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Title: Functional analysis of the SWI/SNF family protein LSH
Author: Myant, Kevin Brian
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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The aims of this study were to characterise the molecular function of LSH and attempt to relate this to its role in DNA methylation in vivo. To address these aims the work described in this thesis uses a variety of approaches to address two key questions. (1) Is LSH an active SNF2 ATPase? (2) How does LSH interaction with, and modulate, the DNA methylation machinery? To determine if LSH is an active SNF2 ATPase, recombinant LSH was purified from insect cells and biochemically characterised. These experiments revealed that LSH can hydrolyse ATP and its activity is stimulated by DNA. However, the rate of ATP hydrolysis is low and LSH does not exhibit chromatin remodelling activity. Thus, either LSH is a relatively weak SNF2 ATPase that cannot remodel chromatin or recombinant LSH may not be fully active. To identify proteins that interact with LSH, biophysical analysis of the native protein was performed. This indicated that the majority of LSH was present as a free monomeric peptide in vivo. However, I was able to demonstrate that LSH is an HDAC transcriptional repressor in vivo. Also, a weak, transient or low abundance complex including LSH, DNMT3B, DNMT1, HDAC1 and HDAC2 was identified. Thus, how LSH interacts with the DNA methylation machinery was demonstrated. Finally, I attempted to investigate how LSH modulates the activity of DNMTs in vitro. These experiments did not identify a role of LSH in stimulating DNMTs in vitro. These studies shed new light on the role of LSH in DNA methylation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available