Cellular stress invokes a protective response in which heat shock factor 1 (HSF1) is activated to increase heat shock protein (Hsp) expression. HSF1 exists as a latent monomer in unstressed cells. Upon stress HSF1 forms homotrimers, increasing its affinity for the heat shock DNA element upstream of all Hsp genes. A second conformational change is required for HSF1 to gain transcriptional competence. During prolonged heat shock or following the resumption of normal conditions HSF1 DNA-binding and transcriptional activities are reduced and HSF1 returns to the monomeric state in a process called attenuation. During the activation/deactivation cycle HSF1 is modified by small ubiquitin-related modifier (SUMO-1) conjugation and undergoes several phosphorylation and dephosphorylation events that modulate HSF1 activity. Hyperphosphorylation of HSF1 is hypothesized to trigger HSF1 transcriptional activity. HSF1 also interacts with a dynamic series of Hsp90/Hsp70-based chaperone heterocomplexes that negatively regulate DNA-binding, and transcriptional activity, and promote attenuation. This thesis was aimed at characterizing the mechanisms regulating HSF1 DNA-binding, and transcriptional activity. Expression of human HSF1 in Xenopus oocytes altered the set-point of DNA-binding in response to heat indicating that both the cellular environment and innate properties of the molecule allow HSF1 to set its activation/deactivation set-point in response to stress in vivo. HSF1 DNA-binding but not transcription was activated in oocytes treated with a high temperature heat shock. Further characterization of this observation determined that HSF1 activated by a brief high temperature heat shock inhibited transcriptionally competent HSF1 from activating transcription. It was hypothesized that this phenomenon exists to ensure the eventual death of the cell due to the accumulation of excessive damage and potential mutation caused by severe stress. The most significant observation made in this thesis is that Hsp expression was detected in oocytes injected with reporter plasmid only during recovery from a high temperature heat shock. These results led to the proposal of a model in which HSF1 trimers are either assembled in a transcriptionally incompetent form or one that has the potential to become transcriptionally competent during stress, prior to DNA-binding. The identity of HSF1-binding proteins that interact with HSF1 at different stages of activation/deactivation was characterized in an effort to assign regulatory roles to these proteins. HSF1 was detected in a high molecular weight complex (350-600 kDa) during all phases of the activation/deactivation cycle. HSF1 at different stages of activation was tested for interaction with specific molecular chaperones by electrophoretic mobility supershift analysis. Hsp90, p23, FKBP52, Hip and Hop are all associated with transcriptionally active and inactive HSF1 suggesting that interaction of HSF1 with any of these molecules does not activate HSF1 transcriptional activity. These results do not exclude the possibility that the function of these molecular chaperones may change during activation of HSF1 transcription or that post-translational modifications may be the primary mechanism that drives HSF1 from a transcriptionally inactive to active form.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-09162003-231622 |
| Date | 17 September 2003 |
| Creators | Mercier, Philippe Arthur |
| Contributors | Roesler, William J., Ovsenek, Nicholas (Nick), Nichol, Helen, Mosser, Dick, Krone, Patrick H. |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-09162003-231622/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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