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Spatial organization of DNA repair within the nucleus

Periodic Reporting for period 4 - 3D-REPAIR (Spatial organization of DNA repair within the nucleus)

Reporting period: 2021-03-01 to 2022-04-30

What is the problem issue being addressed?
Faithful repair of double stranded DNA breaks (DSBs) is essential, as they are at the origin of genome instability that is the hallmark of cancer. The balance between error free and error prone DNA repair pathways must be tightly regulated to preserve genome integrity. Although, we know a lot about the different proteins that repair DSBs, how their action is controlled within the highly structured nuclear environment is unknown. We have previously shown that DNA repair pathway choice is dictated by the spatial organization of DNA in the nucleus. Nevertheless, what determines which pathway is activated in response to DSBs at specific genomic locations is not understood.

Why is it important for society?
Preservation of the integrity or our genome is vital for life. This proposal has significant implications for understanding the mechanisms that determine whether a DNA lesion at a specific position in the nucleus will be repaired in an error free or a mutagenic manner and whether this decision will affect fitness.

What are the overall objectives?
We aim to understand how nuclear compartmentalization, chromatin structure and genome organization impact on genome integrity maintenance. We first aim to study how the nuclear position affects the balance between the error prone and error free repair pathways by identifying factors that are recruited at specific nuclear compartments in the presence of DNA damage. We will then focus on repetitive elements that form heterochromatin as their integrity is vital for chromosome segregation. Finally we will investigate the role of genome 3D folding in the kinetics of DNA damage response and repair.
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.
Objective 1: We have identified for the first time proteins that are associated with LADs and NPC chromatin. We find that there are many common but also distinct proteins that are bound to these compartments. We have also developed an experimental system to specifically induce DSBs at Lamina associated domains and Nuclear pore chromatin and we have identified the factors that are recruited to these specific compartments revealing for the first time how the epigenetic environment affects DNA repair pathways choice and how it suppresses homologous recombination at the LAD repetitive sequences.

Objective 2: i. DNA repair pathway choice at pericentromeric heterochromatin
We have identified the mechanism by which DSBs in pericentromeric heterochromatin in mouse are repaired. We have also studied the spatial regulation of DNA repair in human heterochromatin and we have identified fundamental differences in the activation of Homologous recombination between human and mouse. We have identified that centromeres are a special heterochromatic environment which allow activation of HR in G1 phase of the cell cycle and the mechanism by which centromeric DSBs activate HR in G1.

Objective 3: For this objective we have followed for the first time by two independent mapping methods the kinetics of DSB repair to map in high resolution the locations of DSBs as they get repaired. We identified fragile regions of the genome that are broken even in the absence of exogenous DNA damage and they are located in promoters of active genes. We also identified new break points after exogenous damage that are more located in intergenic regions. These approaches revealed break hotspots of the genome either due to replication or due to exogenous damage.
DNA damage induction at centromeres in mouse cells