Rewiring of the pluripotency enhancer network during early mammalian development

早期哺乳动物发育过程中多能性增强子网络的重新布线

• 批准号: 9403957
• 负责人: Robert Blelloch
• 金额: $ 41.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-07 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9403957
• 关键词: Acetylation; Address; Alpha Cell; Attention; Automobile Driving; Avidin; Binding; Biochemical; Bioinformatics; Biological; Biological Process; Biotinylation; Cell Line; Cell Lineage; Cells; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Data; Development; Disease; Embryo; Embryonic Development; Employee Strikes; Enhancers; Epiblast; Epithelial; Epithelial Cells; Evaluation; Event; Fostering; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genome; Genomic approach; Goals; Growth and Development function; Health; Human; In Vitro; Individual; Knock-out; Knowledge; Maintenance; Measures; Mesenchymal; Methods; Methylation; Mission; Molecular; Molecular Profiling; Monitor; Nucleosomes; Organism; Overlapping Genes; Post-Translational Protein Processing; Preparation; Primitive Streaks; Proteins; Public Health; Recruitment Activity; Regulation; Regulator Genes; Research; Role; Stem cells; Techniques; Technology; Testing; Therapeutic; Transcriptional Regulation; United States National Institutes of Health; cell type; cohesin; embryonic stem cell; epigenome; experimental study; flexibility; gastrulation; improved; in vitro Model; in vivo; interest; mammalian genome; natural Blastocyst Implantation; novel; pluripotency; premature; programs; promoter; public health relevance; single cell sequencing; stem; transcription factor

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.

项目摘要： 多能性是单个细胞产生哺乳动物身体结构的每种细胞类型的非凡能力。 多能细胞存在于早期植入胚胎的外胚层中 有两种已被充分描述的多能细胞。 类型，早期与晚期外胚层的类型，可以在体外建模为幼稚胚胎干细胞与 这些细胞分别在表达谱上差异很小，但在表达谱上差异很大。 特别是，就其表观基因组而言，很大程度上不同的增强子驱动相同基因的表达。 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，但初步结果似乎是早期哺乳动物发育的一个关键方面。 通过追踪单个转录因子 Grhl2 的功能来解决这个问题。 胚胎干到外胚层细胞的转变，并且能够诱导先前潜在的增强子完全活跃 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揭示了 事实上，Grhl2 可能是 Klf2/4/5 相关转录因子的基因调节因子。 正如 Klf2/4/5 下调一样，Klf2/4/5 调节更大的基因网络。 幼稚状态比 Grhl2 在启动状态下更明显，因此，Grhl2 似乎控制了 a。 转变过程中 Klf2/4/5 目标的子集，其他转录因子必须承担对其他转录因子的控制 这些发现导致了这样的假设：在早期到晚期。 外胚层转变，大型幼稚调控网络被分解为更小的启动调控网络 网络，为晚期外胚层细胞提供了分化不同体细胞谱系的灵活性， 在原肠胚形成时，紧接着外胚层晚期阶段，然后每个较小的网络都可以形成。 事实上，Grhl2 网络被排除在原始网络之外。 条带，同时在周围的外胚层细胞中保持表达为了检验该假设，存在三种情况。 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。 分别在胚胎干细胞和外胚层细胞状态下启动子相互作用，以便直接确定 Grhl2 是否会导致 Klf2/4/5 靶标子集之间增强子-启动子相互作用的重新连接。 目标 2，使用生物信息学和新颖的生化方法来揭示额外的外胚层细胞转录 重新连接 Klf2/4/5 驱动网络的其他子集的因素 在目标 3 中，野生型的单细胞测序。 敲除胚胎用于探索增强子在体内重新布线的生物学作用。 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式 调节细胞命运和细胞独特的发育潜力。