DNA interstrand crosslink repair and chromatin remodelling

Project Information

ICL CHROM

Grant agreement ID: 845448

DOI

10.3030/845448

Project closed

EC signature date 15 April 2019

Start date 1 February 2020

End date 26 April 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 224 933,76

EU contribution

€ 224 933,76

Coordinated by

UNIVERSITY OF DUNDEE
United Kingdom

Periodic Reporting for period 1 - ICL CHROM (DNA interstrand crosslink repair and chromatin remodelling)

Reporting period: 2020-02-01 to 2022-01-31

Summary of the context and overall objectives of the project

Human cells are continuously exposed to insults that damage our DNA. DNA damage can come into many different flavours, from mutations in the sequence of DNA, to DNA cross-links that glue together two DNA strands, to breaks into the DNA molecule. To fix DNA damage, our cells are equipped with several mechanisms that identify the damaged DNA and repair it. Malfunctions in the DNA repair pathways, as in the case of human diseases such as Fanconi anemia, Bloom syndrome and Ataxia Telangectasia, results in increased cancer susceptibility and neurodevelopmental disorders. Understanding how DNA repair pathways are regulated and which proteins contribute to DNA stability is therefore essential for human health.

In our cells, DNA is organized into a structure called chromatin, where the DNA molecule is wrapped around proteins called histones. Chromatin provides stability and compaction to DNA, allowing the approximately two meters of DNA of each human cells to fit into the cell nucleus. Chromatin is a very plastic and complex structure, and is roughly divided into ‘active’ compartments and ‘silenced’ compartments, which regulate the expression of genes that make up the different cell types of our body. A wide range of proteins are required to assemble, maintain and regulate chromatin, such as chromatin remodelling enzymes, histone chaperones and histone modifying enzymes.

Chromatin can however also act as a barrier to proteins that want to access and bind DNA, including DNA repair proteins. Therefore, when a DNA lesion occurs, the chromatin structure has to be relaxed so that DNA repair enzymes can access the lesion and repair it. A number of chromatin remodelling enzymes, histone chaperones and modifiers have been found to function during DNA repair. This list is though likely far from being complete and there are some types of DNA repair, such as the repair of DNA inter-strand crosslinks, whose interplay with chromatin is still quite unclear.

In this proposal, we aimed at identifying all the chromatin remodelling enzymes and histone chaperones that are recruited to DNA damage sites, and to characterize how they function together with DNA repair pathways. This will provide a better understanding of how DNA repair is enacted in a complex chromatin environment.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The proposal was divided into two overall parts with complementary approaches to identify and characterise novel chromatin proteins involved in DNA repair.

First, we proposed to develop a new technique to profile all the proteins that are recruited to DNA inter-strand crosslinks. The technique takes advantage of a novel compound (synthetised in-house) which forms DNA inter-strand crosslinks upon shining UVA light on the cells. This compound can be biochemically isolated from cells along with all the proteins that bind in the vicinity of the DNA lesion, and these proteins can then be identified by mass spectrometry. The technique has been optimised in most of its aspects and, while the last step is still to be improved, the technique is at a very promising stage.

The second part of the proposal took a screening approach. We proposed to test by live cell imaging whether known histone chaperone and chromatin remodelling enzyme are recruited to DNA lesions, to find novel chromatin factors that act at DNA damage sites. For this, we constructed several human cell lines, each one expressing a histone chaperones tagged with a GFP fluorophore that can be observed by live cell imaging, and we induced DNA damage in specific regions of the cell nucleus. Proteins that are recruited to DNA lesions can be therefore visualized as accumulating in the damaged area. Using this approach, we identified a novel DNA damage-responsive histone chaperone. This histone chaperone was very poorly studied and we selected it for further characterisation.

We embarked in a multi-approach, collaborative study, where we studied the function of the candidate histone chaperone in both human cells and in a developmental model system, the nematode C. elegans. We observed that C. elegans worms lacking this histone chaperone have defects in their chromatin structure that results in the worms becoming sterile. This first part of the work is in preparation. We also teased out the molecular mechanism whereby the histone chaperone is recruited to DNA damage sites, which includes an interaction with DNA damage signalling pathways.

In terms of dissemination, I have presented this work at both national as well as international meetings. We are currently writing a manuscript on the function of the candidate histone chaperone, which will be published under open access.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Collectively, this project has provided new insights in the chromatin factors that respond to DNA lesions. We have identified and characterised a novel DNA damage-responsive histone chaperone. We have studied its function across different metazoan organisms, showing that it controls chromatin structure and promotes organism fertility. We have also shown how the histone chaperone is recruited to DNA damage sites in human cells and are working on understanding its function during DNA repair. We are also at an advanced stage in the development of a novel technique to profile all proteins recruited to DNA inter-strand crosslink damage in a chromatin context.

While more work will be needed to address all these important aspects, the results obtained so far lay the foundations for future projects. Since the crosstalk between DNA repair pathways and chromatin regulation is essential to maintain genome integrity, the work carried out so far, as well as its future developments, could provide important tools in preventing and curing human disease.