The Effects of SUMO modification of the Tumour Suppressor p53
thesisposted on 2016-04-21, 08:50 authored by Diana Mota Marouco
p53 is a major tumour suppressor protein implicated in many cellular processes, regulating cell cycle arrest, apoptosis and DNA repair. The regulation of p53 can be achieved by post-translational modifications (PTMs), which alter the function of the protein in the cell. Among such PTMs, p53 can be modified via sumoylation – the covalent binding of SUMO (Small Ubiquitin MOdifier) protein – on lysine 386. This work aimed to define the role of sumoylation in regulation of p53 activity. By implementing a MS-proteomics approach, I identified a number of potential binding partners of sumoylated p53, and immunoprecipitation confirmed the transcriptional repressor lysine specific demethylase 1 (LSD1) as a specific interactor of SUMO-modified p53. The overexpression of wild type p53 protein fused with the sumoylation conjugating enzyme ubc9 (p53-ubc9) in p53-null cells H1299 shows a reduced ability to activate p53-target genes, when compared to the sumoylation deficient mutant (p53K386R-ubc9). Furthermore, sumoylation of p53 with both SUMO-1 and SUMO-2 significantly decreases the transcriptional activity of p53 towards its target genes p21 and PUMA, in both U2OS and HCT116 cell lines. Interestingly, ChIP analysis indicate that rather than preventing p53 from binding, sumoylation leads to the stabilization of p53 to these promoters. Moreover, p53 sumoylation led to the recruitment of HDAC2 to target-promoters, which correlated with an increase in H3K9me and a decrease in H3 acetylation, suggesting that p53 sumoylation leads to the recruitment of co-repressor complexes to the nearby chromatin. In addition, in vitro assays showed that p53 acetylation by CBP/p300, as well as the methylation of lysine 372 by SET7/9, is affected by the presence of sumoylation of lysine 386. Lastly, immunofluorescence microscopy showed increased localization of SUMO-p53, but not K386R mutant, to the nuclear membrane and cytoplasm, implicating SUMO on p53 nuclear export. These results highlight the importance of sumoylation as a modulator of p53 activity, and the point to existence of interplay between post-translational modifications in p53 protein to regulate its activity.
Date of award2016-04-18
Author affiliationDepartment of Molecular and Cell Biology
Awarding institutionUniversity of Leicester