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Title: Investigations on the Roles of Ubiquitin in the Regulation of Heat Shock Gene HSP70B'
Keywords: Transcription, Ubiquitin, Proteosome Inhibitor, Heat Shock Gene, HSP70B', Mediator
Issue Date: 3-Aug-2011
Source: EE THIAM HEE, GARY (2011-08-03). Investigations on the Roles of Ubiquitin in the Regulation of Heat Shock Gene HSP70B'. ScholarBank@NUS Repository.
Abstract: In this project we investigated the role of ubiquitin in the regulation of the heat shock gene HSP70B?. Through RNA interference of SKP1, a component of an ubiquitin ligase, we demonstrated that the loss of ubiquitination can severely reduce the induction of HSP70B? by heat shock. However this effect is not necessarily the result of a loss of proteosomal degradation affecting a transcription factor. The application of a proteosome inhibitor induces the expression of HSP70B? and RNA interference of SKP1 likewise can reduce this increase in expression. This would imply that ubiquitination signaling, rather than proteosomal degradation, is a key factor in the regulation of HSP70B?. Split-ubiquitin screens performed to discover if Skp1 has interacting partners outside of its native ubiquitin ligase complex found that Mediator component Srb7 interacts with Skp1 in vitro. In vivo evidence for this interaction was found in a co-immunoprecipitation where human Skp1 could immunoprecipitate Med21, the human homologue of Srb7, and vice versa. RNA interference of MED21 could reduce the induction of HSP70B? by heat shock or proteosomal inhibition much in the same manner as RNA interference targeted at SKP1 did. This implies a functional relationship between these proteins in the regulation of HSP70B?. Co-immunoprecipitation experiments showed that components of the SCF (Skp1-Cullin-F-box protein) complex that Skp1 belongs to can immunoprecipitate components of the Mediator Complex and that the reverse is also true. This could indicate that these two complexes have a hereinto uncharacterized relationship in the regulation of transcription. Subsequent investigations found that the heat shock transcription factor Hsf1 is a regulator of HSP70B? expression; RNA interference targeted at HSF1 causes a strong reduction in HSP70B? induction that can be ameliorated by the application of proteosome inhibitor. Co-immunoprecipitation experiments demonstrated that Hsf1 has abundant ubiquitinated species in vivo. Overall our data favors a model where ubiquitination is a moiety that activates the transcription factor Hsf1 to drive HSP70B? expression. Over time, this ubiquitinated Hsf1 is polyubiquitinated and degraded, limiting its active lifespan which accounts for the activity of both proteosome inhibitors and siRNA targeted at SKP1. The findings of our project imply a functional relationship between Skp1 and Med21 where one was not previously suspected, furthermore our data implies that this relationship might extend to the SCF complex and Mediator complexes as a whole. This has various ramifications for the field of transcriptional regulation as it hints at a more involved partnership between these two complexes than was previously known. We have also provided evidence that the activation of HSP70B? is in accordance to the principles of the Second Generation Timer model and that Hsf1 is likely the activator of this gene in cases of thermal as well as proteotoxic stress brought about by heat shock and proteosome inhibitors respectively. This furthers our understanding of Hsp70B? regulation and possibly the regulation of heat shock proteins by Hsf1 as a whole. This has various implications for human health and disease as these proteins are involved in the pathology of several ailments including neurodegenerative diseases and oncogenesis.
Appears in Collections:Ph.D Theses (Open)

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EeTHG 03 Ee Thiam Hee, Gary - Thesis Main Body.pdf4.47 MBAdobe PDF



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