Date of Award
Doctor of Philosophy (PhD)
V(D)J recombination, the process of antigen receptor gene diversification in lymphocytes, is critical for the immune system, but can have potentially dangerous consequences. DNA double-strand breaks (DSBs) generated by Rag during V(D)J recombination, if improperly repaired, can result in chromosomal translocations and cancer. The DNA damage response, mediated by Atm, normally prevents DSBs from progressing to translocations. In the absence of Atm, DSBs accumulate and form translocations at a high frequency. Atm-deficient cell lines were used to characterize the genomic targets of chromosomal aberrations resulting from mis-repaired Rag-induced DSBs at a retroviral substrate. Of 1903 sequenced targets, 81% mapped to the immunoglobulin heavy and light chain (Ig) loci, at which Rag DSBs are also generated. Additional DSBs induced by ionizing radiation were able to compete with DSBs at Ig loci as targets, as shown by the reduction from 81% to 19% in aberrations targeting Ig loci. Interestingly, most chromosomal aberration targets mapped to the same chromosome as the Rag DSB (60%), suggesting that DSBs in cis are preferentially joined. Furthermore, regions within 200 kb of the Rag DSB were preferentially targeted. This data demonstrates that chromosomal aberrations tend to form between pre-existing DSBs, and implies that there are mechanisms that preferentially join DSBs in cis.
An additional consequence of V(D)J recombination is that 2/3 of rearrangements are out-of-frame (OF), resulting in a nonsense mutation. Nonsense transcripts are normally degraded by nonsense-mediated decay (NMD) to prevent the expression of truncated proteins with potentially detrimental effects. Disruption of the classical pathway of NMD in vitro eliminates the degradation of nonsense T-cell receptor β (TCRβ) transcripts, while nonsense immunoglobulin kappa light chain (IgL) transcripts are degraded in vivo despite lacking features required to activate the classical pathway. We have used gene targeting to generate a modified TCRβ allele (TCRβF ) that is defective in activating classical NMD. Nonsense TCR transcripts accumulate in TCRβF/F thymocytes compared to control thymocytes, though nonsense transcripts are still downregulated three-fold. Despite the accumulation of nonsense transcripts, thymocyte development is normal in TCRβF/F mice, and peripheral T cells with OF rearrangements are not selected against.
Chair and Committee
Paul Allen, Kenneth Murphy, Andrey Shaw, Sheila Stewart, Wojciech Swat
Mahowald, Grace Kao, "Characterization of Genomic Targets of Chromosomal Aberrations and an In Vivo Model of Disrupted Nonsense Mediated Decay" (2011). Arts & Sciences Electronic Theses and Dissertations. 77.
Available for download on Friday, May 15, 2111