Skip to main content

Spatial organization of DNA repair within the nucleus

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

Reporting period: 2019-09-01 to 2021-02-28

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 at a given locus 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 not studied in detail. 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. Furthermore, the impact of 3D-genome folding on the kinetics and efficiency of DSB repair is completely unknown.

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 viability and cell fitness.

What are the overall objectives?
With this proposal 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. Our next goal is to induce DNA damage specifically in LADs and NPC chromatin and identify the specific proteome upon DNA damage. Once in place this experimental system will allow us to identify factors that are recruited to different nuclear compartments after DNA damage and start the validation phase of this objective. These results will unravel for the first time the mechanism behind the specificity of DNA repair factor recruitment in specific nuclear compartments.

Objective 2: How is DNA repair organized in different heterochromatin structures?
i. DNA repair pathway choice at pericentromeric heterochromatin
To further extend our knowledge on the impact of nuclear compartments in DNA repair, we decided to investigate DNA repair pathway choice at the pericentromeric heterochromatin compartment. We have already demonstrated that in pericentromeric heterochromatin the spatial arrangement of DSBs is highly connected to the DNA repair pathway choice. Most importantly, 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. The main difference between mouse and human HC is that mouse pericentromeric repeats of different chromosomes cluster to form the chromocenters, whereas in human cells the breaks are induced mainly on the pericentromeric sat3 repeats of chromosome 9, that does not cluster with a similar domain. We therefore hypothesized that in mouse cells the breaks relocate to the periphery of the HC domain to avoid recombination between identical sequences from different chromosomes leading to chromosomal translocations. Interestingly and in line with this hypothesis, when DSB relocation is inhibited, a dramatic increase in translocations and genomic instability (increased breaks) originating from HC repeats is observed. The results we have obtained so far are intriguing and suggest that the spatial arrangement of repetitive elements at different species determines the way they will repair. We are currently exploring the mechanism by which DNA repair factors like RAD51 sense the heterochromatin repeat clustering and after that we will be ready to publish these results.

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 prepare these results for publication. We have therefore explored in detail and mechanisms by which 3 different heterochromatin structures (1. LADs 2. pericentromeric heterochromatin and 3. centromeric heterochromatin) repair DNA lesions. With the tools and experience we gathered we are in the position to move to the last heterochromatin structure proposed in objective 2 that is retroelements. Once we have complete picture about heterochromatin we will compare these results with euchromatin. We have also started to develop tools to identify by Mass spectrometry the protein composition of major and minor satellites in the presence of DNA damage. To this end, we have coupled the CRISPR/Cas9 system to the Bio-ID technology, capable of revealing weak or transient protein-protein interactions and we have coexpressed it with the guide RNA for minor or major satellites. We are currently comparing these lists and we are moving to the validation step. We expect to find factors that are uniquely recruited to heterochromatin after DNA damage.

Objective 3: What is the role of 3D genome organization in the kinetics of DNA repair and DNA repair pathway choice?
The goal of this objective is to understand the influence of 3D genome organization on DNA repair. We have used DNA end detection methods based on parallel sequencing 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). This was also corroborated by analysis of Lamin-associated domains (LADs), which showed a lower incidence of damage, suggesting that constitutive damage is present preferentially in active chromatin. Interestingly, we observed that a higher proportion of the constitutive break regions are present downstream of transcription start sites. This correlates with the findings that the DNA damage response signalling mechanism is also essential for transcriptional elongation. We also found that repair starts much further away from a transcription start site and then moves towards the start site. After peak annotation of the ChIP-Seq data, it was seen that after induction of damage, a higher percentage of damage persistence was observed in the intergenic and intronic regions. The next step is to validate these results in single cells by superresolution microscopy and we are developing methods to pursue this goal.
Objective 1: What determines the DNA repair specificity in the different nuclear compartments?
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 are developing a system to induce damage and identify the proteins specifically recruited in each compartment after damage. Once we identify them we will explore the specific function of the most interesting proteins. This approach will reveal for the first time factors that uniquely recruited at specific compartments after DNA damage to dictate pathway choice at this compartment.

Objective 2: How is DNA repair organized in different heterochromatin structures?
i. DNA repair pathway choice at pericentromeric heterochromatin
We have indentified the mechanism by which DSBs in pericentromeric heterochromatin in mouse are repaired and we have published these results (Tsouroula et al., 2016). We have also compared DSB repair in mouse and human heterochromatin and found fundamental differences. We are currently investigating the basis of these differences and we expect to have the complete mechanism in a year from now. We have also investigated the mechanism by which centromeric DSBs activate HR in G1 phase of the cells cycle and we are preparing these results for publication. Therefore we have explored in detail and for the first time in mammalian cells the mechanisms by which 3 different heterochromatin structures (1. LADs 2. Pericentromeric heterochromatin and 3. Centromeric heterochromatin) repair DNA lesions. We have also developed Mass spectrometry approaches to identify proteins from the above heterochromatic compartments and we will discover for the first time in the literature the proteome of different types of heterochromatin in the presence of DNA damage.

Objective 3: What is the role of 3D genome organization in the kinetics of DNA repair and DNA repair pathway choice?
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 (Task 3.1 and 3.2). These approaches revealed break hotspots of the genome either due to replication or due to exogenous damage.