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Nuclear architecture in DNA repair and formation of chromosomal translocatons

Periodic Report Summary 1 - NADRCT (Nuclear architecture in DNA repair and formation of chromosomal translocatons)

2. Publishable summary
Acronym :
NADRCT
Marie Curie International Reintegration grant, project IRG 256622, mid-term report.
Title:
Nuclear architecture in DNA repair and formation of chromosomal translocatons
Researcher:
Dr. Evi Soutoglou, IGBMC, Illkirch France. evisou@igbmc.fr

Project summary
Cells continuously experience stress and damage from exogenous sources, such as UV light or irradiation, and endogenous sources, such as oxidative by-products of cellular metabolism that endanger genome stability. Several pathways have been evolved to detect DNA damage, signal its presence and mediate its repair. Double Strand Breaks (DSBs) are the most harmful DNA breaks because their unfaithful repair can lead to chromosomal translocations. Cells respond to DSBs by initiating a signaling cascade called DNA damage response (DDR) that activates checkpoints and promotes repair of the broken ends. Although, there has been much progress in identifying key factors of DDR and DNA repair, only recent cell -biological approaches have started to reveal how they function in the context of local high-ordered chromatin structure and nuclear space. Based on key preliminary findings, our hypothesis suggests that nuclear compartmentalization may contribute significantly to the mechanism linking DDR and DNA repair. This project aims to understand the role of nuclear architecture in the sensing and repair of DSBs and its implications in the production of chromosomal translocations. The three elements of nuclear architecture on which this work will focus on will be higher-ordered chromatin structure, nuclear compartmentalization, and genome organization. We have developed an experimental system to induce a DSB at specific genomic location and follow the fate of the break in relation to the surrounding chromatin structure and its position in the nucleus. Using this system, we first aim to explore whether chromatin decondensation is involved in the propagation of DNA damage response. Subsequently, we will assess whether specific chromatin changes are required for efficient repair of DSBs in heterochromatin or euchromatin. We will also visualise how breaks are recognized and repaired in different nuclear compartments containing distinct chromatin structures and protein compositions. The knowledge acquired from these studies, will then be used to directly test the role of nuclear organization in the formation of chromosomal translocations. The insights gained from this project may significantly advance our understanding of how the organization of repair in the intact nucleus contributes to preventing loss of genome stability.


Project progress for the reporting period
Objective N°1- Investigate the role of the repair factor MDC1 in chromatin decondensation -Tasks achieved for the reporting period:
Using the experimental system described above, we have shown that binding of the Mediator of check-point 1 MDC1 to chromatin provokes decondensation. Interaction studies revealed two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and load MERIT40 and BRCA1 to promote Homologous Recombination and check point activation. We also show that TNKSs counteract the MDC1 dependent decondensation and in turn facilitate the bridging of distal broken DNA ends. Our results suggest a step-wise model whereby upon occurrence of DSBs, MDC1 is first recruited to induce chromatin opening and allow efficient DDR and TNKSs are subsequently recruited by MDC1 to restore chromatin to the original state and facilitate DNA repair.

Objective N°2- Investigate the role of chromatin in the repair of DSBs-Tasks achieved for the reporting period:
We undertook a systematic and unbiased approach to identify novel chromatin related proteins that are involved in the repair of DSBs. We performed a siRNA screen to identify novel chromatin related proteins that their downregulation results in persistent and urepaired DSBs.This approach revealed several novel proteins that we are currently working on.

Objective N°3- Determine the role of nuclear organization in DNA damage response -Tasks achieved for the reporting period:
We have developed an experimental system to induce a DSB at specific genomic location and follow the fate of the break in relation to the surrounding chromatin structure and its position in the nucleus. Using this system we have visualized how breaks are recognized and repaired in the two different subcompartments of the nuclear periphery: the nuclear lamina and the nuclear pores. We show that the DNA damage response (DDR) induced by a break inflicted at the nuclear lamina is delayed and repair by Homologous Recombination (HR) is drastically impaired due to failure of polymerization of the recombination protein RAD51. Interestingly, global as well as local chromatin decondensation rescues these defects. On the other hand, the nuclear pores appear an activating microenvironment for DDR and repair. Our data point to a role of chromatin structure rather than specific proteins at the periphery as the determinant of the differential regulation of DDR and repair at the two distinct compartments of the nuclear periphery and reveal a new level of regulation of DSB repair by spatial organization of DNA in the nucleus.


Reintegration objective

The reintegration objectives as stated in the Marie Curie International Reintegration grant are already fully met for the mid-term reporting period. Thanks to the Marie Curie International Reintegration grant support, Evi Soutoglou was able to establish her research at the internationally renowned biomedical research institute IGBMC (France) in the Developmental Biology and Stem Cells Department. 8 young researchers have joined Evi Soutoglou’s research project: 3 PhD students, and 4 post docs and one technician. Thanks to the Marie Curie Reintegration funding, people in lab were able to perform their research in excellent working conditions and to participate to meetings, workshops and classes. Evi Soutoglou applied to the French national research agency CNRS in 2009 for a research scientist position. The recognition of Evi Soutoglou’s research potential impact on human health through the Marie Curie FP7 European program contributed to her success in getting a tenure position and she was ranked first for a competitive CNRS CR1 position. She joined CNRS in 2010 as an independent group leader to position her scientific career at the interface with clinical research.