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

Descrizione del progetto

Regolazione dello stress torsionale del DNA a livello di lamina nucleare

La trascrizione o replicazione del DNA crea uno stress torsionale nella doppia elica. L’enzima che rimuove questo stress è la DNA topoisomerasi I (Top1), uno dei bersagli della chemioterapia antitumorale. La perdita di attività di Top1 provoca l’accumulo di stress torsionale in geni trascrizionalmente attivi e può portare alla formazione di strutture ibride DNA/RNA non canoniche, denominate loop R. Recentemente, il team del progetto ha dimostrato che l’esaurimento di Top1 porta alla stabilizzazione del loop R, in particolare nei geni ancorati alla lamina nucleare. Questa osservazione suggerisce fortemente che l’attaccamento del DNA alla lamina nucleare potrebbe impedire la dissipazione dello stress torsionale. Il progetto TorsionAtLamina, finanziato dall’UE, indaga la relazione tra stress torsionale, Top1, loop R e attaccamento alla lamina nucleare, impiegando tecniche di genomica sviluppate nel laboratorio ospitante.

Obiettivo

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.