Structural and functional analyses of Nanog and Sox2 in ESCs

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