The role of histone demethylases in embryonic stem cell pluripotency: An epigenetic study

Embryonic stem cells are derived from the inner cell mass of the pre-implanted blastocyst-stage embryo. These cells are characterized by the capability of unlimited self-renewal and the capacity to differentiate into all the lineages of a developing organism. These qualities are established and maintained by multiple regulatory pathways which operate at several levels in the cell. The roles of many proteins in these pathways are largely unknown. We conducted a functional RNAi screen to suppress the expression of each of the known histone demethylases (HDMs) and thereby identify candidates essential for mouse embryonic stem cell identity. We confirmed effective knockdown for the majority of candidate HDMs through Western blot analysis. Upon depletion of the Jmjd1a, Jmjd2b, and Jmjd2c candidate HDMs, lineage-specific developmental genes were induced and stem cell-specific genes were down-regulated. We observed a differentiation phenotype in these cells, indicating that these HDMs are necessary for the maintenance of the pluripotent state. We then generated embryonic stem cell lines expressing the candidate HDMs with a FLAG and biotin tag. This combinational FLAG-biotin tag will be subjected to affinity purification for protein-protein and protein-DNA interaction studies in order to elucidate the binding partners of the candidate HDMs and thereby determine their molecular mechanisms. The FLAG-biotin tagging method has advantages and disadvantages when compared to its constituent tagging methods; a discussion of its utility is a key component of this thesis. Upon generating embryonic stem cell lines expressing FLAG-biotin tagged versions of the candidate HDMs, we verified their presence through Western blot and immunoprecipitation analyses. In summary, we generated embryonic stem cell lines expressing FLAG-biotinylated versions of Jmjd1a, Jmjd2b, and Jmjd2c and determined that these HDMs are necessary for stem cell identity. Future work entails using these embryonic stem cell lines to perform the aforementioned interaction studies to understand how the candidate HDMs fit into the network that maintains embryonic stem cell identity.Biological Sciences, School o