Rewiring of the pluripotency enhancer network during early mammalian development

项目摘要： 多能性是指单个细胞产生哺乳动物身体计划的每一种细胞类型的非凡能力。 植入早期胚胎的外胚层中存在多能细胞。有两个众所周知的多能细胞 早期和晚期外胚层的类型，在体外可以被模拟为幼稚的胚胎干细胞与 分别诱导外胚层细胞。这些细胞在表达谱方面的差异很小，但在 它们表观基因组的术语。特别是，在很大程度上不同的增强子驱动相同基因在 两个州。在没有基因表达变化的情况下，广泛的增强子重新布线的原因是 未知，但似乎是早期哺乳动物发育的关键方面。初步结果开始 通过遵循单个转录因子Grhl2的功能来解决这个问题。Grhl2在过程中上调 胚胎干细胞向外胚层细胞转化并能够诱导先前潜伏的增强子完全激活 状态驱动近端基因的表达。然而，这些基因在转变过程中不会改变表达。 对调节胚胎干细胞中相同基因的潜在增强子的评估发现 KLF2/4/5相关转录因子可能是处于幼稚状态的基因的调节因子。事实上，Grhl2是 就像KLF2/4/5下调一样。然而，KLF2/4/5调控着一个更大的基因网络 这种天真的状态比Grhl2在启动状态下的状态要好。因此，Grhl2似乎承担了对 在过渡期间KLF2/4/5靶子的子集，以及其他转录因子必须承担对其他 KLF2/4/5超大型网络的一部分。这些发现导致了一种假设，即在早期到晚期 副总统过渡，大型幼稚的监管网络被分解为更小的初级监管 网络，为晚期的上胚层细胞提供了向下分化的灵活性，这些分化的体细胞谱系 在原肠胚形成时，紧跟在外胚层晚期之后。那么每个较小的网络都可以 在不同的血统中选择性地保持。事实上，Grhl2网络被排除在原语之外 条纹，但仍在周围的上胚层细胞中表达。为了检验这一假设，有三种方法 明确的目标。在目标1中，使用尖端技术来识别所有KLF2/4/5和Grhl2驱动的增强子 在胚胎干细胞和外胚层细胞状态下启动子的相互作用 Grhl2是否导致KLF2/4/5靶子之间增强子-启动子相互作用的重新连接。在……里面 目的2，使用生物信息学和新的生化方法来发现额外的外胚层细胞转录 KLF2/4/5驱动网络的其他子集重新布线的因素。在目标3中，野生型的单细胞测序 基因敲除胚胎被用来探索体内增强子重连的生物学作用。成功 该项目的完成将具有非常重要的意义，因为它将揭示基因控制的新范例 调节细胞的命运和细胞的独特发展潜力。

PROJECT ABSTRACT: Pluripotency is the remarkable ability of a single cell to give rise to every cell type of the mammalian body plan. Pluripotent cells exist in epiblast of early implantation embryos. There are two well-described pluripotent cell types, those of the early versus late epiblast that can be modeled in vitro as naïve embryonic stem cells versus primed epiblast cells respectively. These cells differ minimally in terms of their expression profiles, yet vastly in terms of their epigenomes. In particularly, largely distinct enhancers drive expression of the same genes in the two states. The reason for the extensive enhancer rewiring in the absence of gene expression changes is unknown, but appears to be a critical aspect of early mammalian development. Preliminary results begin to address this problem by following the function of a single transcription factor Grhl2. Grhl2 is upregulated during embryonic stem to epiblast cell transition and is able to induce previously latent enhancers to a fully active state driving expression of proximal genes. Yet, these genes do not change expression during the transition. Evaluation of potential enhancers regulating the same genes in the embryonic stem cells uncovered the Klf2/4/5- related transcription factors as likely regulators of the genes in the naïve state. Indeed Grhl2 is upregulated just as Klf2/4/5 is downregulated. However, Klf2/4/5 regulates a much larger network of genes in the naive state than Grhl2 does in the primed state. Therefore, it appears that Grhl2 assumes control of a subset of Klf2/4/5 targets during the transition and that other transcription factors must assume control of other parts of the very large Klf2/4/5 network. These findings led to the hypothesis that during the early to late epiblast transition, large naïve regulatory networks are broken down into much smaller primed regulatory networks, providing the late epiblast cells the flexibility to differentiate down the divergent somatic lineages that form at gastrulation, immediately following the late epiblast stage. Then each of the smaller networks can be selectively maintained among the different lineages. Indeed, the Grhl2 network is excluded from the primitive streak while remaining expressed in the surrounding epiblast cells. To test the hypothesis, there are three specific aims. In aim 1, cutting edge technologies are used to identify all Klf2/4/5 and Grhl2 driven enhancer promoter interactions in the embryonic stem and epiblast cell states respectively in order to directly determine whether Grhl2 results in the rewiring of enhancer-promoter interactions among a subset of Klf2/4/5 targets. In aim 2, bioinformatics and novel biochemical methods are used to uncover additional epiblast cell transcription factors that rewire other subsets of the Klf2/4/5 driven network. In aim 3, single cell sequencing of wild-type and knockout embryos is used to explore the biological role for enhancer rewiring in vivo. Successful completion of this project will be highly significant as it will uncover novel paradigms of gene control that regulate cell fate and a cell’s unique development potential.