Regulation of embryonic stem cell self-renewal by the Zscan4 family of Zinc finger proteins

Stem cells are present within our bodies throughout our lives and are very important because they make sure that as damaged and worn out cells die, there is a supply of new cells to replace them. Some organs and tissues have a tremendous capacity to replace cells, e.g. the blood, the liver and the gut. However, organs such as the brain and heart have very limited capacity for repair and regeneration. Stem cells have been isolated from a variety of adult sources including bone marrow, gut and muscle and also from early stage embryos. Research has shown that these stem cells have two remarkable properties. They can divide to form two identical stem cells (self-renew themselves) or they can form many other types of cells (differentiate). There is currently great interest in harnessing these unique properties because by using stem cells it may be possible to generate cells in the laboratory, that can be used to either replace damaged tissues inside the body or to identify safer and more effective medicines. Many chronic diseases cannot currently be effectively treated because loss of cells is the underlying cause, e.g. type 1 diabetes. Parkinson?s disease and stem cell-based therapies offer exciting alternative treatments for disease sufferers. However, much more research needs to be carried out to understand stem cell behaviour before such advances will be brought into modern day medical practice.

The overall objective of this research proposal is to understand in greater detail the processes that control the ability of stem cells to renew themselves. Several signals within stem cells are known to be important in controlling self-renewal and our recent studies have identified a new regulator of this process. We identified this new regulator by analysing genes whose expression changes at early times after embryonic stem cells start to differentiate. This gene is predicted to control the regulation of other genes and we wish to now investigate this in more detail. Using cellular, genetic and biochemical experiments we will determine how this gene works in embryonic stem cells. By understanding the processes that control self-renewal of stem cells in more detail we should be able to apply this knowledge to the exploitation of stem cells in regenerative medicine and drug discovery. Stem cell-based strategies offer real hope for the sufferers of many chronic diseases and the research proposed here could be of real benefit in the medium to longer-term.

Embryonic stem (ES) cells are pluripotent, i.e. they have the ability to differentiate into all cell types comprising the adult organism. Pluripotency is underpinned by self-renewal, defined in ES cells as division accompanied by the suppression of differentiation to generate two undifferentiated daughters. The ability to maintain and expand pluirpotent ES cells is essential if the therapeutic potential offered by ES cell-derived progeny to regenerative medicine and drug discovery are to be realised, hence understanding regulation of self-renewal is critical. Previously we have demonstrated a role for phosphoinositide 3-kinase-dependent signalling in optimal maintenance of self-renewal of murine ES cells. Using global expression profiling we have now defined the PI3K-dependent transcriptome in murine ES cells and identified a cluster of 6 genes whose expression was significantly down-regulated within 24h of PI3K inhibition. Loss and gain of function studies of one of these genes, Gm397, demonstrate that it plays a role in regulation of mES cell self-renewal. Gm397 belongs to a family of closely related SCAN-domain-containing zinc-finger proteins, collectively termed Zscan4. Based on our functional analyses, and supported by work published recently, we propose that members of the Zscan4 family are novel regulators of ES cell self-renewal and mediate their activity via actions as transcriptional regulators. We will test this hypothesis by investigating the mechanisms of ZScan4 action, focusing on the following specific objectives:
1. Investigation of the significance of the mosaic expression of Zscan4 in mES cells
2. Definition of the expression profile of Zscan4 genes in mES cells.
3. Examination of the aspects of ES cell biology controlled by Zscan4 members that contribute to regulation self-renewal.
4. Determination of the ability of Zscan4 family proteins to regulate gene transcription.
5. Assessment of the functional importance of ZSCAN4 in human ES cells.
We will use a combination of cellular, molecular, biochemical and immunological approaches in these investigations. The significance and basis of Zscan4 mosaic expression will be investigated. We will define those Zscan4 members expressed by ES cells and their relative levels of expression. Loss and gain of function approaches (Tet-regulated) will be used to examine in detail the aspects of ES cell biology regulated by Zscan4 proteins and the activities of different Zscan4 members compared. Complementary approaches, including transcriptional analyses and gene profiling, will be used to define genes regulated by Zscan4. We will also assess the functional importance of ZSCAN4 in human ES cells.