Geminin Is Required for Zygotic Gene Expression at the Xenopus Mid-Blastula Transition

In many organisms early development is under control of the maternal genome and zygotic gene expression is delayed until the mid-blastula transition (MBT). As zygotic transcription initiates, cell cycle checkpoints become activated and the tempo of cell division slows. The mechanisms that activate zygotic transcription at the MBT are incompletely understood, but they are of interest because they may resemble mechanisms that cause stem cells to stop dividing and terminally differentiate. The unstable regulatory protein Geminin is thought to coordinate cell division with cell differentiation. Geminin is a bi-functional protein. It prevents a second round of DNA replication during S and G2 phase by binding and inhibiting the essential replication factor Cdt1. Geminin also binds and inhibits a number of transcription factors and chromatin remodeling proteins and is thought to keep dividing cells in an undifferentiated state. We previously found that the cells of Geminin-deficient Xenopus embryos arrest in G2 phase just after the MBT then disintegrate at the onset of gastrulation. Here we report that they also fail to express most zygotic genes. The gene expression defect is cell-autonomous and is reproduced by over-expressing Cdt1 or by incubating the embryos in hydroxyurea. Geminin deficient and hydroxyurea-treated blastomeres accumulate DNA damage in the form of double stranded breaks. Bypassing the Chk1 pathway overcomes the cell cycle arrest caused by Geminin depletion but does not restore zygotic gene expression. In fact, bypassing the Chk1 pathway by itself induces double stranded breaks and abolishes zygotic transcription. We did not find evidence that Geminin has a replication-independent effect on transcription. We conclude that Geminin is required to maintain genome integrity during the rapid cleavage divisions, and that DNA damage disrupts zygotic gene transcription at the MBT, probably through activation of DNA damage checkpoint pathways.

在多数生物中，早期发育受母源基因组（maternal genome）调控，合子基因表达（zygotic gene expression）会延迟至囊胚中期转换（mid-blastula transition, MBT）阶段。随着合子转录（zygotic transcription）启动，细胞周期检验点（cell cycle checkpoints）被激活，细胞分裂速率随之放缓。目前学界对MBT阶段激活合子转录的调控机制尚未完全阐明，但该方向具有重要研究价值，因为其调控机制或与干细胞停止分裂并发生终末分化的过程存在相似性。不稳定调控蛋白Geminin（Geminin）被认为可协调细胞分裂与细胞分化的进程。Geminin是一种双功能蛋白：它可通过结合并抑制必需的复制因子Cdt1，阻断S期与G2期内DNA发生二次复制；同时还可结合并抑制多种转录因子与染色质重塑蛋白，被认为能够维持分裂细胞的未分化状态。我们此前的研究发现，Geminin缺陷型非洲爪蟾（Xenopus）胚胎的细胞会在MBT结束后即刻于G2期发生周期停滞，随后在原肠胚形成初期发生裂解。本研究证实，此类胚胎同时无法表达绝大多数合子基因。该基因表达缺陷具有细胞自主性，且可通过过表达Cdt1或将胚胎置于羟基脲（hydroxyurea）环境中得以重现。Geminin缺陷型胚胎与经羟基脲处理的胚泡细胞会以DNA双链断裂的形式积累基因组损伤。绕过Chk1通路可克服Geminin缺失引发的细胞周期停滞，但无法恢复合子基因的正常表达。事实上，仅单独绕过Chk1通路本身就会诱导DNA双链断裂的产生，并阻断合子转录进程。我们未发现Geminin对转录存在不依赖于DNA复制的调控效应的相关证据。综上，Geminin对于快速卵裂阶段维持基因组完整性至关重要；而DNA损伤或通过激活DNA损伤检验点通路，干扰MBT阶段的合子基因转录。