Jk DNA GAGA MOTIFS ARE REQUIRED FOR LOCAL NUCLEOSOME REMODELING AND Vk-Jk RECOMBINATION [RNA-Seq]

Immunoreceptor gene recombination requires complementary 12 bp and 23 bp recombination signal sequences (RSSs). In addition, the RSSs that assemble the RAG proteins, recombination centers, must be accessible yet flanked by a 5' nucleosome decorated with H3K4me3. In Drosophila, DNA GAGA motifs play an important role in nucleosome positioning. Herein, we report that 5' to each functional Jk RSS is a DNA GAGA motif conserved across mammalian species. In mice, the GAGA motif 5' to Jk1 regulated local RSS accessibility and 5' nucleosome placement. Furthermore, it was required for Vk-Jk1 recombination. Murine Jk3 is nonfunctional, having mutations in both RSS and GAGA motifs. Restoring both GAGA and RSS motifs rescued Vk-Jk3 recombination. In contrast, restoring the RSS alone did not. Genome-wide, strong cryptic 23 RSSs were preferentially bound to nucleosomes. Furthermore, evolutionary selection against cRSS only occurred in the A Compartment of B lymphocytes, not embryonic stem cells. These data indicate that in developing B cells, nucleosome positioning both enables and restricts recombination to Jk. Furthermore, our data suggest an expanded definition of recombination center-associated RSSs to include a 5' GAGA sequence that dictates the local epigenetic state required for gene recombination. Overall design: RNA-seq of flow-sorted small pre-b cells from WT and Jk1-GAGAmut mice.