Objective 1: What determines the DNA repair specificity in the different nuclear compartments?
A previous study from my lab revealed that when a DSB is occurring at the chromatin that is associated to the nuclear lamina is not repaired in a faithful manner on contrary to a lesion induced at the chromatin (LADs) associated with the nuclear pores a region next to the nuclear lamina that is faithfully repaired. To understand what determines DNA repair pathway choice specificity in these two compartments, we have successfully employed quantitative proteomic approaches and we have identified the proteins that reside in Lamina Associated chromatin and NPC chromatin in the absence of DNA damage. These approaches revealed a set of unique but also common proteins in both nuclear compartments.
Objective 2: How is DNA repair organized in different heterochromatin structures?
i. DNA repair pathway choice at pericentromeric heterochromatin
Here we investigated DNA repair pathway choice at the pericentromeric heterochromatin compartment. We have already demonstrated that in pericentromeric heterochromatin the spatial arrangement of DSBs is connected to the DNA repair pathway choice. We showed that DSBs in repeats like heterochromatin relocate to this periphery to avoid inter-repeat recombination suggesting that the DNA repair pathway regulates the position of the breaks within heterochromatin structures. These results were published by Tsouroula et al. Mol Cell 2016 after the ERC was granted but before the official start date. To further investigate whether DSB repair is conserved in human HC, we used the CRISPR technology and we specifically induced beaks at he satellite 3 repeats. We observed that these repeats do not relocate and recombination is activated inside the core domain of the sat3 granules, demonstrating fundamental differences between mouse and human HC DSB repair.
These results were published at Ioanna Mitrentsi, et al. (2022). Heterochromatic repeat clustering imposes a physical barrier on homologous recombination to prevent chromosomal translocations. Moleculal Cell Apr 13:S1097-2765(22)00289-1
ii. DNA repair pathway choice at centromeric heterochromatin.
Our previous results by Tsouroula et al., 2016 also revealed indicated that DSBs at centromeric heterochromatin exceptionally activate Homologous recombination (HR) even in G1 stage of the cell cycle that is normally supressed. We have investigated the mechanism by which this is allowed in G1 at centromeres and found that the histone variant CENPA together with H3K4me2 are important for this process by recruiting the DUB enzyme USP11 that allows the specific recruitment of HR factors at this structure. We have published these results in Yilmaz, D. et al., (2021). Activation of homologous recombination in G1 preserves centromeric integrity. Nature 600, 748-753.
Objective 3: What is the role of 3D genome organization in the kinetics of DNA repair and DNA repair pathway choice?
The goal here is to understand the influence of 3D genome organization on DNA repair. We have used DNA end detection methods like BLESS and ChIP-Seq and followed the kinetics of DSB repair to map in high resolution the locations of DSBs as they get repaired. This mapping revealed that there are fragile regions non-randomly distributed in the mouse genome which could be classified into distinct categories based on their occurrence and repair profiles. We observed that in untreated cells, constitutive damage occurred in active chromatin (H3K4me3, H3K36me3).
The role of chromatin in DNA repair was published in Ghodke I., et al.,( 2021). AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation. Molecular Cell, Jun 17;81(12):2596-2610.e7 and
Evangelista FM,et al., (2018). Transcription and mRNA export machineries SAGA and TREX-2 maintain monoubiquitinated H2B balance required for DNA repair. The Journal of cell biology 217: 3382-3397 *Corresponding authors.
