Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597711
Title: HP1 and its interaction with chromatin
Author: Clark, J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2009
Availability of Full Text:
Full text unavailable from EThOS. Please contact the current institution’s library for further details.
Abstract:
Heterochromatin protein 1 (HP1) is a highly conserved and abundant non-histone chromosomal protein involved in heterochromatin packaging. All HP1 isoforms contain a chromo domain (which interacts with methylated lysine 9 of H3), a flexible hinge region and a chromo shadow domain (which mediates dimerisation of HP1 and binding to many other proteins). The Neurospora H3K9 methyltransferase DIM-5 was used in vitro to methylate H3 with the aim of making highly methylated chromatin for HP1 binding studies. DIM-5 could trimethylate H3 in solution but not H3 bound to chromatin/DNA. Mass spectrometry was used to investigate the efficiency of methylation, which was found to be high. mHP1β was unable to bind to chromatin in vitro, whereas mHP1α and mHP1γ could both bind, with mHP1α binding best. mHP1α and mHP1γ were able to bind mononucleosomes without the core histone N-terminal regions, suggesting that their binding site was not methylated H3K9. The secondary structure of the hinge regions of mHP1α and mHP1γ as isolated peptides was investigated by CD spectroscopy. This confirmed that they were globally unstructured, but showed some α-helix in the presence of trifluoroethanol, a high concentration of perchlorate ions, or DNA. This propensity for α-helix formation was confirmed for the mHP1α hinge region by NMR spectroscopy. The ability of the mHP1α and mHP1γ hinge regions to bind chromatin and DNA was also studied; only the mHP1α hinge region was found to bind. It seems that the binding of mHP1α for chromatin was mostly due to the effect of its hinge region binding. Cross-linking experiments showed that all three mouse HP1 isoforms were able to bind to H1, but not through their hinge regions. The interaction between mHP1β and H1 was further examined by analytical gel-filtration and NMR. It was found that both the chromo and the chromo shadow domains were able to bind H1.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.597711  DOI: Not available
Share: