In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel assay to evaluate V(D)J recombination activity on thousands of RSSs where the 12-RSS heptamer and adjoining spacer region contained randomized sequences. While the consensus heptamer sequence (CACAGTG) was marginally preferred, V(D)J recombination was highly active on a wide range of non-consensus sequences. Select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site were generally preferred. In addition, while different coding flanks and nonamer sequences affected recombination efficiency, the relative dependency on the purine/pyrimidine motifs in the RSS heptamer remained unchanged. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions.

RAG2 of the V(D)J recombinase is essential for lymphocyte development. Within the RAG2 noncore region is a plant homeodomain (PHD) that interacts with the modified histone H3K4me3, and this interaction is important for relieving inhibition of the RAG recombinase for V(D)J recombination. However, the effect of the noncore region on RAG2 localization and dynamics in cell nuclei is poorly understood. Here, we used cell imaging to measure the effect of mutating the RAG2 noncore region on properties of the full length protein. We measured GFP-labeled full length RAG2 (FL), the RAG2 core region alone (Core), and a T490A mutant in the noncore region, which has unique regulatory properties. This showed that FL, T490A, and Core localized to nuclear domains that were adjacent to DAPI-rich heterochromatin, and that contained the active chromatin marker H3K4me3. Within the RAG2-enriched regions, T490A exhibited greater colocalization with H3K4me3 than either FL or Core. Furthermore, colocalization of H3K4me3 with FL and T490A, but not Core, increased in conditions that increased H3K4me3 levels. Superresolution imaging showed H3K4me3 was distributed as puncta that RAG2 abutted, and mobility measurements showed that T490A had a significantly lower rate of diffusion within the nucleus than either FL or Core proteins. Finally, mutating Trp 453 of the T490A mutant (W453A,T490A), which blocks PHD-dependent interactions with H3K4me3, abolished the T490A-mediated increased colocalization with H3K4me3 and slower mobility compared to FL. Altogether, these data show that Thr 490 in the noncore region modulates RAG2 localization and dynamics in the pre-B cell nucleus, such as by affecting RAG2 interactions with H3K4me3.

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RAG2 is essential for V(D)J recombination in developing lymphocytes, yet its localization and dynamics in cell nuclei is poorly understood. Here, we used single-cell imaging to interrogate RAG2 interactions with transcriptionally active chromatin containing H3K4me3. We measured GFP-labeled full length RAG2 (FL), the RAG2 core region alone (Core), and a T490A mutant of RAG2. Each RAG2 construct localized to nuclear regions that were adjacent to DAPI-rich heterochromatin, where the T490A RAG2 exhibited greater colocalization with H3K4me3 than either FL or Core. Furthermore, mobility measurements showed that T490A had a significantly lower rate of diffusion within the nucleus than either FL or Core proteins. Finally, mutating W453 in the T490A mutant (W453A, T490A), which blocks interactions between the RAG2 PHD region and H3K4me3, decreased the RAG2 colocalization with H3K4me3 and increased its mobility to values similar to that of FL. Altogether, these data show that Thr490 modulates RAG2 localization and dynamics in the pre-B cell nucleus, such as by affecting PHD-dependent interactions between RAG2 and H3K4me3.

In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel V(D)J recombination assay to evaluate RAG1/2 activity on thousands of RSSs. We focused our study on the RSS heptamer and adjoining spacer region, as this region undergoes extensive conformational changes during RAG-mediated DNA cleavage. While the consensus heptamer sequence (CACAGTG) was marginally preferred, RAG1/2 was highly active on a wide range of non-consensus sequences. RAG1/2 generally preferred select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions. Further investigation of RAG1/2 specificity using this new approach will help elucidate the genetic instructions guiding V(D)J recombination.Summary StatementPartially conserved recombination signal sequences (RSSs) govern antigen receptor gene assembly during V(D)J recombination. Here, a massively parallel analysis of randomized RSSs reveals key attributes that allow DNA sequence diversity in the RAG1/2 active site and that contribute to the differential utilization of RSSs in endogenous V(D)J recombination. Overall, these results will assist identification of RAG1/2 off-target sites, which can drive leukemia cell transformation, as well as characterization of bona fide RSSs used to generate antigen receptor diversity.

Developing B‐ and T‐cells produce a diverse antigen receptor repertoire by rearranging antigen receptor genes during VDJ recombination. Recombination activating proteins, RAG1 and RAG2, catalyze VDJ recombination by cleaving DNA between recombination signal sequences (RSS) and antigen receptor gene segments, and the double‐strand breaks are repaired by nonhomologous end‐joining machinery. The RSS is defined as containing a consensus heptamer and a nonamer sequence separated by a less conserved spacer sequence. However, many RSSs deviate from the consensus sequence. Therefore, RAG1/2 must be promiscuous to facilitate recombination of poorly conserved RSSs, but it must also be precise to avoid off‐target cryptic RSSs. Recent cryo‐EM and X‐ray crystallographic studies on RAG1/2 showed the RSS heptamer undergoes a dramatic structural transition in the RAG1/2 active site. Before cleavage, the heptamer region untwists by 180°, so RAG1/2 sidechains contacting the major groove switch to contacting the minor groove and vice‐versa. RAG1/2 also makes few base‐specific contacts prior to nicking. Therefore, we hypothesized that sequence‐specific structural properties of the RSS heptamer plays a role in RAG1/2 specificity by facilitating these structural transitions in the RAG1/2 active site. To test this hypothesis and better characterize the DNA sequence specificity of RAG1/2, we modified an episomal‐based VDJ recombination assay to quantify RAG1/2 activity in an unbiased approach using a large sequence‐diverse set of RSSs. While the consensus heptamer sequence was highly preferred, RAG1/2 showed similarly high activity on many different sequences. Notably, purine/pyrimidine (R/Y) content of RSS heptamers was a strong predictor of RAG1/2 activity, preferring alternating R/Y sequence motifs at nucleotide positions 5‐7 of the RSS heptamer. RAG1/2 also favored A+T base‐pairs in the first two positions of the RSS spacer. These data show RAG1/2 is highly active on many DNA sequences deviating from the consensus RSS, and R/Y content of RSS heptamers is a good predictor of RAG activity. Molecular dynamics simulations of RSSs using the parmbsc1 force field showed that DNA sequences preferred by RAG1/2 had unique twist distributions, particularly between nucleotide positions 5‐7. In conclusion, these data support our hypothesis that RAG1/2 can recognize many different DNA sequences, and sequence‐specific structural features of alternating R/Y nucleotides promote RAG1/2 cleavage. This mechanism of promiscuously selecting sequences for VDJ recombination yields a diverse antigen receptor repertoire, but it also highlights the constant threat of off‐target VDJ recombination events. Further investigation of RAG1/2 specificity will help elucidate the genetic instructions guiding VDJ recombination to antigen receptor gene segments.