Nanog is a divergent homeodomain protein with the capacity to direct constitutive self-renewal in the absence of otherwise obligatory cytokine stimulation. <i>Nanog</i> is expressed in the early mouse embryo and is essential for the specification of pluripotent cells. However the mechanism by which Nanog governs pluripotency is incompletely understood. In this thesis experiments are presented that further the functional characterisation of Nanog. In the mouse embryo, <i>Nanog</i> is normally down regulated in cells prior to de-lamination and ingression through the primitive streak. To address the consequence of <i>Nanog</i> over-expression <i>in vivo</i>, a revertible <i>Nanog</i> over-expressing cell line has been generated which can be tracked in the embryo. Results show that the modest 2-3 fold increase in <i>Nanog</i> expression does not cause any overt phenotype at this stage and <i>Nanog</i> over-expressing cells can be detected in the mesoderm of mouse embryos. Nanog is shown to exist in multimers in ES cells. The domain mediating multimerisation is identified as a tryptophan repeat motif and the functional consequence of deletion of this domain is investigated. To identify Nanog partner proteins, a biotinylation tagging system in ES cells has been designed, constructed, and implemented. This led to the identification of putative Nanog partner proteins via mass-spectrometry. Two Nanog partner proteins, Esrrb and HDAC2 have been confirmed by co-immunoprecipitation. In addition, the SLQQ motif within the Nanog homeodomain is shown to be the site of interaction between Nanog and Sall4. This SLQQ motif is found at a similar location in only one other homeodomain protein, Oct4. Consistent with these observations Sall4 is also shown to bind Oct4.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:664115 |
| Date | January 2007 |
| Creators | Yates, Adam |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14712 |
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