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Title: SUMOylation as a regulatory mechanism for the Sox3 transcription factor
Author: Alfassam, Haifa
ISNI:       0000 0004 8502 3569
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2019
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Evidence of the essential role played by Sox3 in cell proliferation and cancer survival is increasing, highlighting the importance of understanding the mechanisms that regulate the function of the Sox3 protein. It has been shown that Sox3 plays a crucial role in embryonic and adult neural stem cells by slowing down proliferation to regulate the stem cells state. Sox3 exerts these functions by working as transcriptional activator and repressor of target genes. It is still not clear how these two functions of Sox3 are regulated. In this study, I was interested in understanding how Sox3 can switch between these two functions, investigating whether this change in activity is linked to SUMOylation. Cell biological analyses via alamar blue, cell counting and apoptosis assays show that cells over-expressing wt Sox3 grow at a slower rate than negative control cells. Cells over-expressing SUMOylated Sox3 grow at an even slower rate. These data suggest that SUMOylation of Sox3 increases the anti-proliferative function of Sox3. Biochemical analysis of Sox3 SUMOylation via western blot showed that co-transfection of either Sox3wt or a single SUMOylation site mutant form of Sox3 along with E2/Ubc9 and SUMO constructs induced a rapid degradation of Sox3 protein. Treatment of the transfected HeLa cells with the proteasome-specific inhibitor, MG132, showed that Sox3 could be degraded by the ubiquitin-proteasome system (UPS). This evidence suggests that SUMO can act as a signal to ubiquitinate SUMOylated proteins and target them for proteasomal degradation. The data in this study provide evidence, for the first time, that Sox3 can be a target of a SUMOylation-dependent degradation process that might regulate the function of Sox3 by controlling its intracellular level. Moreover, either of the potential SUMO attachment sites in the Sox3 protein was found to be sufficient to initiate SUMOylation-dependent degradation. A mutant Sox3 construct which should not be able to bind to DNA, Sox3N40I, was prepared using site-direct mutagenesis. Comparison between the protein level of Sox3N40I and Sox3wt in cells co-transfected with SUMO1 and E2/Ubc9 revealed that SUMOylation-dependent degradation require DNA binding. Taken together, the evidence in this study reveals a new mechanism, SUMOylation-dependent degradation, which appears to regulate the function of Sox3 at the protein level by controlling the amount of Sox3 protein in cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH573 Cytology