UC San Diego

Placental mammals begin life as free-living embryos that implant into the uterus. Before implantation, the embryo differentiates into two lineages, the trophectoderm and the inner cell mass (ICM). The ICM contains undifferentiated cells that give rise the adult animal. This ability to produce all the cells of the adult animal is called pluripotency. This dissertation consists of work to understand the molecular makeup of pluripotent cells. Previous work on mRNA content of pluripotent cells lead to important discoveries of factors necessary and sufficient for pluripotency. I reasoned that a quantitative analysis of the proteome of pluripotent cells would be useful in confirming mRNA observations, identifying protein markers of the pluripotent and differentiated state and identifying post-transcriptional regulation. This thesis describes my work toward that goal. I describe the quantitative proteomes of two systems also used for transcriptome studies of pluriopotent cells: mouse ES cells and mouse EC cells. I compared of the two protein datasets to each other to identify conserved proteins associated with pluripotency and to mRNA datasets to identify putative cases of post-transcriptional regulation. I confirmed putative cases of post- transcriptional regulation by western blot and RT-PCR, and confirmed the role of one of these proteins in ES cell colony formation by knockdown. I describe my semi- quantitative analyses of the phosphoproteomes of the same cell types, allowing me to identify phoshoproteins associated with the undifferentiated and differentiated states. I tested the phosphorylation of the transcription factor UTF1 for changes in the protein's function, and found no difference between wild-type and phosphomutant proteins. Finally, I describe my work to combine the two datasets in order to identify changes in phosphorylation that are independent of changes in underlying protein levels. I identified factors associated with alternative splicing that become phosphorylated after differentiation. I inhibited a candidate kinase, Clk1, that phosphorylates splicing factors and performed a phosphoproteome on the inhibited cells. These studies represent a deep, quantitative representation of the protein makeup of pluripotent cells and result in new markers of pluripotency and differentiation as well as new phosphorylation events on factors known to be necessary and sufficient for pluripotency