Epigenetic control of Microhomology Mediated End Joining (MMEJ) in heterochromatin of Lamina Associated Domains.

Cells are subjected to tens of thousands of DNA lesions a day, which require both rapid and high-fidelity repair to avoid deleterious genetic mutations and genomic rearrangements. Such genetic aberrations can deregulate gene expression and lead to diseases such as cancer. Since the discovery that the DNA is an unstable molecule, genetic screens in model organisms and detailed biochemical analyses have dissected the main pathways that repair DNA breaks. Although, there has been much progress in identifying key factors of DDR and DNA repair, it is not understood how they function in the nuclear space. A key feature of the mammalian cell nucleus is the non-random arrangement of the genome. Chromosomes are confined in discrete territories and within them further levels of spatial organisation are imposed on the chromatin. Our recent data show that DNA repair efficiency and pathway specificity is not the same everywhere in the nucleus and are in line with recent results that suggest that differential DNA repair is the cause of mutation variation across the genome. More specifically we find that DNA lesions in parts of the genome which associate with the periphery of the nucleus utilise erroneous DNA Repair in expense of error free. Whether the differential usage of error prone or error free pathways in different genomic locations is cause of mutation and genomic aberration variation around the genome and whether this is linked to the propensity of genomic regions to translocate is not at all clear. This projects aims to decipher the mechanism behind this phenomenon and the consequences for genome integrity and nuclear function. The questions we address here are of significant importance for human health because abnormalities in the regulation of this highly complex network of interactions can give rise to genetic diseases and/or cancer. For example after chemotherapy or radiotherapy, cancer patients often develop recurrent secondary tumors later in life. Greater knowledge of the role of 3D genome organization in regulating DNA repair efficiency and pathway choice will reveal the regions of the genome that are susceptible to genomic instability and help us understand why certain mutations and translocations are recurrent in cancers. Therefore, this project is very timely and will give groundbreaking insight into the compartmentalization of DNA repair, a largely unaddressed but very important gap in our knowledge to understand how nuclear architecture impinges on genome stability.

Cells are subjected to thousands of DNA lesions a day, which require high-fidelity repair to avoid genetic mutations and genomic rearrangements. These deleterious effects of DNA damage can contribute to tumour formation, and they are frequently exploited in anticancer therapies. A key feature of the mammalian cell nucleus is the non-random arrangement of the genome within the nuclear space. It is becoming increasingly clear that the nuclear organization of genomic DNA affects how cells cope with DNA damage by determining the balance of error free and error prone DNA repair. DSBs at heterochromatin and late replicate domains such as Lamina Associated domains (LADs), are prone to highly erroneous DNA repair which is in line with the high mutation rates detected in heterochromatin of cancer genomes. What shifts the balance towards mutagenic DNA repair in heterochromatin of LADs in expense of an error free DNA repair pathway and what are the consequences of this for the integrity of LADs and nuclear lamina are largely unexplored.
We have developed novel experimental tools that enable us to induce specific DSBs at LADs in a tightly controlled manner, to princely measure the kinetics of different DNA repair pathways in real time and to identify the proteome of LADs in the absence and presence of DNA damage. These tools will allow us to carefully dissect the epigenetic mechanisms that promote the activation mutagenic DNA repair at LADs and how these impact on maintenance of heterochromatin and nuclear lamina. Furthermore, they will allow us to understand how we can modify chromatin to shift DNA repair pathways choice and exploit it for cancer treatment. We will employ cutting-edge approaches such as Dam-ID, Bio-ID, TIDE/NGS and HT-microscopy to provide the first systematic account of DSB repair dynamics and fidelity in LADs, answering long-standing questions on the causative or passenger role of the underlying chromatin structure in genome stability.