Stem cells are defined by their pluripotent and self-renewing properties. A significant amount of research is focused on unlocking these properties in differentiated cells in order to create patient-derived stem cells that can be used to treat disease. However, the process of creating patient-derived stem cells relies heavily on expressing pluripotency factors within the nucleus; using techniques that often render stem cells too dangerous for regenerative medicine. This proposal takes a unique approach to understand how cytoplasmic components called germ granules regulate cellular stemness. The ultimate goal is to be able to manipulate germ-granule pluripotency factors within the cytoplasm to improve the quality and safety of patient-derived stem cells. Germ granules are a heterogeneous mix of RNA and RNA-binding proteins, and they are conserved in the germline from worms to humans. The transparency and sophisticated genetics of C. elegans offers an opportunity to study germ-granule function in a way that is not feasible in more complex model organisms. Our recent studies have shown that germ-granules create a cytoplasmic microenvironment that facilitates post- transcriptional regulation events that are central to the pluripotent and self-renewing properties of the germline. We have also shown that depleting germ granules causes germ cells to lose pluripotency, initiate somatic differentiation, and express transcripts associated with spermatogenesis. Our preliminary data suggests that germ granules maintain pluripotency by 1) silencing somatic mRNAs that are stochastically expressed in the germline, and 2) repressing the function of associated proteins required for sperm-specific mRNA expression. We will test these ideas by inducing the expression of mRNAs that exhibit high germ-granule affinity, and then we will compare changes in their distribution, accumulation, and translation in the presence and absence of germ granules (Aim 1). We will also determine how and which sperm-promoting pathways are negatively regulated by germ granules (Aim 2). And finally, we examine a specific germ-granule protein, CSR-1, that has the capacity to recognize and promote the expression of germline mRNAs, and will determine how it and its cofactors function in germ granules to promote germ-cell pluripotency (Aim 3). These findings will provide new approaches to safely induce cellular pluripotency when it is needed and shut off pluripotency when it is not.
The promise of regenerative medicine is to replace defective cell types with patient-derived stem cells; however, current techniques to create stem cells often render them too dangerous for this purpose. In this proposal we will determine how special structures called germ granules promote stem-cell properties in reproductive cells. By determining how germ granules function, we can apply mechanisms naturally used by reproductive cells to safely generate patient-derived stem cells.
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|Kelly, Ashley L; Senter-Zapata, Michael J; Campbell, Anne C et al. (2015) A Forward Genetic Screen for Suppressors of Somatic P Granules in Caenorhabditis elegans. G3 (Bethesda) 5:2209-15|
|Strome, Susan; Updike, Dustin (2015) Specifying and protecting germ cell fate. Nat Rev Mol Cell Biol 16:406-16|
|Campbell, Anne C; Updike, Dustin L (2015) CSR-1 and P granules suppress sperm-specific transcription in the C. elegans germline. Development 142:1745-55|