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
