Periodic Reporting for period 4 - DNAendProtection (DNA end protection in Immunity and Cancer)
Reporting period: 2020-03-01 to 2021-06-30
Accurate repair of both accidental and programmed DNA double-strand breaks (DSBs) is a physiological and beneficial aspect of DNA metabolism since it ensures preservation of the somatic genome on one side, and guarantees generation of diversity during meiotic recombination and lymphocyte receptor gene rearrangement reactions on the other. Indeed, defective DSB repair is responsible for several human genetic disorders characterized by developmental and neurological defects, cancer predisposition, and immunodeficiency. A deep understanding of the mechanisms regulating DSB repair outcome is therefore of the outmost importance for the characterization of the molecular basis of several human pathologies including cancer and immunodeficiency.
DNA end protection activity against DNA resection is a crucial determinant of DSB repair outcome. In mature B lymphocytes undergoing Class Switch Recombination (CSR), DNA end protection is beneficial as it ensures productive repair events leading to antibody gene diversification by class switching. However, in the absence of a functional BRCA1 protein, DNA end protection prevents appropriate repair of DNA replication-associated breaks, and promotes aberrant repair reactions leading to genomic instability. The investigation of the molecular mechanism(s) that mediate DNA end protection is crucial for our understanding of the regulation of DSB repair outcome.
DNA end protection activity against DNA resection is a crucial determinant of DSB repair outcome. In mature B lymphocytes undergoing Class Switch Recombination (CSR), DNA end protection is beneficial as it ensures productive repair events leading to antibody gene diversification by class switching. However, in the absence of a functional BRCA1 protein, DNA end protection prevents appropriate repair of DNA replication-associated breaks, and promotes aberrant repair reactions leading to genomic instability. The investigation of the molecular mechanism(s) that mediate DNA end protection is crucial for our understanding of the regulation of DSB repair outcome.
DSBs are cytotoxic DNA lesions that arise as a by-product of DNA replication, but also as a physiological intermediate during antigen receptor diversification in the immune system. DNA end processing is a major determinant of DSB repair outcome. Resection of DNA ends is a prerequisite for physiological repair of replication-associated breaks by homologous recombination, but detrimental for productive end-joining events during immunoglobulin CSR in B lymphocytes. We and others had previously shown that the DNA repair factor 53BP1 plays a crucial role in protecting DNA ends against resection, and consistent with this function, 53BP1 is essential for CSR, but also responsible for aberrant repair of replication-associated DNA damage. Furthermore, we identified the first DNA end protection effector downstream 53BP1, the DSB repair protein RIF1, which is recruited to the break sites in a manner dependent on the DSB-induced phosphorylation of 53BP1.
In this grant, we proposed to study the molecular machinery that mediates protection of DNA ends, and the end resection-promoting factors that are antagonized by this activity. To do so, we employed mature B lymphocytes as our primary model system since in addition to experiencing programmed DNA breaks during CSR, these cells are highly proliferative and therefore susceptible to stochastic replication-associated damage, thus allowing us to comprehensively investigate the mechanisms ensuring genome diversity and stability, and their relationship.
A major goal of the project was to dissect the mechanisms underlying the role of key DNA repair factors in CSR and DSB end protection. This was achieved first via the analysis of the molecular determinants of these factors in term of domains and post-translational modifications (PTMs) required for their function during repair of CSR DSBs. As a secondary approach, we aimed to identify their functional partners. To do so, we have developed and optimized SILAC-based proteomics approaches optimized for the identification of bona fide and likely transient/labile interactors of DNA repair factors in primary cultures of splenocytes (B lymphocytes). Using this proteomics approach, we have defined their interactomes in B lymphocytes undergoing CSR. In parallel, we have set up robust loss-of-CSR as well as gain-of-proliferation assays in the B cell lymphoma line CH12 and derived genotypes, and applied it to functionally screen the interactome lists to dissect the contribution of the newly-identified factors in the repair of both programmed CSR breaks as well as replication-associated DSBs.
The second major goal was to define the landscape of DNA end resection-promoting factors in B cells by exploiting the relationship between DNA end protection and CSR. To do so, we have designed a screening workflow for genome-wide analysis of pathways promoting resection of CSR breaks in activated B cells. The strategy design took advantage of unexpected findings we uncovered during the initial phase of the project execution, which unveiled a yet-to-be-defined function of 53BP1 that is mediated by its capability to form higher-order oligomers and is dominant over its DNA end protection activity during CSR.
In this grant, we proposed to study the molecular machinery that mediates protection of DNA ends, and the end resection-promoting factors that are antagonized by this activity. To do so, we employed mature B lymphocytes as our primary model system since in addition to experiencing programmed DNA breaks during CSR, these cells are highly proliferative and therefore susceptible to stochastic replication-associated damage, thus allowing us to comprehensively investigate the mechanisms ensuring genome diversity and stability, and their relationship.
A major goal of the project was to dissect the mechanisms underlying the role of key DNA repair factors in CSR and DSB end protection. This was achieved first via the analysis of the molecular determinants of these factors in term of domains and post-translational modifications (PTMs) required for their function during repair of CSR DSBs. As a secondary approach, we aimed to identify their functional partners. To do so, we have developed and optimized SILAC-based proteomics approaches optimized for the identification of bona fide and likely transient/labile interactors of DNA repair factors in primary cultures of splenocytes (B lymphocytes). Using this proteomics approach, we have defined their interactomes in B lymphocytes undergoing CSR. In parallel, we have set up robust loss-of-CSR as well as gain-of-proliferation assays in the B cell lymphoma line CH12 and derived genotypes, and applied it to functionally screen the interactome lists to dissect the contribution of the newly-identified factors in the repair of both programmed CSR breaks as well as replication-associated DSBs.
The second major goal was to define the landscape of DNA end resection-promoting factors in B cells by exploiting the relationship between DNA end protection and CSR. To do so, we have designed a screening workflow for genome-wide analysis of pathways promoting resection of CSR breaks in activated B cells. The strategy design took advantage of unexpected findings we uncovered during the initial phase of the project execution, which unveiled a yet-to-be-defined function of 53BP1 that is mediated by its capability to form higher-order oligomers and is dominant over its DNA end protection activity during CSR.
By elucidating the molecular mechanisms underlying DSB end processing in B lymphocytes, these studies have provided a catalogue of factors that have the potential to advance our understanding of the molecular basis of immunodeficiencies and cancer predisposition in lymphoma and solid tumors. Furthermore, the investigation of the mechanisms and pathways that are responsible for the regulation of DNA end processing have uncovered unanticipated findings linked to other aspects of antibody diversification and the preservation of genome integrity in B cells, including the involvement of key DNA repair factors or their interactors in other steps of CSR, and a potential cross-talk between pathways that control the generation of programmed DNA damage in immunoglobulin genes and the ability of B cells to adjust to the cellular stress imposed by their activation.