Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616849
Title: Unravelling the proteome of chromatin bound RNA polymerase II using Proteome-ChIP in murine stem cells
Author: Natarajan, Kedar Nath
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Abstract:
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.
Supervisor: Pombo, Ana ; Barahona, Mauricio Sponsor: European Commission ; Medical Research Council ; Imperial College London
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.616849  DOI: Not available
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