Research statement:
Understand the mechanisms controling the inter-individual effects following DNA damage

General introduction:
Except for surgery, treatment of human tumors relies on selective killing of tumor cells. The killing is usually achieved by induction of DNA damage, either though radiation or chemical agents. The resulting DNA damage will elicit different DNA repair systems, many which have been rather well characterized. The components of the DNA repair machinery show relatively little variation between people (Svensson et al, PLoS Med, 2006; Fry, Svensson et al, Genes&Dev, 2008), with some exceptions (patients with rare disorders such as Ataxia teleangiactasia, Xeroderma pigmentosum, …). However, the anti-cancer treatment shows that both individuals and tumors show a great deal of variation in their sensitivity to different damaging agents. One possibility is that this variation is caused by the many genes and proteins that are needed in addition to the actual repair proteins. Studies have shown that as much as 30% of the proteins are needed for cellular survival after treatment with DNA damaging agents (Begley et al, Mol Cell 2004)

Research questions:
In the lab, we are focusing on the cellular mechanisms that are triggered by the induction of damage but surrounds the actual restoration of the DNA. Currently, we are focusing on two steps: 1) the consequences of histone H2B ubiquitination following DNA damage 2) the role of autophagy in clearance of compromised cellular components With sufficient amount of damaged DNA the tumor cells will die. However, complicated repair systems the cells may restore the genome and evade death. To induce the cellular survival mechanisms, specific genes needs to be activiated (and de-activated), requiring modification of the chromosomal structure at specific positions to allow for changed levels of transcription. Mutations in genes that affect the histone H2B ubiquitination affects the cellular survival to DNA damaging agents such as MMS and 4-NQO (figure 1).


Figure 1: (left) Fission yeast mutants show differential survival when growing on plates containing DNA damaging agent MMS (Abdul Mateen Rajput). (middle) Starved yeast cells (transfected with a GFP-ATG( construct) have induced autophagy and cleaved the C-terminal of ATG8, resulting in free GFP.(right) Autophagy detection in human 293T cells expressing fluorescently labelled LC3 (Laia Sadeghi).

H2B ubiquitination and genotoxic damage
Post-translational modification of histones is a common way to modify the accessibility of chromatin. Histone H2B is one of the core histones making up the nuclesome. Ubiquitination of at the C-terminus (K119 in S.pombe and K120 in mammals) is associated with actively transcribed genes and it is also a prerequisite for histone H3 K4 methylation, found in promoters of transcribed genes. Recent evidence link H2B ubiquitination to cellular survival after treatment with alkylating agents. We are studying the mechanisms behind this, using both classical and genome-wide techniques.

Damage-induced autophagy
Most anti-cancer agents do not only target DNA, but also other cellular targets are affected, such as proteins, RNA and lipids. A way to rescue the cells from death is to degrade the damage proteins or organelles. This can happen through a process known as autophagy. DNA damage induced autophagy has recently turned out to be an important cellular mechanism to rescue damaged cells. We are studying the differences between starvation-induced and DNA damage-induced autophagy. Currently, we are setting up a system that allows rapid and quantitative identification of individual cells with induced autophagy.

Model systems
In the lab, we are using both human cell lines and fission yeast as model systems. Fission yeast, Schizosaccharomyces pombe, is used as a model organism as it shows resemblance to the human chromosome structure with centromeres and telomeres, yet its genome is compact and easily studied. The compact genome of S. pombe makes it ideal to study genome-wide chromatin modifications.

Development of Computational analysis tools
To detect exact positions of modifications, chromatin IP-chip is used. To allow extensive analysis we are developing computational tools that specifically suits our needs. In collaboration with the lab of Karl Ekwall, we are setting up a Positioning Database and Analysis Tool (Podbat). In Podbat, users can upload chromosome position or RNA expression data which will be mapped onto the genome and visualized. Specific regions of interest can be computationally identified and the deviation between experimental conditions can be quantified. Comparisons between different datasets are relatively easy to make and lists of genes or regions can be queried for correlations between experiments or enrichment of functional categories.
Podbat is a Java-based tool that is currently under development but can be downloaded and tested from www.podbat.org.

Figure 2: Screenshot of the Podbat tool with a RNA expression dataset loaded (Ingela Djupedal).