Periodic Reporting for period 1 - TorsionAtLamina (TWISTING THE BOUNDARIES: ROLE OF TOPOISOMERASE 1 AT THE NUCLEAR LAMINA)
Période du rapport: 2020-09-01 au 2022-08-31
The DNA contained in one single cellular nucleus is 2 meters longs when stretched on a linear scale. Thus, fitting in a nucleus that is usually not bigger than 2 micrometers, requires that DNA has a high degree of compaction (1000000 times!!) and to achieve this the genome needs to be organized at several levels inside the nucleus. A correct genome organization is crucial to guarantee its proper function and stability. One crucial aspect of genome organization is anchoring of DNA to the nuclear lamina (NL), a scaffold that provides mechanical sustain to the nucleus. What controls the interaction between DNA and the NL is still a mystery. Among several proteins proposed to have a role in genome organization DNA topoisomerases have recently received particular attention. These enzymes can promote the relaxation of DNA that usually undergoes to substantial torsional stress during physiological processes inside the nucleus. By removing torsion from DNA, Topoisomerases can also control high order chromatin structures and favor and disfavor DNA compaction. DNA Topoisomerases are currently targeted in chemotherapy, thus understanding its biological function and interaction partners, especially in the context of 3D genome organization is very important to guarantee the development of new therapeutical approaches and identify potential additional targets to achieve more effective combinatorial therapies.
The goal of this project was to understand the relationship between DNA Topoisomerases and genome interaction with the nuclear lamina. DNA Topoisomerases were investigated for a potential role in controlling DNA-NL interaction using a specific technique called pA-DamID. I also tried to directly measure the torsional stress on chromatin, identifying the effects of both local and genome wide chromatin context in modulation of torsional stress and DNA structures that are topology dependent.
The goal of this project was to understand the relationship between DNA Topoisomerases and genome interaction with the nuclear lamina. DNA Topoisomerases were investigated for a potential role in controlling DNA-NL interaction using a specific technique called pA-DamID. I also tried to directly measure the torsional stress on chromatin, identifying the effects of both local and genome wide chromatin context in modulation of torsional stress and DNA structures that are topology dependent.
To better understand the roles of DNA topoisomerases in controlling contact between DNA and the nuclear lamina I performed topoisomerase depletion experiments followed by genome-wide mapping of DNA-NL interactions. I first targeted Top1, which was the first suspect for this proposal. However, several models of Top1 depletion showed that this topoisomerase is responsible for controlling DNA-NL contacts only at very minimal level. I decided to extend my analysis to two additional topoisomerases (Top2A and Top2B) and Identified Top2B as a key regulator of DNA-NL interactions. Indeed, after Top2B depletion DNA-NL contacts were remarkably reshaped with very big portion of the genome that moved toward the NL and some others that detach from it. I found that this depended on specific chromatin features, suggesting that Top2B could modulate these interactions according to the chromatin context. Finally, I found that Top2B modulated DNA-NL interaction in an almost identical way to LBR (lamin B receptor) a nuclear envelope tether which has a very important role in genome organization. Thus, Top2B and LBR might coordinate to guarantee proper genome organization.
These results were shown to several international conferences and really appreciated from the scientific community. At the end of the action I was collecting them in a manuscript that hopefully will be published in a high impact factor journal. These results will also allow me to open an original research line and apply for fundings that hopefully will allow me to be an independent scientist.
These results were shown to several international conferences and really appreciated from the scientific community. At the end of the action I was collecting them in a manuscript that hopefully will be published in a high impact factor journal. These results will also allow me to open an original research line and apply for fundings that hopefully will allow me to be an independent scientist.
CONCLUSION: I identified and new player in genome organisation, Top2B, which is a DNA relaxing enzyme able to modulate association of the genome. I also identified a new genetic partner of Top2B, LBR, a component of the nuclear envelope that together with Top2B controls DNA/NL association.
This work identified for the first time a new role for DNA Topoisomerase 2B in controlling genome organisation at level of DNA-NL interactions. This will open a new field of investigation. Many questions need to be answered. What are the consequences of this genome reshaping induced by Top2B loss of function? How does this affect genome stability and cell identity? In addition to this, the finding that LBR modulated DNA-NL interactions similarly to Top2B also suggests that I identified a new genetic interaction between these two proteins. This is important because it is very likely, that these two proteins work together in several important process that usually involve massive genome re-organisation like differentiation and oncogene induced senescence. I intend to investigate the role of these proteins in such processes in the near future. Thus, this study will pave the way to future works that can have strong relevance and impact on how we understand and target DNA topoisomerases and their potential genetic partners.
This work identified for the first time a new role for DNA Topoisomerase 2B in controlling genome organisation at level of DNA-NL interactions. This will open a new field of investigation. Many questions need to be answered. What are the consequences of this genome reshaping induced by Top2B loss of function? How does this affect genome stability and cell identity? In addition to this, the finding that LBR modulated DNA-NL interactions similarly to Top2B also suggests that I identified a new genetic interaction between these two proteins. This is important because it is very likely, that these two proteins work together in several important process that usually involve massive genome re-organisation like differentiation and oncogene induced senescence. I intend to investigate the role of these proteins in such processes in the near future. Thus, this study will pave the way to future works that can have strong relevance and impact on how we understand and target DNA topoisomerases and their potential genetic partners.