Rewiring of the pluripotency enhancer network during early mammalian development

项目摘要： 多能性是单个细胞产生哺乳动物身体计划的每种细胞类型的显着能力。 早期植入胚胎的外胚层存在多能性细胞。有两种被充分描述的多能细胞 类型，早期与晚期外胚层，可以在体外模拟为幼稚胚胎干细胞与 致敏的上胚层细胞。这些细胞在表达谱方面差异很小，但在表达谱方面差异很大。 他们的表观基因组。特别地，在很大程度上不同的增强子驱动相同基因在细胞中的表达。 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一个关键方面。初步结果开始， 通过跟踪单个转录因子Grhl 2的功能来解决这个问题。Grhl 2在生长过程中上调， 胚胎干细胞向上胚层细胞的转变，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，这些基因在过渡期间不会改变表达。 对调节胚胎干细胞中相同基因的潜在增强子的评估揭示了 Klf 2/4/5相关转录因子作为幼稚状态基因的可能调节因子。其实Grhl 2是 Klf 2/4/5下调。然而，Klf 2/4/5调节了一个更大的基因网络， 比Grhl 2在启动状态下的反应要快。因此，Grhl 2似乎控制了一个 在过渡期间，Klf 2/4/5靶点的亚群被抑制，并且其他转录因子必须承担对其他转录因子的控制。 Klf 2/4/5网络的一部分。这些发现导致了一种假设，即在早期到晚期， 上胚层过渡，大的幼稚调节网络被分解成小得多的启动调节网络， 网络，为晚期上胚层细胞提供了分化分化不同体细胞谱系的灵活性， 在原肠胚形成时形成，紧接着上胚层晚期。然后每个较小的网络可以 在不同的血统中选择性地维持。事实上，Grhl 2网络被排除在原始网络之外。 条纹，同时在周围的上胚层细胞中保持表达。为了验证这个假设，有三个 明确的目标。在目标1中，使用尖端技术来识别所有Klf 2/4/5和Grhl 2驱动的增强子 分别在胚胎干细胞和上胚层细胞状态下的启动子相互作用，以直接确定 Grhl 2是否导致Klf 2/4/5靶标亚组中增强子-启动子相互作用的重新连接。在 目标2，使用生物信息学和新型生化方法来揭示额外的外胚细胞转录 重新连接Klf 2/4/5驱动网络的其他子集的因素。在aim 3中，野生型的单细胞测序 和敲除胚胎用于探索增强子体内重新布线的生物学作用。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， 调节细胞命运和细胞独特的发育潜力。

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.