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Title: Ubiquitin-ligase-mediated transcription initiation in cellular stress defences
Author: Furniss, James John
ISNI:       0000 0004 6351 5722
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2016
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Accurate regulation of gene transcription is essential for organismal survival, and is orchestrated by myriad transcription factors and cofactors (TFs). Little is known about how the intrinsic activity of TFs is controlled. Recent work has indicated that the selective proteolysis of TFs provided by the ubiquitin-proteasome system (UPS) plays an important role in stimulating gene expression through a ‘destruction-activation’ mechanism, whereby the degradation of a ‘used’ TF is thought to stimulate further ‘fresh’ TF binding and reinitiate gene transcription. TFs are targeted to the proteasome via E3 ligases that mediate the addition of ubiquitin molecules to form a chain on the substrate TF. These polyubiquitin chains may be extended by E4 ligases, which recognize substrates with four or more ubiquitin molecules, amplifying substrate targeting to the proteasome. In plants the immune response to many pathogens is regulated by the hormone salicylic acid (SA), which operates through the transcriptional coactivator NPR1 to induce large scale changes in gene expression. Proteasome-mediated degradation of NPR1 appears to be required for the activation of its target genes. Mutation of the E3 ligase prevents ubiquitination of NPR1, leading its to stabilisation and suppression of transcription. Chapter 3 of this work identifies the first E4 ligase, UBE4, involved in NPR1 regulation. Mutation of UBE4 resulted in reduced capacity to polyubiquitinate substrates and stabilized NPR1. In contrast to E3 ligase mutants, however, mutant ube4 plants displayed increased NPR1 target gene expression. These results suggest that initial ubiquitination of NPR1 may stimulate its ability to initiate transcription and that subsequent ubiquitin chain elongation limits NPR1 activity by targeting it to the proteasome. Chapter 4 describes a ubiquitin-protein-ligase (UPL) which is both novel and crucial to the SA-mediated defence response. Mutation of this UPL leads a large reduction in total cellular polyubiquitinated proteins and was associated with strongly enhanced disease susceptibility. Gene expression profiling of upl mutants revealed an intimate connection between cellular polyubiquitination and appropriate activation of SA-responsive gene expression programmes. Destruction activation was first described in yeast and is required for the regulation of yeast amino acid synthesis TF GCN4. GCN4 requires proteasome-mediated degradation to induce genes involved in amino acid production. Chapter 5 investigates the role of two E4 ligases in GCN4 turnover. While one mutation had little effect of GCN4-mediated transcription a second increased basal transcriptional levels, suggesting that an E4 is required for the prevention of spurious GCN4-mediated transcription. In summary the work presented here describes cellular mechanisms by which global and substrate-specific polyubiqutination are vital to regulation of gene transcription.
Supervisor: Spoel, Steven ; Loake, Gary Sponsor: Biotechnology and Biological Sciences Research Council (BBSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: ubiquitination ; transcription ; Arabidopsis ; immunity