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Abstract

V(D)J recombination is a spatially and temporally restricted process, and depends heavily on epigenetic regulation to provide this control. The failure of V(D)J recombination would result in an immunodeficiency, while the lack of regulation of this process can result in genomic instability and oncogenic transformation. Brwd1 is an epigenetic reader required for normal B cell development, and specifically for Igk recombination. Brwd1 is recruited to activating histone marks, and when binding is coincident with GAGA DNA repeats, it appears to participate in epigenetic remodeling by altering nucleosome positioning and enhancing DNA accessibility. During normal Igk recombination, Brwd1 binds at the Jk gene cluster and repositions nucleosomes 5’ to each Jk segment, exposing the RSS, allowing for RAG recruitment. Here, we examine whether GAGA repeat domains are required for the chromatin remodeling activity of Brwd1. Using CRISPR-Cas9, we removed the 5’ GAGA motif from Jk1 and Jk2 in mice. This resulted in not only a dramatic loss of usage of Jk1 in the Jk1-GAGA-deletion mice, but also in a partial developmental block during Igk recombination and lower overall kappa usage. Furthermore, the architecture of nucleosome positioning is altered in the Jk locus in small pre-B cells from mice with the Jk1 GAGA deletion. The Jk2 GAGA deletion also shows decreased usage of the gene segment proximal to the deletion, but with a less severe overall defect. Our work adds new insights to the accessibility hypothesis of ordered recombination, and demonstrates the importance of absolute primary nucleosome positioning at Jk in cells preparing for recombination. Furthermore, it provides evidence that GAGA motif domains are required for the role of Brwd1 in chromatin remodeling. Finally, our results inspired a new perspective on how cryptic RSSs throughout the genome are shielded from off-target recombination by the RAG recombinase, thus aiding in the maintenance of genomic integrity.

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