The role of the histone tails and their post-translational modifications in nucleosome stability and chromatin folding
| dc.contributor.author | Moore, Susan Catherine | |
| dc.contributor.supervisor | Ausio, Juan | |
| dc.date.accessioned | 2018-11-21T17:52:12Z | |
| dc.date.available | 2018-11-21T17:52:12Z | |
| dc.date.copyright | 2002 | en_US |
| dc.date.issued | 2018-11-21 | |
| dc.degree.department | Department of Biochemistry and Microbiology | |
| dc.degree.level | Doctor of Philosophy Ph.D. | en_US |
| dc.description.abstract | The purpose of this thesis was to characterize the impact of the histone tails and their posttranslational modifications on nucleosome stability and chromatin folding dynamics. This was done with a variety of techniques, particularly sedimentation velocity analysis and circular dichroism as well as a range of other procedures including various restriction enzyme digests, DNase I footprinting, and solubility assays. Until recently the methods used to purify histones and their modifications have been somewhat inadequate, particularly with regards to the separation of individually modified histones. Therefore a method which allows the production of native-like histones and their use in reconstituted nucleosomes and chromatin fibers was developed using RP-HPLC fractionation. This has proven particularly useful for the successful purification of modified histones. The importance of the histone tails in chromatin fiber folding was demonstrated using nucleosome trypsinization and in vitro investigations using reconstituted polynucleosome arrays and sedimentation velocity analysis. Both the H2A–H2B and H3–H4 tails participate in folding events however the H3–H4 tails have the greatest influence. The structural effects of two specific histone modifications were also examined: acetylation, which occurs on the N-terminal tails of all the core histones; and ubiquitination which occurs primarily on the C-terminal tails of histones H2A and H2B. Examination of different nucleosomes (native, acetylated and trypsinized) using circular dichroism demonstrated that the histone tails have α-helical content which is independent of DNA interactions and increases with acetylation. Closer examination of the histone H4 N-terminal tails with different levels of acetylation showed that this α-helical content increase as the level of acetylation increases. Examination of histone H2A ubiquitination in nucleosome and polynucleosomal arrays demonstrated that despite the size of this modification, there were no significant structural changes in chromatin fiber folding or nucleosome structure. The results indicate that contrary to expectations uH2A did not prevent chromatin fiber folding and in fact it increased the aggregation of fibers and possibly even increased the stability of the nucleosome. Together the results illustrate the importance of investigating the structural effects of histones modifications and indicate that such modifications may produce very subtle effects rather than the huge structural disturbances often predicted. | en_US |
| dc.description.scholarlevel | Graduate | en_US |
| dc.identifier.uri | http://hdl.handle.net/1828/10342 | |
| dc.language | English | eng |
| dc.language.iso | en | en_US |
| dc.rights | Available to the World Wide Web | en_US |
| dc.subject | Histones | en_US |
| dc.subject | Posttranslational | en_US |
| dc.subject | Nucleosome | en_US |
| dc.subject | Chromatin folding | en_US |
| dc.subject | Biological sciences | en_US |
| dc.title | The role of the histone tails and their post-translational modifications in nucleosome stability and chromatin folding | en_US |
| dc.type | Thesis | en_US |