RAG-mediated DNA Cleavage Research Articles

Updating the definition of V(D)J recombination signal sequences with high-throughput recombination assays

Abstract V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Antigen receptor diversity is required for adaptive immunity in all jawed vertebrates. 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, and the molecular mechanism for semi-selective V(D)J recombination specificity is unknown. Here, we developed a cell-based, high-throughput V(D)J recombination assay to evaluate RAG1/2 activity on thousands of RSSs in parallel. Specifically, we focused our study on the RSS heptamer and adjoining spacer region because it 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 selected purine/pyrimidine motifs instead of specific bases. Molecular dynamics simulations of RSSs showed that DNA sequences preferred by RAG1/2 had dynamic structural features that may accommodate heptamer unwinding in the RAG1/2 active site. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by dynamic DNA properties dependent on purine/pyrimidine pattern. Further investigation of RAG1/2 specificity will help elucidate the genetic instructions guiding V(D)J recombination to antigen receptor gene segments. National Institutes of Health (AI128137, AI15635); Oklahoma Center for Advancement in Science and Technology (OCAST) [HR21-142]; Presbyterian Health Foundation of Oklahoma City; National Institute of Allergy and Infectious Diseases Immunology training grant [T32 AI7633-16]

Risky business blockade: RAG2 basic region blocks V(D)J recombinase function upon genotoxic stress in DNA damage response

Abstract The V(D)J recombinase, composed of the recombination activating gene (RAG) proteins, RAG1 and RAG2, generate functional antigen receptor genes through the repeated generation of DNA double strand breaks and DNA repair. However, this process can lead to increased genomic instability particularly in the presence of excess DNA breaks. Previously, we showed in pre-B cells that full length RAG2, a nuclear protein, will export to the cytoplasm with DNA damage in an ATM-dependent manner with eventual recovery back into the nucleus following DNA repair. We propose that re-localization of RAG2 prevents RAG-mediated DNA cleavage in the presence of excess DNA ends. Here, by using fluorescently tagged RAG proteins, we show that DNA damage-induced export of the V(D)J recombinase can be recapitulated in non-lymphoid cells. Further, we determined that mutations in the intrinsically-disordered region of RAG2 have distinct effects on this process. Specifically, a basic region mutant of RAG2 (K/R 518–524 A) alters the localization pattern of the V(D)J recombinase, and yet its pre-DNA damage localization pattern is re-established upon DNA repair. Conversely, a T490A mutant blocks relocalization of RAG2 following genotoxic stress for both the single T490A mutant and the combined T490A/basic region mutant. Together, these results demonstrate the role of the intrinsically-disordered region in RAG2 in regulating the mobility and subcellular localization of the V(D)J recombinase upon cellular conditions that may be deemed too risky for continued DNA rearrangements. Supported by NIA T32AG052363, NIH AI128137, OCAST

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity is required for V(D)J recombination.

A whole-genome CRISPR/Cas9 screen identified ATP2A2, the gene encoding the Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2 protein, as being important for V(D)J recombination. SERCAs are ER transmembrane proteins that pump Ca2+ from the cytosol into the ER lumen to maintain the ER Ca2+ reservoir and regulate cytosolic Ca2+-dependent processes. In preB cells, loss of SERCA2 leads to reduced V(D)J recombination kinetics due to diminished RAG-mediated DNA cleavage. SERCA2 deficiency in B cells leads to increased expression of SERCA3, and combined loss of SERCA2 and SERCA3 results in decreased ER Ca2+ levels, increased cytosolic Ca2+ levels, reduction in RAG1 and RAG2 gene expression, and a profound block in V(D)J recombination. Mice with B cells deficient in SERCA2 and humans with Darier disease, caused by heterozygous ATP2A2 mutations, have reduced numbers of mature B cells. We conclude that SERCA proteins modulate intracellular Ca2+ levels to regulate RAG1 and RAG2 gene expression and V(D)J recombination and that defects in SERCA functions cause lymphopenia.

An Allosteric Mechanism for Epigenetic Activation of the V(D)J Recombinase

V(D)J recombination, the process by which antigen receptor genes are assembled from discrete DNA segments during lymphoid development, is responsible for the generation of the primary immune repertoire. Errors in V(D)J recombination have been implicated in the pathogenesis of lymphoid malignancies, including follicular lymphoma, MALT lymphoma and mantle cell lymphoma. V(D)J recombination is initiated by a specialized transposase, RAG, consisting of RAG-1 and RAG-2 subunits. RAG mobilizes participating gene segments in a site-specific fashion by cleaving DNA at conserved recombination signal sequences. The accessibility of these sequences to RAG is subject to locus- and developmental stage-specific control by mechanisms that are as yet poorly understood. Elucidation of these mechanisms is fundamental to our understanding of the off-target events linking RAG activity to tumorigenesis. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). RAG-2 contains a plant homeodomain (PHD) finger that binds specifically to H3K4me3. Disruption of this PHD finger impairs V(D)J recombination in vivo. Peptides bearing H3K4me3 stimulate substrate binding and catalysis of DNA cleavage by RAG. This stimulation is dependent on an intact PHD finger, suggesting that H3K4me3 is an allosteric activator of the V(D)J recombinase. Indeed, binding of H3K4me3 to the RAG-2 PHD induces dynamic conformational changes in RAG-1. Because substrate binding and catalysis are functions of RAG-1, information regarding occupancy of the RAG-2 PHD must be transmitted to the RAG-1 subunit. To understand how the recognition of active chromatin is coupled to the binding and cleavage of recombination signal sequences, we sought to trace the path of allostery from the RAG-2 PHD finger to RAG-1. Our strategy has been: (1) to generate chimeric RAG-2 proteins in which the mouse PHD finger is replaced by the PHD finger of a phylogenetically distant RAG-2; (2) to identify chimeric RAG-2 proteins that are capable of binding H3K4me3 but incapable of allosteric activation; (3) to systematically back-mutate residues in the foreign PHD to the mouse sequence; and (4) to identify back-mutations that rescue allosteric activation. A chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the bamboo shark, C. punctatum, fails to support V(D)J recombination in vivo. This chimeric protein retains the ability to bind H3K4me3 but engagement of H3K4me3 does not result in allosteric activation, suggesting that the allosteric interface of the PHD finger is disrupted. The amino acid sequence differences between mouse and C. punctatum form several clusters, located on the opposite side of the PHD from the H3K4me3 binding site. Each of these clusters in the C. punctatum PHD finger was mutated to the mouse sequence and the corresponding back-mutated chimeric RAG-2 proteins were tested for their ability to support V(D)J recombination. Strikingly, mutation of one such cluster, corresponding to residues 425 - 429, 431 and 433 of mouse RAG-2, was sufficient to rescue recombination activity to the level of wild-type. Taken together, our observations indicate that the binding of H3K4me3 by RAG-2 is itself insufficient to support recombination; rather, information regarding the engagement of H3K4me3 must be transmitted allosterically. Moreover, our mutational analysis has identified a putative allosteric surface within the PHD finger and distinct from the H3K4me3 binding site that is responsible for transmitting the allosteric signal. The requirement for allosteric activation by H3K4me3 may play a role in defining patterns of RAG-mediated DNA cleavage during normal development and in the generation of lymphoid malignancies. DisclosuresDesiderio:Genentech: Consultancy; AbbVie: Consultancy.

Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination.

V(D)J recombination is initiated by the binding of the RAG1 and RAG2 proteins to recombination signal sequences (RSSs) that consist of conserved heptamer and nonamer sequences separated by a spacer of either 12 or 23 bp. Here, we used RAG-inducible pro-B v-Abl cell lines in conjunction with chromatin immunoprecipitation to better understand the protein and RSS requirements for RAG recruitment to chromatin. Using a catalytic mutant form of RAG1 to prevent recombination, we did not observe cooperation between RAG1 and RAG2 in their recruitment to endogenous Jκ gene segments over a 48-h time course. Using retroviral recombination substrates, we found that RAG1 was recruited inefficiently to substrates lacking an RSS or containing a single RSS, better to substrates with two 12-bp RSSs (12RSSs) or two 23-bp RSSs (23RSSs), and more efficiently to a substrate with a 12/23RSS pair. RSS mutagenesis demonstrated a major role for the nonamer element in RAG1 binding, and correspondingly, a cryptic RSS consisting of a repeat of CA dinucleotides, which poorly re-creates the nonamer, was ineffective in recruiting RAG1. Our findings suggest that 12RSS-23RSS cooperation (the "12/23 rule") is important not only for regulating RAG-mediated DNA cleavage but also for the efficiency of RAG recruitment to chromatin.

Histone reader BRWD1 targets and restricts recombination to the Igk locus.

B lymphopoiesis requires that immunoglobulin genes be accessible to the RAG1-RAG2 recombinase. However, the RAG proteins bind widely to open chromatin suggesting that additional mechanisms must restrict RAG-mediated DNA cleavage. Here, we demonstrate developmental downregulation of interleukin 7 (IL-7) receptor signaling in small pre-B cells induced expression of the bromodomain family member BRWD1, which was recruited to a specific epigenetic landscape at Igk dictated by pre-BCR-dependent Erk activation. BRWD1 enhanced RAG recruitment, increased gene accessibility and positioned nucleosomes 5′ to each Jκ recombination signal sequence. BRWD1 thus targets recombination to Igk and places recombination within the context of signaling cascades that control B cell development. Our findings provide a paradigm in which, at any particular antigen receptor locus, specialized mechanisms enforce lineage and stage specific recombination.

A non-B DNA can replace heptamer of V(D)J recombination when present along with a nonamer: implications in chromosomal translocations and cancer

The RAG (recombination-activating gene) complex is responsible for the generation of antigen receptor diversity by acting as a sequence-specific nuclease. Recent studies have shown that it also acts as a structure-specific nuclease. However, little is known about the factors regulating this activity at the genomic level. We show in the present study that the proximity of a V(D)J nonamer to heteroduplex DNA significantly increases RAG cleavage and binding efficiencies at physiological concentrations of MgCl(2). The position of the nonamer with respect to heteroduplex DNA was important, but not orientation. A spacer length of 18 bp between the nonamer and mismatch was optimal for RAG-mediated DNA cleavage. Mutations to the sequence of the nonamer and deletion of the nonamer-binding domain of RAG1 reinforced the role of the nonamer in the enhancement in RAG cleavage. Interestingly, partial mutation of the nonamer did not significantly reduce RAG cleavage on heteroduplex DNA, suggesting that even cryptic nonamers were sufficient to enhance RAG cleavage. More importantly, we show that the fragile region involved in chromosomal translocations associated with BCL2 (B-cell lymphoma 2) can be cleaved by RAGs following a nonamer-dependent mechanism. Hence our results from the present study suggest that a non-B DNA can replace the heptamer of RSS (recombination signal sequence) when present adjacent to nonamers, explaining the generation of certain chromosomal translocations in lymphoid malignancies.

Coupling of V(D)J Recombination to the Cell Cycle Suppresses Genomic Instability and Lymphoid Tumorigenesis

V(D)J gene segment recombination is linked to the cell cycle by the periodic phosphorylation and destruction of the RAG-2 protein at the G1-to-S cell cycle transition. To examine the function of this coupling, we constructed mice in which the phosphorylation site at threonine 490 of RAG-2 was mutated to alanine. The RAG-2(T490A) mutation uncoupled DNA cleavage from cell cycle and promoted aberrant recombination. Similar aberrant recombination products were observed in mice deficient in the Skp2 ubiquitin ligase subunit, which is required for periodic destruction of RAG-2. On a p53-deficient background, the RAG-2(T490A) mutation induced lymphoid malignancies characterized by clonal chromosomal translocations involving antigen receptor genes. Taken together, these observations provide a direct link between the periodic destruction of RAG-2 and lymphoid tumorigenesis. We infer that cell cycle control of the V(D)J recombinase limits the potential genomic damage that could otherwise result from RAG-mediated DNA cleavage.

HMGB1/2 can target DNA for illegitimate cleavage by the RAG1/2 complex

BackgroundV(D)J recombination is initiated in antigen receptor loci by the pairwise cleavage of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins via a nick-hairpin mechanism. The RSS contains highly conserved heptamer (consensus: 5'-CACAGTG) and nonamer (consensus: 5'-ACAAAAACC) motifs separated by either 12- or 23-base pairs of poorly conserved sequence. The high mobility group proteins HMGB1 and HMGB2 (HMGB1/2) are highly abundant architectural DNA binding proteins known to promote RAG-mediated synapsis and cleavage of consensus recombination signals in vitro by facilitating RSS binding and bending by the RAG1/2 complex. HMGB1/2 are known to recognize distorted DNA structures such as four-way junctions, and damaged or modified DNA. Whether HMGB1/2 can promote RAG-mediated DNA cleavage at sites lacking a canonical RSS by targeting or stabilizing structural distortions is unclear, but is important for understanding the etiology of chromosomal translocations involving antigen receptor genes and proto-oncogene sequences that do not contain an obvious RSS-like element.ResultsHere we identify a novel DNA breakpoint site in the plasmid V(D)J recombination substrate pGG49 (bps6197) that is cleaved by the RAG proteins via a nick-hairpin mechanism. The bps6197 sequence lacks a recognizable heptamer at the breakpoint (5'-CCTGACG-3') but contains a nonamer-like element (5'-ACATTAACC-3') 30 base pairs from the cleavage site. We find that RAG-mediated bps6197 cleavage is promoted by HMGB1/2, requiring both HMG-box domains to be intact to facilitate RAG-mediated cleavage, and is stimulated by synapsis with a 12-RSS. A dyad-symmetric inverted repeat sequence lying 5' to the breakpoint is implicated as a target for HMGB1/2 activity.ConclusionWe have identified a novel DNA sequence, called bps6197, that supports standard V(D)J-type cleavage despite the absence of an apparent heptamer motif. Efficient RAG-mediated bps6197 cleavage requires the presence of HMGB1/2, is stimulated by synapsis with a 12-RSS partner, and is directed in part by an inverted repeat sequence adjacent to the DNA cleavage site. These results have important implications for understanding how the RAG proteins can introduce a DNA double-strand break at DNA sequences that do not contain an obvious heptamer-like motif.

Mobilization of RAG-Generated Signal Ends by Transposition and Insertion In Vivo

In addition to their essential roles in V(D)J recombination, the RAG proteins have been found to catalyze transposition in vitro, but it has been difficult to demonstrate transposition by the RAG proteins in vivo in vertebrate cells. As genomic instability and chromosomal translocations are common outcomes of transposition in other species, it is critical to understand if the RAG proteins behave as a transposase in vertebrate cells. To facilitate this, we have developed an episome-based assay to detect products of RAG-mediated transposition in the human embryonic kidney cell line 293T. Transposition events into the target episome, accompanied by characteristic target site duplications, were detected at a low frequency using RAG1 and either truncated "core" RAG2 or full-length RAG2. More frequently, insertion of the RAG-generated signal end fragment into the target was accompanied by deletions or more complex rearrangements, and our data indicate that these events occur by a mechanism that is distinct from transposition. An assay to detect transposition from an episome into the human genome failed to detect bona fide transposition events but instead yielded chromosome deletion and translocation events involving the signal end fragment mobilized by the RAG proteins. These assays provide a means of assessing RAG-mediated transposition in vivo, and our findings provide insight into the potential for the products of RAG-mediated DNA cleavage to cause genome instability.

Both High Mobility Group (HMG)-boxes and the Acidic Tail of HMGB1 Regulate Recombination-activating Gene (RAG)-mediated Recombination Signal Synapsis and Cleavage in Vitro

RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.

Ubiquitylation of RAG-2 by Skp2-SCF Links Destruction of the V(D)J Recombinase to the Cell Cycle

The periodic destruction of RAG-2 at the G1-to-S transition couples V(D)J recombination to the G0 and G1 cell cycle phases and coordinates RAG-mediated DNA cleavage with DNA repair by nonhomologous end joining. To define the mechanism by which this occurs, we reproduced cell cycle-dependent regulation of the V(D)J recombinase in a cell-free system. The ubiquitin-proteasomal pathway carries out destruction of RAG-2 in lysates of S phase cells and during S phase in vivo. Remarkably, the Skp2-SCF ubiquitin ligase, which plays a central role in cell cycle regulation through the destruction of p27, mediates ubiquitylation of RAG-2 in vitro and degradation of RAG-2 in vivo. The regulation of antigen receptor gene assembly by Skp2-SCF provides an unexpected and direct mechanistic link between DNA recombination and the cell cycle.

RAG-1 and RAG-2-dependent assembly of functional complexes with V(D)J recombination substrates in solution.

V(D)J recombination is initiated by RAG-1 and RAG-2, which introduce double-strand DNA breaks at recombination signal sequences (RSSs) of antigen receptor gene segments to produce signal ends, terminating in blunt, double-strand breaks, and coding ends, terminating in DNA hairpins. While the formation of RAG-RSS complexes has been documented, observations regarding the individual contributions of RAG-1 and RAG-2 to RSS recognition are in conflict. Here we describe an assay for formation and maintenance of functional RAG-RSS complexes in the course of the DNA cleavage reaction. Under conditions of in vitro cleavage, the RAG proteins sequester intact substrate DNA in a stable complex which is formed prior to strand scission. The cleavage reaction subsequently proceeds through nicking and hairpin formation without dissociation of substrate. Notably, the presence of both RAG-1 and RAG-2 is essential for formation of stable, functional complexes with substrate DNA under conditions of the sequestration assay. Two classes of substrate mutation are distinguished by their effects on RAG-mediated DNA cleavage in vitro. A mutation of the first class, residing within the RSS nonamer and associated with coordinate impairment of nicking and hairpin formation, greatly reduces the stability of RAG association with intact substrate DNA. In contrast, a mutation of the second class, lying within the RSS heptamer and associated with selective abolition of hairpin formation, has little or no effect on the half-life of the RAG-substrate complex.