着阻止复制的完成。突变胚胎中 Rif1 的缺失缩短了 MBT 后的 S 期，并挽救了因 S 期促进激酶、细胞分裂周期 7 (Cdc7) 耗竭而中断的胚胎细胞周期。我们的工作表明，Rif1 和 S 期激酶组成了一个复制计时器，首先控制晚期复制的发育开始，然后控制 S 期复制的精确时间表。此外，我们描述了晚期复制的开始如何适应发育过程中异染色质的逐渐成熟。大多数动物的早期胚胎中细胞分裂迅速。然而，在称为囊胚中期转变 (MBT) 的保守发育时期，细胞周期急剧减慢。在果蝇胚胎中，基因组复制突然减慢，从而启动细胞周期延长。这是通过晚期复制的开始来实现的，这是一种众所周知的现象，其中基因组的特定序列等待复制，直到其他序列完成很久之后。尽管这种复制的时间程序是 S 期持续时间的主要决定因素，但参与该过程的因素仍然未知。在这里，我们使用遗传学和实时显微镜观察发育中的果蝇胚胎中的复制，并表明蛋白质 Rap1 相互作用因子 1 (Rif1) 介导 MBT 中晚期复制的引入。我们发现，在这个阶段，Rif1 结合并选择性地延迟大块重复 DNA（称为卫星序列）的复制。在 MBT 之前的快速细胞周期中，我们发现细胞周期蛋白依赖性激酶 1 (Cdk1) 通过驱动 Rif1 从染色质中去除来阻止 Rif1 减慢 DNA 复制。 MBT 处 Cdk1 的发育下调使 Rif1 与卫星序列相关联并启动细胞周期减慢。我们的工作为 S 期的时间编程和晚期复制的胚胎起源提供了新的见解。
In preparation for dramatic morphogenetic events of gastrulation, rapid embryonic cell cycles slow at the mid-blastula transition (MBT). In Drosophila melanogaster embryos, down-regulation of cyclin-dependent kinase 1 (Cdk1) activity initiates this slowing by delaying replication of heterochromatic satellite sequences and extending S phase. We found that Cdk1 activity inhibited the chromatin association of Rap1 interacting factor 1 (Rif1), a candidate repressor of replication. Furthermore, Rif1 bound selectively to satellite sequences following Cdk1 down-regulation at the MBT. In the next S phase, Rif1 dissociated from different satellites in an orderly schedule that anticipated their replication. Rif1 lacking potential phosphorylation sites failed to dissociate and dominantly prevented completion of replication. Loss of Rif1 in mutant embryos shortened the post-MBT S phase and rescued embryonic cell cycles disrupted by depletion of the S phase–promoting kinase, cell division cycle 7 (Cdc7). Our work shows that Rif1 and S phase kinases compose a replication timer controlling first the developmental onset of late replication and then the precise schedule of replication within S phase. In addition, we describe how onset of late replication fits into the progressive maturation of heterochromatin during development. Cells divide rapidly in the early embryos of most animals. However, during a conserved period of development known as the mid-blastula transition (MBT), the cell cycle slows down dramatically. In Drosophila embryos, genome duplication abruptly slows to initiate this cell cycle prolongation. This is achieved through the onset of late replication, a well-recognized phenomenon in which specific sequences of the genome await replication until long after other sequences have finished. Even though this temporal program of replication is a major determinant of the duration of S phase, the factors involved in this process remain unknown. Here, we use genetics and real-time microscopy to visualize replication in developing fly embryos and show that the protein Rap1 interacting factor 1 (Rif1) mediates the introduction of late replication at the MBT. We find that at this stage, Rif1 binds to and selectively delays the replication of large blocks of repetitive DNA known as satellite sequences. During the rapid cell cycles before the MBT, we show that the cyclin-dependent kinase 1 (Cdk1) prevents Rif1 from slowing down DNA replication by driving its removal from the chromatin. The developmental down-regulation of Cdk1 at the MBT allows Rif1 to associate with the satellite sequences and initiate cell cycle slowing. Our work provides new insights into the temporal programming of S phase and into the embryonic origin of late replication.
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