Date of Award
Doctor of Philosophy (PhD)
Craig H. Bassing
Developing B and T lymphocytes must rearrange the genomic sequence of antigen receptor genes by V(D)J recombination. The lymphocyte-specific endonuclease RAG, composed of Rag1 and Rag2, initiates this process by cleaving specific sites within antigen receptor loci. RAG expression must be carefully regulated to ensure that V(D)J recombination occurs only under appropriate circumstances. The Bassing laboratory has previously demonstrated that Igκ locus cleavage by RAG in pre-B cells initiates a feedback-inhibition signal suppressing RAG expression. Here, we show that DNA double strand breaks (DSBs) induced by a variety of genotoxic agents have a similar effect in suppressing mRNA expression of Rag1 and Rag2. This effect can be observed in pro-B cells, pre-B cells, and DN thymocytes, but is not found in DP thymocytes. Using primary pre-B cells as a model system, we show that DSBs activate ATM and Nemo to rapidly suppress transcription of Rag1 and Rag2. In pre-B cells, loss of Rag1 and Rag2 mRNA expression leads to loss of Rag1 protein, but Rag2 protein is more stable and persists in the absence of Rag2 mRNA. Suppression of Rag1 expression by DSBs is associated with suppressed RAG-mediated cleavage of the Igκ locus or an artificial recombination substrate in Abelson-transformed pre-B cells. However, simply over-expressing Rag1 does not allow cells to complete V(D)J recombination in the presence of DSBs, suggesting that other factors may also play a role in suppressing V(D)J recombination. Parallel studies indicate that that RAG-induced DSBs created during V(D)J recombination activate this suppressive signal to enforce allelic exclusion of IgH, TCRβ, and Igκ antigen receptor proteins. We discuss the importance of Rag1 and Rag2 suppression in the context of allelic exclusion and propose a role in maintaining genomic stability of developing B and T lymphocytes.
Fisher, Megan Rose, "Dna Double Strand Breaks Suppress Expression Of The Rag Recombinase: Mechanisms And Consequences" (2017). Publicly Accessible Penn Dissertations. 2280.