Distinct Contributions of Tryptophan Residues within the Dimerization Domain to Nanog Function

The level of the transcription factor Nanog directly determines the efficiency of mouse embryonic stem cell (ESC) self-renewal. Nanog protein exists as a dimer with the dimerisation domain composed of a simple repeat region in which every fifth residue is a tryptophan, the tryptophan repeat (WR). Although WR is necessary to enable Nanog to confer LIF-independent self-renewal, the mechanism of dimerisation and the effect of modulating dimerisation strength has been unclear. Here we couple mutagenesis with functional and dimerisation assays to show that the number of tryptophans within the WR is linked to the strength of homodimerisation, Sox2 heterodimerisation and self-renewal activity. A reduction in the number of tryptophan residues leads initially to a gradual reduction in activity before a precipitous reduction in activity occurs upon reduction in tryptophan number below eight. Further functional attrition follows subsequent tryptophan number reduction with substitution of all tryptophan residues ablating dimerisation and self-renewal function completely. A strong positional influence of tryptophans exists with residues at the WR termini contributing more to Nanog function, particularly at the N-terminal end. Limited proteolysis demonstrates that a structural core of Nanog encompassing the homeodomain and the tryptophan repeat can support LIFindependent colony formation. These results increase understanding of the molecular interactions occurring between transcription factor subunits at the core of the pluripotency gene regulatory network and will enhance our ability to control pluripotent cell selfrenewal and differentiation.