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TWISTING THE BOUNDARIES: ROLE OF TOPOISOMERASE 1 AT THE NUCLEAR LAMINA

Project description

Regulation of DNA torsional stress at nuclear lamina

DNA transcription or replication creates torsional stress in the double helix. The enzyme that removes this stress is DNA topoisomerase I (Top1), a target of cancer chemotherapy. Loss of Top1 activity causes torsional stress accumulation in transcriptionally active genes and can lead to the formation of non-canonical DNA/RNA hybrid structures, called R loops. Recently, the project's team showed that depletion of Top1 leads to R loop stabilisation, specifically in genes anchored to the nuclear lamina. This observation strongly suggests that attachment of DNA to the nuclear lamina may prevent the dissipation of torsional stress. The EU-funded TorsionAtLamina project investigates the relationship between torsional stress, Top1, R loops and nuclear lamina attachment, employing genomics techniques developed in the host lab.

Objective

When DNA is transcribed or replicated, torsional stress accumulates on the double helix. This tension must be dissipated by spreading it along the DNA fiber, or it must be removed altogether. One of the main factors responsible for the removal of such stress is DNA Topoisomerase I (Top1), an important target of cancer chemotherapy. When Top1 activity is lost, torsional stress accumulates on transcriptionally active genes and can lead to the formation of non-canonical DNA/RNA hybrid structures called R loops. These structures are emerging as important regulators of genome function and stability. By genome-wide mapping of R loops in human cells, I recently found that depletion of Top1 leads to a marked R loop stabilization, specifically on genes that are anchored to the nuclear lamina. This strongly suggests that attachment of DNA to the nuclear lamina may prevent dissipation of torsional stress, but how this works is still largely unclear. I propose to investigate the causal relationships between torsonal stress, Top1, R-loops and nuclear lamina attachment, taking advantage of a suite of unique genomics techniques developed in the host lab. Specifically, I will: 1) Develop two novel reporter assays to probe the effects of chromatin context (in particular lamina associated chromatin) and Top1 on torsional stress and R loop formation, at thousands of locations in the human genome. 2) Investigate if and how Top1 regulates DNA/nuclear lamina contacts by by means of a novel version of the powerful genome-wide DamID mapping method with much-improved time resolution. My expertise in Top1, R-loops and DNA topology combined with the unique genomics methodologies in the host lab, as well as their expertise in lamina-associated DNA, will lead to a unique synergy that should result in new insights into the relationship between nuclear organization, torsional stress and R loop formation. Moreover, it will yield new methods that will boost scientific progress in this field.

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Coordinator

STICHTING HET NEDERLANDS KANKER INSTITUUT-ANTONI VAN LEEUWENHOEK ZIEKENHUIS
Net EU contribution
€ 175 572,48
Address
Plesmanlaan 121
1066 CX Amsterdam
Netherlands

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Region
West-Nederland Noord-Holland Groot-Amsterdam
Activity type
Research Organisations
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Other funding
€ 0,00