Control and coordination of the maternal-to-zygotic transition

 描述（由申请人提供）：从单个受精卵发育成成体多细胞生物体需要大量细胞的增殖和分化。在许多物种中，早期受精后分裂快速而同步地发生，没有生长阶段和细胞周期检查点。这些早期胚胎几乎完全没有转录活性，因此由母体提供的RNA驱动。在囊胚中期转换（MBT），胚胎启动合子基因组的大规模转录，细胞获得生长阶段和检查点。先前的工作表明，MBT是由DNA与细胞质的比例增加引起的，这是由于重复的DNA复制和细胞分裂而没有细胞生长。这导致了这样的假设，即胚胎中存在的抑制因子的逐步滴定允许合子转录的启动。使用无细胞系统，在体外重演合子基因组激活，我们纯化了非洲爪蟾卵细胞质中存在的转录抑制活性，并确定组蛋白H3/H4作为MBT的DNA滴定抑制剂。操纵组蛋白水平定量转移合子转录和细胞周期延长在体内的发病，表现出特定的作用，染色质状态的MBT启动。这就提出了一个问题：组蛋白滴定和染色质状态如何与转录和细胞周期持续时间机械地联系起来。为了解决这一问题，我们将通过控制和组蛋白操纵的胚胎的早期发育，以前所未有的时间分辨率测量合子转录和核小体占据全基因组。我们将确定连接DNA复制和组蛋白水平的机制。这些目标的成功完成将确定全球组蛋白水平可用于协调转录和细胞分裂与发育的机制。由于激活合子转录是人类胚胎受精后的第一个主要转变，我们的工作确定如何染色质为基础的机制调节转录的启动也将提供洞察全球基因调控和早期脊椎动物胚胎发育重编程。

 DESCRIPTION (provided by applicant): The development of an adult multi-cellular organism from a single fertilized egg requires the proliferation and differentiation of a large number of cells. In many species, the early post-fertilization divisions occur rapidly and synchronously without growth phases and cell cycle checkpoints. These early embryos are almost entirely transcriptionally inactive and therefore driven by maternally supplied RNAs. At the Mid-Blastula Transition (MBT), the embryo initiates large-scale transcription from the zygotic genome and cells gain growth phases and checkpoints. Previous work suggested that the MBT is initiated by the increased DNA-to-cytoplasmic ratio resulting from repeated rounds of DNA replication and cell division without cell growth. This led to the hypothesis that the progressive titration of an inhibitory factor present in the embryo allows the initiation of zygotic transcription. Using a cel free system that recapitulates zygotic genome activation in vitro, we purified the transcriptional inhibitory activity present in the Xenopus egg cytoplasm and identified histones H3/H4 as DNA-titrated inhibitors of the MBT. Manipulating histone levels quantitatively shifts the onset of zygotic transcription and cell cycle lengthening in vivo, demonstrating a specific role for chromatin state in MBT initiation. This raises the question as to how histone titration and chromatin state are mechanistically linked to transcription and cell cycle duration. To address this, we will measure zygotic transcription and nucleosome occupancy genome wide at unprecedented temporal resolution through early development in control and histone manipulated embryos. We will determine mechanism linking DNA replication and histone levels. Successful completion of these aims will identify the mechanism through which global histone levels can be used to coordinate transcription and cell division with development. Since activating zygotic transcription is the first major transition after fertilization in human embryos our work determining how chromatin based mechanisms regulate the initiation of transcription will also provide insight global gene regulation and developmental reprogramming in the early vertebrate embryo.