Embryonic stem (ES) cells have the remarkable ability to differentiate into all cells comprising the three germ layers of the developing embryo. It is this pluripotency that makes them attractive for use in regenerative medicine. However, in order to harness this potential, we must understand the molecular mechanisms regulating the ability of ES cells to self-renew and thereby generate identical pluripotent daughter ES cells. The Welham laboratory has previously described a requirement for PI3K signalling in maintaining self-renewal of murine ES (mES) (Paling et al., 2004; Storm et al., 2007). To identify the molecular mechanisms involved in regulating mES cell self-renewal downstream of PI3K signalling, an Affymetrix microarray screen was carried out prior to the start of this PhD. For the screen, mES cells were grown in the presence of LIF and treated with the reversible PI3K inhibitor LY294002 (LY) or a DMSO control for 24, 48 and 72 hours. A total of 646 statistically significant transcriptional changes were detected and subsequently divided into 12 clusters using k-means clustering.Experiments using pharmacological inhibitors suggest that genes within the same cluster are regulated by common mechanisms. To identify potential candidates involved in regulation of mES cell pluripotency, further analyses concentrated on transcription factors and genes with unknown functions. In our microarray data Zscan4c, a member of a SCAN-domain containing Zinc finger protein family, is one of the earliest down-regulated probe-sets. Loss-of-function experiments using siRNA approaches highlight a role for Zscan4 downstream of PI3Ks in regulation of ES cell self-renewal. Immunohistochemical staining of cells overexpressing Zscan4c showed nuclear accumulation of the protein. This, together with the fact that Zscan4c was mainly detectable in the nuclear protein fraction, strengthens a role of Zscan4c in transcriptional regulation. Potential Zscan4c protein interaction partners were identified by applying a combined immunoprecipitation (IP) - mass spectrometry strategy. Interestingly, the majority of potential Zscan4c interacting proteins identified are associated with functions related to transcriptional regulation and DNA damage response, all characteristics linked with Zscan4. Furthermore, the Class IA PI3K catalytic isoforms were genetically activated in mES cells, and liberation of the requirement for LIF was found upon over-expression of an activated p110 catalytic subunit.
|Date of Award||1 Oct 2011|
|Supervisor||Melanie Welham (Supervisor)|