Regulation of gene expression is critical to govern distinct transcriptional programs
for a cell type, lineage specification and developmental stage. Transcription is the
first step in gene expression wherein RNA Polymerase II (RNAPII) transcribes
protein-coding genes. Transcription is a highly coordinated process that involves a
range of chromatin interactions including transcription machinery, chromatin
remodellers and co-transcriptional RNA processing. Embryonic stem (ES) cells are
pluripotent, self-renewing cells that can differentiate to give rise to all lineages making
them an invaluable tool to study early development and in therapy. Genome-wide
analysis in murine mES cells has identified 30% of known genes harbouring bivalent
chromatin modifications along with repressive Polycomb complexes and a novel
variant of RNAPII (modified as S5p+S7p-S2p-) with mechanistic implications in stem
cell pluripotency, differentiation potential and lineage specification.
To explore chromatin composition associated with different variants of RNAPII, I
developed an unbiased method, ‘Proteome-ChIP’ (pChIP) wherein crosslinked
chromatin is purified by immunoprecipitation followed by protein extraction and
identification by Mass Spectrometry. Using an unbiased comprehensive experimental
strategy and a novel systems biology approach, I qualitatively and quantitatively
dissect the proteome composition and dependencies on RNAPII modifications during
different stages of the transcription cycle. The work done in this thesis provides an
invaluable resource of RNAPII chromatin interactions. We identify known and novel
components of the co-transcriptional machinery, chromatin remodelling and RNA
processing machinery. The work also uncovers novel processes associated with
unusual RNAPII (S5p+S7p-S2p-) including DNA replication, Polycomb proteins and
chromatin remodellers; many of these processes critical for stem cell viability and
regulation.
Extending the RNAPII-pChIP analysis on low complexity samples by Native-pChIP
and Gradient-pChIP highlights the versatility of robustness of our method. The work
described in this sheds light on regulatory chromatin processes specific to mES cells,
which informs our understanding of stem cell biology and reprogramming.