Structural and functional analyses of Nanog and Sox2 in ESCs

ESC 中 Nanog 和 Sox2 的结构和功能分析

• 批准号: 2107313
• 金额: --
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2107313
• 关键词: Structural; functional; analyses; Nanog; Sox2

Embryonic stem cells (ESCs) are cells which derive from the inner cell mass (ICM) from blastocysts. They are defined by the ability to self-renew retaining their ability to proliferate as well as being pluripotent; able to differentiate into cells from the 3 germ layers. Various factors are involved in maintaining the pluripotency of ES cells including signalling molecules such as cytokine Leukemia inhibitory factor (LIF) which activate downstream ES critical effectors such as Sox2 and Nanog. Nanog, Sox2 and Oct4 are known to be central transcription factors in regulation of ESCs by controlling genes resulting in self-renewal and pluripotency. Although these proteins are thought to be key in this process, they are known to interact with many other transcription factors in cooperative regulation (Pan and Thomson, 2007).When looking at the interactions of these transcription factors, the focus has mainly been on the interaction between Oct4 and Sox2 through its DNA binding domains. Nanog is known to bind Sox2 independently of DBD, instead through the Tryptophan Repeat (WR) region which is a domain containing 10 pentapeptide repeats each with a tryptophan as the first residue. When these tryptophan residues are mutated to alanine, the interaction between the two proteins does not occur suggesting this region is required for Nanog association with Sox2. The interacting motif on Sox2 is a serine rich region containing three repeats of the SXT/SY sequence. Mutation of the first and third repeat to contain alanine residues was shown to remove almost all Nanog binding capabilities. Specifically, the tyrosine residues are critical for interaction through stacking of the aromatic rings with the tryptophan residues in Nanog (Gagliardi et al, 2013).Nanog dimerization is critical to the self-renewal properties of ESCs and is also mediated by the WR region. In high concentrations Nanog can cause ES cells to retain their self-renewal properties regardless of LIF signalling. Overexpression of monomeric Nanog causes the ESCs to differentiate in the absence of LIF whilst the Nanog dimer retains the ES morphology (Wang et al, 2008). Similarly, deletion of the WR caused ES cells to lose self-renewal capabilities and differentiate showing Nanog dimerises through this region (Mullin et al, 2010).As Nanog forms homodimers as well as interacts with Sox2 through its WR region, mutation of the WR should cause loss of the ES morphology and identify genes involved in differentiation and proliferation. By comparing single cell RNA sequencing (scRNA-seq) data of wild type Nanog and Nanog with mutated WR, we should be able to identify genes which are underexpressed in the absence of Nanog dimerization or Nanog-Sox2 interaction. Use of CRISPR/Cas9 will allow production of multiple cell lines with mutations in the Nanog WR and the Sox2 SXT/SY sequence. In addition to the scRNA-seq, these lines will also be used in self-renewal assays to investigate the effect of loss of the Nanog-Sox2 interaction on rate of differentiation in ESCs

胚胎干细胞（ESCs）是从囊胚的内细胞团（ICM）中获得的细胞。它们的定义是自我更新的能力，保留了它们增殖的能力以及多能性；能够从3个胚层分化成细胞。多种因素参与维持胚胎干细胞的多能性，包括信号分子，如细胞因子白血病抑制因子（LIF），它激活下游的胚胎干细胞关键效应物，如Sox2和Nanog。已知Nanog， Sox2和Oct4是通过控制导致自我更新和多能性的基因来调节ESCs的中心转录因子。虽然这些蛋白质被认为是这一过程的关键，但已知它们在协同调节中与许多其他转录因子相互作用（Pan和Thomson， 2007）。当观察这些转录因子的相互作用时，重点主要放在Oct4和Sox2通过其DNA结合域之间的相互作用上。已知Nanog可以独立于DBD结合Sox2，而是通过色氨酸重复（Tryptophan Repeat， WR）区域，该区域包含10个五肽重复序列，每个重复序列以色氨酸作为第一个残基。当这些色氨酸残基突变为丙氨酸时，两种蛋白之间的相互作用不会发生，这表明该区域是Nanog与Sox2结合所必需的。Sox2上的相互作用基序是一个富含丝氨酸的区域，包含三个重复的SXT/SY序列。含有丙氨酸残基的第一个和第三个重复的突变被证明可以去除几乎所有的Nanog结合能力。具体来说，酪氨酸残基是Nanog中通过芳香环与色氨酸残基堆叠相互作用的关键（Gagliardi et al, 2013）。纳米二聚化对ESCs的自我更新特性至关重要，也由WR区域介导。高浓度的Nanog可以使胚胎干细胞保持其自我更新的特性，而不受LIF信号的影响。单体Nanog的过表达导致ESCs在缺乏LIF的情况下分化，而Nanog二聚体保留了ES的形态（Wang et al, 2008）。同样，WR的缺失导致胚胎干细胞失去自我更新能力并分化，通过该区域显示Nanog二聚体（Mullin等，2010）。由于Nanog形成同型二聚体，并通过其WR区与Sox2相互作用，因此WR的突变会导致ES形态的丧失，并识别与分化和增殖有关的基因。通过比较野生型Nanog和突变WR的Nanog的单细胞RNA测序（scRNA-seq）数据，我们应该能够识别在没有Nanog二聚化或Nanog- sox2相互作用的情况下低表达的基因。使用CRISPR/Cas9将允许生产具有Nanog WR和Sox2 SXT/SY序列突变的多个细胞系。除了scRNA-seq，这些细胞系还将用于自我更新试验，以研究Nanog-Sox2相互作用缺失对ESCs分化率的影响