Functional consequences of phosphorylation of pluripotency transcription factors

Embryonic stem cells (ESCs) are pluripotent and give rise to cells of all three primary germ layers. In suitable culture conditions ESCs self-renew indefinitely. ESC self-renewal is regulated by a network of transcription factors (TFs) centred on the master regulators Nanog, Sox2 and Oct4 [1]. However, the mechanisms by which these TFs work remains unclear. Such an understanding is critical to the successful manipulation of these systems in the field of regenerative medicine. All three TFs are phosphoproteins but in most cases there is no information as to the function of the modification, how the patterns of phosphorylation change during differentiation or across the cell cycle and which kinases phosphorylate individual sites.

Aims
1. Monitoring of changes in phosphorylation during differentiation
We propose to culture ESC in naïve (2i), primed (LIF/FCS) and differentiating media (N2B27), purify the TFs by immunoprecipitation and identify changes in the phosphorylation patterns by mass spectrometry (IP-MS). This will identify residues which are potentially central to the signaling pathways in ESCs. These residues will be mutated and the consequences of the alterations investigated in ESC functional assays. The combined expertise of the Chambers lab in ESC culture/TF immunoprecipitation and the Findlay lab in phosphorylation analysis makes us ideally placed to perform these analyses.

2. Identification of kinases and determination of function of phosphorylation
Once sites that change are identified, phosphospecific antibodies will be raised to these sites. These antibodies will be able to detect phosphorylated factors by immunohistochemical staining and will then be used in an in-cell, high throughput screen of large kinase inhibitor libraries. Inhibitors that block phosphorylation will prevent binding of anti-phosphorylation antibodies which will allow identification of the responsible kinases. Using the identified inhibitors in functional ESC assays will probe the role of the kinases. The expertise of the Findlay lab in high throughput kinase inhibitor screening [2] and the Chambers lab proficiency in ESC functional assays will be combined.

3. Identification of cryptic phosphorylation sites in Nanog

The C-terminal half of the Nanog protein contains only a single trypsin target residue. As our preliminary studies to identify phosphosites within Nanog used trypsin to digest the protein before mass spectrometry the coverage of Nanog was approximately 50%. The region of Nanog that is currently not covered includes the tryptophan repeat (WR), a region known to mediate protein-protein interactions with several partners [3]. Within the 50 amino acid WR there are 16 potentially phosphorylatable residues. The phospho-analysis of Nanog will be completed by a dual approach. First, we will perform IP-MS of Nanog from ESC under the conditions described in aim 1 using proteases with broader specificity ranges to cleave the protein prior to MS, using chymotrypsin initially. Mutagenesis will also be used to introduce trypsin target residues into the C-terminal half of Nanog. A variant, but functional Nanog protein contains a lysine residue within the WR and will provide a starting point. Additional mutants will be screened for function in ESC self-renewal assays prior to IP-MS as required.