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.

Analysis of pluripotency transcription factor interactions reveals preferential binding of NANOG to SOX2 rather than NANOG or OCT4

“…In contrast, when a GFP-Nanog fusion in which the tryptophan residues in the tryptophan repeat (WR; Nanog-W10A) was expressed in CHO cells alongside (FLAG)3-Nanog, incubation of nuclear lysate with anti-FLAGconjugated beads yielded no fluorescence ( Fig 1B). This is consistent with previous reports of a critical role for WR tryptophan residues in homodimerisation of Nanog (Mullin, Yates et al 2008, Wang, Levasseur et al 2008, Mullin, Gagliardi et al 2017). In addition, incubation of beads with (FLAG)3-Nanog and GFP-Sox2, resulted in localisation of fluorescence to the bead surface ( Fig 1B).…”

“…The dimerisation of Nanog may also be affected by additional Nanog partner proteins. While the residues involved in Nanog homodimerisation and heterodimerisation with Sox2 have been determined (Mullin, Yates et al 2008, Gagliardi, Mullin et al 2013, Mullin, Gagliardi et al 2017, how Nanog interacts with additional partners remains to be shown. For example, while Nanog has been reported to bind Oct4 (Wang, Levasseur et al 2008), other studies did not report an interaction between Nanog and Oct4 (van den Berg, Snoek et al 2010, Gagliardi, Mullin et al 2013.…”

A Family of Vertebrate-Specific Polycombs Encoded by the LCOR/LCORL Genes Balance PRC2 Subtype Activities

“…In order to further dissect the PIP domain of PALI1 and PALI2, we closely analyzed the conservation and charges of its constituent amino acids ( Figure 3C). Two highly conserved tryptophans (W1125 and W1186) were selected for mutation due to the potential importance of aromatic residues in protein-protein interactions and for structural stability within a modular domain (Mullin et al, 2017). Mutagenesis of these tryptophans to alanines individually did not disrupt the interaction with EZH2 ( Figure 3D).…”

TET1 interacts directly with NANOG via independent regions containing hydrophobic and aromatic residues

“…Alanine substitution mutagenesis identified aromatic and hydrophobic residues that mediate the interaction between TET1 and NANOG. In NANOG, tryptophans within the WR are critical for the biological function of NANOG and mediate NANOG homodimerization and binding to SOX2 aromatic residues [49,50,30,51]. Tryptophan residues within the NANOG WR also interact with aromatic residues in the TET1 C-terminus, suggesting an interaction by aromatic stacking.…”