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: 9754842
• 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/9754842
• 关键词: Acetylation; Address; 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; Enhancers; Epiblast; Epithelial; Epithelial Cells; Evaluation; Event; Expression Profiling; Fostering; Gene Expression; Gene Expression Regulation; 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; Monitor; Nucleosomes; Organism; Overlapping Genes; Post-Translational Protein Processing; Preparation; Primitive Streaks; Proteins; Public Health; 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; recruit; 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计划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层细胞中。有两个描述的多能细胞 类型，早期和晚期的尤其是可以在体外建模为幼稚的胚胎干细胞而不是类型的类型 分别启动的其中膜细胞。这些细胞在其表达曲线方面差异很小，但在 他们的表观人体学条款。特别是，在很大程度上不同的增强剂驱动相同基因的表达 两个州。在没有基因表达变化的情况下，大量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的功能来解决此问题。 GRHL2在 胚胎茎到层细胞过渡，能够诱导以前的潜在增强子 近端基因的状态驱动表达。然而，这些基因在过渡过程中不会改变表达。 评估在胚胎干细胞中调查相同基因的潜在增强剂发现 KLF2/4/5-相关转录因子与幼稚状态的基因调节剂一样。确实是Grhl2 正如KLF2/4/5的上调一样。但是，KLF2/4/5调节了更大的基因网络 与Grhl2相比，天真状态在启动状态下。因此，似乎GRHL2假设了 过渡过程中KLF2/4/5目标的子集，其他转录因子必须控制其他人 非常大的KLF2/4/5网络的一部分。这些发现导致了以下假设 大型幼稚的调节网络被分解为较小的启动调节 网络，为晚期的尤其细胞提供了区分不同的躯体谱系的灵活性 在e术后期，e肉阶段后期立即形成。那么每个较小的网络都可以 有选择地维护在不同的线路之间。实际上，GRHL2网络被排除在原始 在周围的其中类细胞中保持表达的同时。为了检验假设，有三个 具体目标。在AIM 1中，最先进的技术用于识别所有KLF2/4/5和GRHL2驱动器增强器 为了直接确定胚胎茎和e纤维细胞状态中的启动子相互作用 GRHL2是否导致KLF2/4/5目标子集之间增强子促销相互作用的重新布线。在 AIM 2，生物信息学和新型生化方法用于揭示其他尤其细胞转录 重新连接KLF2/4/5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 敲除胚胎用于探索在体内重新布线的生物学作用。成功的 该项目的完成将非常重要，因为它将发现基因控制的新型范式 调节细胞命运和细胞的独特发育潜力。