ChroSoDSB · Chromatin Study of DNA Double Strand Breaks
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2017-07-01 → 2019-06-30
- EU contribution
- €200,195
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
Lines connect the coordinator with its partners.
Results in brief
Chromatin Study of DNA Double Strand Breaks
Our DNA contains all the necessary information to develop from a single cell to a functional organism. However, cellular DNA is neither static nor de facto safe, and it constantly faces threats coming from inside and outside sources. To keep chromosomes and their DNA intact, life has evolved appropriate protective mechanisms, known as the DNA Damage Response (DDR). The most threatening form of DNA damage is probably DNA double strand breaks (DSBs), as they are difficult to accurately repair. If DSBs persist or their repair is inaccurate, DNA mutations gradually emerge and the onset of pathological conditions and diseases, such as cancer, premature ageing and neurodegeneration, becomes much more likely. While scientists have made giant leaps in comprehending how our cells respond to DNA damage, and DSBs in particular, we still need to develop tailor-made tools and nuanced approaches to perform focused studies in specific cellular contexts. By understanding in detail and specific cellular contexts, how the DDR and DNA repair pathways act, we may be able to interpret better how complex diseases, such as cancer, establish a foothold and we may be able to develop more specific drugs in order to target them. In this project, my main objective was to develop a cellular system, resembling as close as possible physiological conditions, in which we could induce a specific number DSBs, in a specific cellular compartment, in a controllable manner. By combining such a system with an unbiased, systematic way of screening protein complexes, my objective was to identify new proteins with a key role in the DSB reponse and to then functionally characterise their role. These objective have been met to a satisfactory extent, though more work remains to be done. More specifically, we were able to generate an untransformed cell-line, in which we can induce DSBs in a controllable, uniform and specific manner. We combined this cell-line with high-content microscopy and siRNA-mediated protein depletion, that is we removed one-by-one proteins in an independent fashion. This allowed us to identify new candidate proteins that control the response to DSBs. We then validated that some of those factors by showing that they accumulate at sites of DNA damage and that they affect cell survival. We are now trying to expand our screens to test as many proteins as possible, and to further pinpoint at which stage of the DSB response the already validated proteins exert their action.
Data: CORDIS, © European Union
Project objective
In stark contrast to common belief, cellular DNA is not de facto safe as it constantly encounters numerous perils. To keep chromosomal DNA intact and to prevent the onset of pathological conditions, such as cancer and premature ageing, which are driven by DNA damage-induced genomic instability, life has evolved appropriate protective mechanisms, collectively referred to as the DNA Damage Response (DDR). A vital branch of the DDR is the response to DNA double-strand breaks (DSBs), arguably the most cytotoxic and mutagenic lesions. DSBs lead to the modification of their local chromatin environment to allow scaffolding of downstream protein factories, comprised of signalling, chromatin remodelling and DNA repair proteins. These DSB processing factories assemble in the context of chromatinised DNA, making chromatin architecture crucial for DSB restoration. Though our understanding of how chromatin status affects the DSB response has significantly progressed in recent years, we are only starting to identify factors regulating this intricate interplay. Moreover, current ways to induce DSB result in random, heterogeneous DNA damage, therefore not allowing the study of the DSB response in a specific chromatin compartment. In order to overcome this technical hurdle and to identify new regulators of DSB signalling and repair, I will use a powerful new method, combining the generation of limited, well-distributed DSBs, exclusively located in either euchromatin or heterochromatin, with specific purification of DSB-containing chromatin protein-complexes. Subsequently, I will select, validate and functionally characterise novel factors that have not yet been implicated in the DSB response, but behave similarly to known DSB-related proteins. By performing this multidisciplinary project, I will obtain new scientific and transferrable skills, thus taking a huge step towards true scientific independence and better career opportunities.
Original text from CORDIS.
Participants
- KOBENHAVNS UNIVERSITET · KOBENHAVNCoordinatorDenmark
Links
Data: CORDIS, © European Union