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The handle http://hdl.handle.net/1887/28538 holds various files of this Leiden University dissertation
Author: Schimmel, Joost
Title: Regulation of genome stability and cell cycle progression by SUMOylation
Issue Date: 2014-09-09
Aim and outline of the thesis
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Aim and outline of the thesis
It has been well established that SUMOylation regulates a wide variety of cellular processes, either on its own or in coordination with other post translational modifications such as ubiquitination. Currently, little is known about the cooperative actions of different PTMs and quite often we lack knowledge on how global SUMOylation contributes to cellular processes.
Therefore, the main aims of this thesis are to study the crosstalk between SUMOylation and ubiquitin, identify SUMO target proteins and their acceptor sites and to obtain more insight in the role that SUMOylation plays in maintaining genome stability.
The current concepts in SUMOylation are summarized and reviewed in Chapter 1.
An overview of the SUMOylation machinery is given and several examples of crosstalk between SUMOylation and other PTMs are presented. Furthermore, the role of SUMOylation in cell cycle progression and DNA repair is discussed as well as the current state of SUMO based proteomic studies.
In Chapter 2 we have investigated the crosstalk between SUMOylation and ubiquitination.
Using proteasome inhibition we identified a subset of SUMO2 target proteins that are subsequently ubiquitinated and degraded. Furthermore we have found that the ubiquitin- proteasome system is essential for the recycling of unconjugated SUMO2 proteins and that SUMO2 is a direct target for ubiquitination.
Chapter 3 describes the identification of a protein that can reverse the ubiquitination of SUMOylated proteins. The ubiquitin specific protease protein USP11 was identified to interact with the SUMO targeted ubiquitin ligase RNF4. In vitro assays revealed that USP11 can counteract RNF4 activity by removing ubiquitin proteins from SUMO-ubiquitin hybrid chains; this activity depends on four SUMO interacting motifs in USP11. Functionally, USP11 stabilizes PML nuclear bodies by preventing RNF4 mediated ubiquitination and degradation.
Studying SUMOylation is often challenging due to a lack of information on the modified lysines in proteins. Therefore we have developed a strategy to map SUMO acceptor sites in cells; this is described in Chapter 4. Site specific identification of SUMO2 target proteins enabled the discovery of an inverted SUMO consensus site and the identification of a hydrophobic cluster SUMOylation motif. Furthermore, we found direct evidence for crosstalk between phosphorylation and SUMOylation on several proteins.
In Chapter 5 we have analyzed global SUMOylation dynamics during cell cycle progression.
Cell cycle synchronization experiments enabled us to identify and quantify SUMOylation of proteins at different phases of the cell cycle. Bioinformatics revealed that transcription
Aim and outline of the thesis
factors belong to the largest SUMO regulated group of proteins, including transcription factor Forkhead box protein M1 (FoxM1). Follow-up studies showed that FoxM1 SUMOylation mainly takes place during G2 and M phase. During these phases, SUMOylation enhances transcriptional activity of FoxM1 by counteracting autorepression. Functionally, FoxM1 SUMOylation contributes to the maintenance of genome stability by reducing the risk of developing polyploidy.
Chapter 6 focuses on the SUMOylation of the Cockayne Syndrome B protein (CSB). Mass spectrometry based experiments showed that CSB is specifically and rapidly SUMOylated upon UV induced DNA damage. Although this process is dispensable for cells to survive after DNA damage, global SUMOylation events seem to contribute to efficient DNA repair. We show that the recruitment of the Cockayne Syndrome A (CSA) E3 ubiquitin ligase complex to sites of DNA damage induces the destabilization of SUMOylated CSB, potentially via the ubiquitination and degradation of RNA polymerase II.
The work presented in this thesis is summarized and discussed in Chapter 7.
