Functions of DNA Damage Response Factors in Lymphocyte Development and Transformation

DNA double strand breaks (DSBs) can activate cell cycle checkpoints or apoptosis, and lead to genomic alterations that drive malignant transformation. The H2AX core histone variant is phosphorylated in chromatin around DSBs by kinases such as ATM and DNA-PKcs. However, how H2AX suppresses chromosome breaks and translocations in cells and prevents tumorigenesis in mice and humans is not well understood. V(D)J recombination is a genetically programmed DNA damage and repair process that assembles the variable region exons of antigen receptor genes in developing lymphocytes. Using an inducible V(D)J recombination system, I found that H2AX is phosphorylated along cleaved antigen receptor loci DNA strands, prevents their irreversible separation in G1 phase, and reduces chromosome breaks and translocations in subsequent cell cycles. Consistent with H2AX functions in DSB repair, I also demonstrated that conditional H2AX deletion results in accumulation of genomic instability in cells, but delays tumor onset in a mouse thymic lymphoma model, presumably due to increased death of cells with synthetic loss of multiple repair factors. To further test this possibility, I generated cells and mice deficient in both H2AX and ATM to examine whether ATM-independent H2AX functions downstream of other kinases are essential for proper DSB repair. I found that thymocyte-specific ablation of H2AX in ATM-deficient mice results in a 50% reduction in thymus cellularity but does not accelerate or delay tumorigenesis. My results suggest that the outcomes of functional interactions between DNA damage response factors likely depend on the cellular context. Additionally, I discovered a novel function of ATM in regulating mono-allelic recombination at the immunoglobulin light chain locus. ATM could orchestrate the signaling pathways enforcing allelic exclusion to provide a time window to test whether the rearrangement on the first allele is productive. In summary, my work has provided novel mechanistic insights into how DNA damage and repair factors coordinately regulate V(D)J recombination, lymphocyte development and neoplastic transformation