|dc.description.abstract||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
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
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.||en