Assessing the functions of histone ADP-ribosylation during DNA repair – AROSE

ADP-ribosylation signaling by PARP1 is a key early event of the DNA damage response. While recent works have elucidated the role of PARP1 auto-ADP-ribosylation, less is known about the impact of the ADP-ribosylation of histones, which are the second main target of this signaling pathway after PARP1 itself. In this project, we aim to uncover the functions of histone ADP-ribosylation in three key steps of the DNA repair process known to be regulated by this signaling pathway: i) modulation of the chromatin architecture, ii) recruitment of repair factors and iii) control of the timely release of PARP1 from the sites of damage. To study these different components of the repair mechanism, we set up an interdisciplinary consortium of 5 research teams with complementary expertise in the fields of ADP-ribosylation signaling, chromatin architecture and repair protein dynamics. The chromatin architecture will be characterized both in vitro and in living cells using cutting edge microscopy methods that will give us access to the chromatin structure and dynamics at the single nucleosome level. These tools will be applied in cells lacking specific components of the ADP-ribosylation signaling pathway to be able to delineate the impact of histone ADP-ribosylation on the chromatin architecture. We will also investigate two alternative mechanisms by which histone ADP-ribosylation could promote the recruitment of repair factors at sites of damage. First, by combining advanced proteomics assays with live cell microscopy, we intend to identify factors displaying binding specificity to ADP-ribosylated histones. Second, we will study whether histone ADP-ribosylation could promote the accumulation of repair proteins to DNA lesions via liquid-liquid phase separation mechanisms, focusing on the two key actors 53BP1 and BRCA2. Using complementary assays to characterize the dynamics of these two proteins at the population and single-molecule levels in living cells, we will be able to distinguish between accumulation mechanisms driven by specific binding to the damaged chromatin, and confinement within liquid droplets surrounding the lesions. Then, we will analyze the dynamics of 53BP1 and BRCA2 at DNA lesions in cells lacking histone ADP-ribosylation compared to control cells to establish the specific impact of this histone mark on the recruitment mechanism. Finally, we will monitor PARP1 recruitment kinetics and turnover at sites of damage to define how histone ADP-ribosylation could contribute to the rapid dissipation of PARP1 from the DNA lesions. Altogether, building-up on the interdisciplinary expertise of the consortium established for this project and the use of high-end quantitative methods, this project will provide a precise description of the roles of histone ADP-ribosylation in the context of the DNA damage response. We anticipate that these findings will deepen our understanding of the complex histone-based epigenetic landscape by dissecting the functions of one of its least characterized components.