Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - DNAendProtection (DNA end protection in Immunity and Cancer)

Teaser

This proposal addresses a fundamental question in biology: What are the molecular mechanisms that ensure both the integrity and diversity of our genome by steering DNA double-strand break repair towards the appropriate physiological outcome? Accurate repair of both accidental...

Summary

This proposal addresses a fundamental question in biology: What are the molecular mechanisms that ensure both the integrity and diversity of our genome by steering DNA double-strand break repair towards the appropriate physiological outcome? Accurate repair of both accidental and programmed DNA double-strand break 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 DNA double-strand break 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 DNA double-strand break 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 DNA double-strand break repair pathway choice and outcome. In mature B cells undergoing Class Switch Recombination, 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 pathological repair reactions leading to genomic instability. A deep understanding of the molecular mechanism(s) that mediate DNA end protection is crucial for our understanding of the regulation of DNA double-strand break repair outcome.

This proposal employs B lymphocytes as primary model system to study DNA double-strand break repair pathway choice, since they allow for the analysis of both physiological (Class Switch Recombination) and pathological (genomic instability) consequences of DNA end protection activity.

The proposed Specific Aims investigate complementary aspects of DSB end metabolism: DNA end protection versus resection. Specifically, the findings that will originate from these studies will inform us about: 1) the identity of the key components of the DNA end protection machinery; 2) the resection-promoting factors that are antagonized by such activity; and 3) how their functional relationships influence DNA double-strand break repair outcomes.

Work performed

During these first 18 months from the beginning of the project, we have successfully met key milestones for the implementation of our specific research goals:

- We have generated clonal derivatives of murine B cell lymphoma lines deficient for known Class Switch Recombination and DNA double-strand break factors to be employed as an additional model system in parallel with primary B cells. These cell lines are amenable to long-term selection procedures, thus considerably extending the range of experimental protocols that can be implemented for the project. The clonal cell line derivatives were generated via implementation of the CRISPR-Cas9 gene targeting technology in a high-throughput approach, and characterized at the genomic scar, protein levels, and Class Switch Recombination capabilities. These cell lines will be employed as key control cell lines and/or model systems for screening purposes throughout the project.

- We have optimized and performed proteomics-based strategies for the identification of novel Class Switch Recombination and DNA double-strand break factors in primary B cells.

- In parallel, robust loss-of-Class Switch Recombination and gain-of-proliferation screens have been set up and optimized for the purpose of screening newly identified candidates for their involvement in DNA double-strand break repair. Top hits from these functional screens are currently being validated and their role in Class Switch Recombination and DNA double-strand break repair is currently being investigated.

- Furthermore, we have designed and validated a novel DNA end resection assay to directly study the involvement of newly-identified Class Switch Recombination and DNA double-strand break factors in DNA end protection. The assay will be also used for the characterization of novel DNA end resection-promoting factors identified via our screens.

Final results

The studies proposed in this application will clarify the molecular mechanisms that control the delicate balance between DNA double-strand break end resection and protection. The implications of the findings from these studies are far-reaching:

- These mechanisms ensure productive DNA repair and safeguard the genome against toxic repair reactions or aberrant DNA end processing leading to chromosome instability and carcinogenesis. Inappropriate resection of DNA breaks can lead to deletion of genetic information. These events can have potentially oncogenic consequences if resection affects genome regions that might encode essential housekeeping or tumor suppressor genes. Indeed, chromosome deletions are hallmarks of a variety of cancer genomes. Therefore, the identification of novel end resection factors in B lymphocytes will be highly valuable to our understanding of the mechanisms that contribute to the loss of genetic information during potentially oncogenic chromosome rearrangements in mammalian cells.

- DNA double-strand break end protection activity is a major determinant of genomic instability in BRCA1-mutated cells. The characterization of the mechanisms governing this function will shed light on the molecular factors that predispose BRCA1-mutant carriers to carcinogenesis, and have important implications for the treatment of hereditary breast and ovarian cancers.

- The mechanisms of DNA double-strand break formation and repair are of particular relevance to B lymphocyte biology and lymphomagenesis. The characterization of the mechanisms underlying DNA end protection in the context of switching B lymphocytes will result in the discovery of novel factors required for Class Switch Recombination. These findings will not only broaden our basic knowledge of this process, but will also provide additional candidates for the identification of causative mutations in patients affected by genetically undefined Class Switch Recombination deficiencies.

- Furthermore, the high proliferative capability and the programmed DNA damage incurred during Class Switch Recombination render mature B cells particularly susceptible to aberrant DNA double-strand break repair reactions leading to chromosomal translocations, which are considered primary oncogenic events in the genesis of human germinal center lymphoma and multiple myeloma. Therefore, the identification of novel players in DNA double-strand break repair in the context of switching B cells will provide crucial insights into the mechanisms that predispose mature B lymphocytes to malignant transformation, and ultimately open new avenues to the prevention and therapy of lymphoma and multiple myeloma.