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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - ChroSoDSB (Chromatin Study of DNA Double Strand Breaks)

Teaser

Summary

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.

Work performed

During my project, we first established a novel cell system to induce DNA damage, more specifically DNA Double Strand Breaks (DSBs) in a controllable, reversible and specific manner. We did so by generating a new cell-line, in which we could express the Cas9 nuclease by the addition of the specific compounds. In parallel, we designed specific guide RNAs (sgRNAs), that function as drivers of the Cas9 nuclease, targetting it at specific places in the genome. In this way, DSBs are induced only at the targetted locations, which we can predict and control. Next, we validated that the combination of our cell-line and our designed sgRNAs indeed produced DNA damage at the expected locations. We further characterised this system by monitoring how fast DSBs are formed, removed and if their removal is dependent on known DNA repair factors. Subsequently, we removed specific proteins one-by-one, in an independent from one another fashion, and we checked if their removal had any effect on the rate DSBs were repaired by high-content microscopy. Using this approach we identified with confidence 4 proteins that regulate the response to the specific type of induced DSBs; those proteins were never previously reported to play a role in the DSB response. We then tried to independently validate those 4 proteins, by checking whether they assemble at DNA breaks and by investigating if their removal also affects cell survival. We were happy to see that indeed one of them checked all those boxes and we are currently trying to characterise the mechanistic details of its involvement in the DSB response.
In terms of exploitation/dissemination to the scientific community: (1) we have strived to communicate our findings to our colleagues, both by talks, presentations and posters in local and international meetings, (2) we will publish our work in open-access journals so that itâ€™s readily available to everyone, (3) we have used my project to promote international mobility of younger scientists by recruiting and training master students, especially from southern European countries, where scientific opportunities are rarer. With respect to dissemination to the general audience: (1) I have presented our work to diverse audiences in public outreach events, as part of the EU-funded ENABLE symposium, the annual Researcher night and other local events, (2) I have tried to promote the general spirit of the Marie SkÅ‚odowska-Curie actions, by helping organise the Greek Chapter of the Marie Curie Alumni Association, by applying to the pairing-with-MEP program organised by the EU parliament and by being an in-house â€œeditorâ€ of other colleaguesâ€™ applications.

Final results

Thanks to years-long, outstanding research, scientists have come a long way in understanding how DNA damage affects our cells, and broader our bodies. It is now well understood that lingering or incorrectly repaired DNA damage results in mutations in the DNA; in time, these mutations accumulate and promote the onset or progress of diseases and conditions, such as cancer, neurodegeneration and ageing. Along the same lines, because excessive DNA damage kills cells, one of our first approaches to fight cancer cells is giving chemotherapeutic drugs that induce overwhelming DNA damage. The caveat is that generic DNA damage affects both cancer and normal cells, and it further induces bystander DNA damage that promotes secondary tumour formation. To overcome these issues, scientists in our field are striving to elucidate how cells locally respond to DNA damage: what proteins play key roles, what pathways are activated, how we can exploit differences between normal and cancer cells, or even within different cells in the tumour micro-environment. by shedding as much light as possible to the DNA damage response, we may find new druggable targets and new ways of targeting subsets of cells, so that they are specifically hit by DNA damage in a â€œstealthâ€ manner.
The long-term scientific, and consequently societal, implications of my project are relatively straightforward: (1) by designing more elegant systems to study DNA damage in highly-specific contexts, we can acquire a more detailed understanding on how cells respond to targeted DNA damage. This in turn can help us comprehend the origins of tumourigenic cells, (2) via our developed system, we can identify new proteins that regulate the DNA damage response, a few of them might indicate druggable pathways, (3) we can discriminate between proteins playing a generic or highly-specific role in the DNA damage response. The potential of all three previous points is that we can try to focus on specific cellular contexts that sensitise specific cells or cells to specific conditions. We can then try to mimic these conditions by specific drugs, thereby inducing a kind of DNA damage that may target only a certain subset of cancer cells, with minimal side effects.