Functional analysis of the role of the Nanog Tryptophan Repeat in ES cells
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Nanog is a transcription factor that plays a central part in the gene regulatory network that maintains and induces pluripotency of embryonic stem cells (ESCs). However, the molecular basis by which Nanog achieves its functions is not fully understood. At the centre of C-terminal domain of Nanog a tryptophan repeat (WR) is located, comprising 10 penta-peptide repeats each starting with a tryptophan. A mutant form of Nanog (Nanog-W10A) in which all 10 tryptophan residues have been substituted by alanine has an impaired capacity to drive LIF-independent self-renewal and a reduced efficiency in reprogramming primed epiblast stem cells to naïve pluripotency. To understand how the WR contributes to Nanog function, Nanog-W10A-ERT2 was introduced into Nanog null cells. Upon hydroxytamoxifen addition, the Nanog-ERT2 fusion proteins were detected on chromatin within 1 hour, allowing a comparison of genome-wide transcriptional responses to Nanog and Nanog-W10A by microarray. When treated with LIF, Nanog-W10A can activate most of Nanog targets as efficiently as Nanog. In contrast, Nanog-W10A did not efficiently repress most Nanog targets, including Otx2 and Tcf15 that were previously suggested to prime ESCs for differentiation. The microarray experiments performed in the absence of LIF signalling showed that Nanog and LIF co-regulate an extensive list of targets, including Klf4 and Mras. When LIF is absent, wildtype Nanog can still activate pro-self-renewal factors, including Esrrb and repress differentiation-priming factor, such as Tcf15 and Otx2. In contrast, in the absence of LIF, the activation of pro-self-renewal factors Klf4 and Mras is reduced. In addition, activation of Esrrb by Nanog-W10A induction delays but does not prevent differentiation. These effects allow the de-repression of Otx2 and Tcf15 by Nanog-W10A to dominate. Therefore, the function of Nanog is not only mediated by the activation of pro-self-renewal genes, but also repression of pro-differentiation signals. The functional significance of the repression of Nanog targets was further exemplified by the robust capacity of Otx2 to dominate over the self-renewal signals and to drive differentiation. The Otx2 protein is a direct interacting partner of Nanog that binds the Nanog WR tryptophan residues. The previously identified Otx2 “tail domain” comprises two imperfectly aligned repeats and aromatic residues of each repeat align with aromatic residues of the Sox2 “SXS/TY” motif previously identified to mediate the interaction between Sox2 and Nanog. Aromatic residues of Otx2 were demonstrated to directly interact with both Nanog and Sox2. The interactions between Otx2, Nanog and Sox2 are essential for Otx2 functions in driving ESCs differentiation, as Otx2 mutants with alanine substitutions of the aromatic residues in both or either of the repeats have reduced efficiency to drive differentiation. As Nanog and Sox2 may co-occupy many loci important in maintaining ESC self-renewal, Otx2 may be able to “read” the Nanog/Sox2 co-binding sites to dissolve the pluripotent networks. In summary, the repression function of Nanog is located within the Nanog WR region and represents an important module of Nanog in fine-tuning the balance between self-renewal and differentiation. This module involving Nanog WR can also be recognised by differentiation-priming factor Otx2 and may represent an initial step during the exit of differentiation.