Regulation of V(D)J Recombination: Mechanisms that Govern Locus Accessibility and Mitigate Against Genomic Instability
Embargo until
Date
2020-07-22
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Johns Hopkins University
Abstract
V(D)J recombination is the process that generates the primary antigen receptor repertoire of the adaptive immune system. This process involves the ordered rearrangement of gene segments within antigen receptor loci and is initiated by the V(D)J recombinase, comprised of the recombination activating gene 1 and 2 proteins (RAG-1 and RAG-2). V(D)J recombination involves the programmed introduction of DNA double stranded breaks in the genome and is therefore tightly regulated to ensure genomic stability. Epigenetic regulation of V(D)J recombination by trimethylation at lysine 4 of histone 3(H3K4me3) is modulated through the plant homeodomain (PHD) of RAG-2. Binding of the RAG-2 PHD to H3K4me3 couples V(D)J recombination to active transcription and H3K4me3 allosterically activates RAG. In this dissertation, we demonstrate that the binding and allosteric activation of H3K4me3 are separable functions of the RAG-2 PHD.
Temporal regulation of V(D)J recombination is an additional method of preserving genomic integrity. RAG-2 undergoes periodic destruction at the G1-to-S transition through phosphorylation at T490 and subsequent degradation by the ubiquitin-proteasome pathway. This limits RAG activity to the G1 cell cycle phase, during which nonhomologous end joining (NHEJ) is the predominant form of DNA double stranded break repair. In this dissertation, we employ a mouse model in which RAG-2 protein is constitutively expressed and DNA double stranded breaks accumulate throughout the cell cycle. To characterize genomic rearrangements that result from misregulated RAG activity, we developed a capture-sequencing approach. We demonstrate that this method is capable of identifying endogenous V(D)J rearrangements and novel translocations involving antigen receptor loci.
Description
Keywords
V(D)J recombination, Allostery, PHD