Structural and functional analyses of Nanog and Sox2 in ESCs

胚胎干细胞（Embryonic stem cells，ESCs）是由囊胚内细胞团（inner cell mass，ICM）分化而来的细胞。它们被定义为自我更新的能力，保留其增殖能力以及多能性;能够从3个胚层分化成细胞。维持ES细胞的多能性涉及多种因素，包括信号传导分子，如细胞因子白血病抑制因子（LIF），其激活下游ES关键效应子，如Sox 2和Nanog。已知Nanog、Sox 2和Oct 4是通过控制导致自我更新和多能性的基因来调节ESC的中心转录因子。虽然这些蛋白质被认为是这一过程中的关键，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，焦点主要是Oct 4和Sox 2之间通过其DNA结合结构域的相互作用。已知Nanog独立于DBD结合Sox 2，而是通过色氨酸重复序列（WR）区域结合，该区域是含有10个五肽重复序列的结构域，每个五肽重复序列具有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，两种蛋白质之间的相互作用不会发生，这表明该区域是Nanog与Sox 2结合所必需的。Sox 2上的相互作用基序是富含丝氨酸的区域，其含有SXT/SY序列的三个重复。第一个和第三个重复突变为含有丙氨酸残基，显示去除几乎所有的Nanog结合能力。具体地，酪氨酸残基对于通过芳环与Nanog中的色氨酸残基的堆叠的相互作用是关键的（Gagliardi等人，2013）Nanog二聚化对于ESC的自我更新性质是关键的，并且也由WR区域介导。在高浓度下，Nanog可以使ES细胞保持其自我更新特性，而不管LIF信号传导。单体Nanog的过表达导致ESC在不存在LIF的情况下分化，而Nanog二聚体保留ES形态（Wang等人，2008）。类似地，WR的缺失导致ES细胞失去自我更新能力并分化，显示Nanog通过该区域二聚化（Mullin等，2010）Anog形成同源二聚体以及通过其WR区域与Sox 2相互作用，WR的突变应导致ES形态的丧失并鉴定参与分化和增殖的基因。通过比较野生型Nanog和Nanog与突变WR的单细胞RNA测序（scRNA-seq）数据，我们应该能够鉴定在不存在Nanog二聚化或Nanog-Sox 2相互作用的情况下表达不足的基因。使用CRISPR/Cas9将允许产生在Nanog WR和Sox 2 SXT/SY序列中具有突变的多个细胞系。除了scRNA-seq之外，这些细胞系也将用于自我更新测定，以研究Nanog-Sox 2相互作用的丧失对ESC分化速率的影响

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