Targeted proteomic analysis of human chromatin: discovery and functional characterization of REQQL5 helicase as a
novel negative RNAP II transcription factor.
Doctoral thesis, UCL (University College London).
RNA Polymerase II (RNAP II) performs transcription in the context of chromatin, which is the physiological substrate of all DNA-mediated processes. While several independent studies investigated the proteins associated with the high-salt soluble form of human RNAP II, the proteomic composition of the native chromatinassociated RNAP II was unknown. In this work, a procedure to purify native chromatinassociated RNAP II has been developed and the DNA helicase RECQL5 was identified as a novel RNAP II-associated protein. Reciprocal proteomic analysis of RECQL5- associated proteins revealed that RECQL5 co-purifies with RNAP II complex, as well as RNAP II elongation and initiation factors. Reconstitution of RNAP II transcription with highly purified transcription factors revealed that RECQL5 inhibits both the early initiation and processive elongation stages of RNAP II transcription. Moreover, RECQL5 can stimulate cleavage of nascent transcripts associated with stalled RNAP II elongation complexes. Importantly, all these effects require a stable RECQL5-RNAP II interaction. Genome-wide association analysis by using a ChIP-Seq approach revealed that RECQL5 is enriched in promoters and coding regions of several RNAP II transcription units in the human genome. These results collectively suggest that human RECQL5 protein is a bona fide RNAP II transcription factor and plays multiple roles in RNAP II transcription cycle. Finally, proteomic analyses of other chromatin-associated proteins suggest that the initial purification approach developed for RNAP II can be utilized as a generic strategy. Together, the observations presented in this work illustrate that the previous view of human RNAP II transcription machinery was far from complete, and analysis of native chromatin-associated proteins may expand the current knowledge about other DNA-mediated processes in human cells as well.
|Title:||Targeted proteomic analysis of human chromatin: discovery and functional characterization of REQQL5 helicase as a novel negative RNAP II transcription factor|
|Additional information:||Authorisation for digitisation not received|
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