title: Distinct Contributions of Tryptophan Residues within the Dimerization Domain to Nanog Function.
abstract: The level of the transcription factor Nanog directly determines the efficiency of mouse embryonic stem cell self-renewal. Nanog protein exists as a dimer with the dimerization 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 dimerization and the effect of modulating dimerization strength have been unclear. Here we couple mutagenesis with functional and dimerization assays to show that the number of tryptophans within the WR is linked to the strength of homodimerization, Sox2 heterodimerization 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 dimerization 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 LIF-independent 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 self-renewal and differentiation.
authors.affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK. Electronic address: nick.mullin@ed.ac.uk.
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Mullin
authors.affiliation: Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, 675 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3.
authors.forename: Alessia
authors.initials: A
authors.lastname: Gagliardi
authors.affiliation: Department of Anatomy and Embryology, PhD Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Le Tran Phuc
authors.initials: LTP
authors.lastname: Khoa
authors.affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK.
authors.forename: Douglas
authors.initials: D
authors.lastname: Colby
authors.affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK.
authors.forename: Elisa
authors.initials: E
authors.lastname: Hall-Ponsele
authors.affiliation: NCMH Business Centre, School of Biosciences, University of Nottingham, Sutton Bonington, Leicestershire LE12 5RD, UK.
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Rowe
authors.affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK. Electronic address: ichambers@ed.ac.uk.
authors.forename: Ian
authors.initials: I
authors.lastname: Chambers
mesh_terms: D000818:Animals; D004252:DNA Mutational Analysis; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000502:physiology*; D000071317:Nanog Homeobox Protein / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D055748:SOXB1 Transcription Factors / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
chemical_list: D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D014364:Tryptophan
keywords: Sox2; aromatic interactions; dimerization; self-renewal; structural core