RAG Recombinase Research Articles

RAG genomic variation causes autoimmune diseases through specific structure-based mechanisms of enzyme dysregulation

Interpreting genetic changes observed in individual patients is a critical challenge. The array of immune deficiency syndromes is typically caused by genetic variation unique to individuals. Therefore, new approaches are needed to interpret functional variation and accelerate genomics interpretation. We constructed the first full-length structural model of human RAG recombinase across four functional states of the recombination process. We functionally tested 182 clinically observed RAG missense mutations. These experiments revealed dysfunction due to recombinase dysfunction and altered chromatin interactions. Structural modeling identified mechanical and energetic roles for each mutation. We built regression models for RAG1 (R2= 0.91) and RAG2 (R2= 0.97) to predict RAG activity changes. We applied our model to 711 additional RAG variants observed in population studies and identified a subset that may impair RAG function. Thus, we demonstrated a fundamental advance in the mechanistic interpretation of human genetic variations spanning from rare and undiagnosed diseases to population health.

Different sea urchin RAG-like genes were domesticated to carry out different functions.

The closely linked recombination activating genes (RAG1 and RAG2) in vertebrates encode the core of the RAG recombinase that mediates the V(D)J recombination of the immunoglobulin and T-cell receptor genes. RAG1 and RAG2 homologues (RAG1L and RAG2L) are present in multiple invertebrate phyla, including mollusks, nemerteans, cnidarians, and sea urchins. However, the function of the invertebrates' RAGL proteins is yet unknown. The sea urchins contain multiple RAGL genes that presumably originated in a common ancestral transposon. In this study, we demonstrated that two different RAG1L genes in the sea urchin Paracentrutus lividus (PlRAG1La and PlRAG1Lb) lost their mobility and, along with PlRAG2L, were fully domesticated to carry out different functions. We found that the examined echinoid RAGL homologues have distinct expression profiles in early developmental stages and in adult tissues. Moreover, the predicted structure of the proteins suggests that while PlRAG1La could maintain its endonuclease activity and create a heterotetramer with PlRAG2L, the PlRAG1Lb adopted a different function that does not include an interaction with DNA nor a collaboration with PlRAG2L. By characterizing the different RAG homologues in the echinoid lineage, we hope to increase the knowledge about the evolution of these genes and shed light on their domestication processes.

Direct regulation of TCR rearrangement and expression by E proteins during early T cell development.

γδ T cells are widely distributed throughout mucosal and epithelial cell-rich tissues and are an important early source of IL-17 in response to several pathogens. Like αβ T cells, γδ T cells undergo a stepwise process of development in the thymus that requires recombination of genome-encoded segments to assemble mature T cell receptor (TCR) genes. This process is tightly controlled on multiple levels to enable TCR segment assembly while preventing the genomic instability inherent in the double-stranded DNA breaks that occur during this process. Each TCR locus has unique aspects in its structure and requirements, with different types of regulation before and after the αβ/γδ T cell fate choice. It has been known that Runx and Myb are critical transcriptional regulators of TCRγ and TCRδ expression, but the roles of E proteins in TCRγ and TCRδ regulation have been less well explored. Multiple lines of evidence show that E proteins are involved in TCR expression at many different levels, including the regulation of Rag recombinase gene expression and protein stability, induction of germline V segment expression, chromatin remodeling, and restriction of the fetal and adult γδTCR repertoires. Importantly, E proteins interact directly with the cis-regulatory elements of the TCRγ and TCRδ loci, controlling the predisposition of a cell to become an αβ T cell or a γδ T cell, even before the lineage-dictating TCR signaling events. This article is categorized under: Immune System Diseases > Stem Cells and Development Immune System Diseases > Genetics/Genomics/Epigenetics.

Structural insights into the evolution of the RAG recombinase.

Adaptive immunity in jawed vertebrates relies on the assembly of antigen receptor genes by the recombination activating gene 1 (RAG1)-RAG2 (collectively RAG) recombinase in a reaction known as V(D)J recombination. Extensive biochemical and structural evidence indicates that RAG and V(D)J recombination evolved from the components of a RAG-like (RAGL) transposable element through a process known as transposon molecular domestication. This Review describes recent advances in our understanding of the functional and structural transitions that occurred during RAG evolution. We use the structures of RAG and RAGL enzymes to trace the evolutionary adaptations that yielded a RAG recombinase with exquisitely regulated cleavage activity and a multilayered array of mechanisms to suppress transposition. We describe how changes in modes of DNA binding, alterations in the dynamics of protein-DNA complexes, single amino acid mutations and a modular design likely enabled RAG family enzymes to survive and spread in the genomes of eukaryotes. These advances highlight the insight that can be gained from viewing evolution of vertebrate immunity through the lens of comparative genome analyses coupled with structural biology and biochemistry.

ETV6-RUNX1 and RUNX1 directly regulate RAG1 expression: one more step in the understanding of childhood B-cell\xa0acute lymphoblastic leukemia leukemogenesis

HighlightsETV6-RUNX1 and RUNX1 directly promote RAG1 expression.ETV6-RUNX1 and RUNX1 preferentially bind to the −1200 bp enhancer of RAG1 and the −80 bp promoter of RAG1 gene respectively, and compete for these bindings.ETV6-RUNX1 and RUNX1 induce an excessive RAG recombinase activity.ETV6-RUNX1 participates directly in two events of the multi-hit ALL leukemogenesis: as an initiating event and as an activator of RAG1 expression.

RAG enhances BCR-ABL1-positive leukemic cell growth through its endonuclease activity in vitro and in vivo.

BCR‐ABL1 gene fusion associated with additional DNA lesions involves the pathogenesis of chronic myelogenous leukemia (CML) from a chronic phase (CP) to a blast crisis of B lymphoid (CML‐LBC) lineage and BCR‐ABL1 + acute lymphoblastic leukemia (BCR‐ABL1 + ALL). The recombination‐activating gene RAG1 and RAG2 (collectively, RAG) proteins that assemble a diverse set of antigen receptor genes during lymphocyte development are abnormally expressed in CML‐LBC and BCR‐ABL1 + ALL. However, the direct involvement of dysregulated RAG in disease progression remains unclear. Here, we generate human wild‐type (WT) RAG and catalytically inactive RAG‐expressing BCR‐ABL1 + and BCR‐ABL1 − cell lines, respectively, and demonstrate that BCR‐ABL1 specifically collaborates with RAG recombinase to promote cell survival in vitro and in xenograft mice models. WT RAG‐expressing BCR‐ABL1 + cell lines and primary CD34+ bone marrow cells from CML‐LBC samples maintain more double‐strand breaks (DSB) compared to catalytically inactive RAG‐expressing BCR‐ABL1 + cell lines and RAG‐deficient CML‐CP samples, which are measured by γ‐H2AX. WT RAG‐expressing BCR‐ABL1 + cells are biased to repair RAG‐mediated DSB by the alternative non–homologous end joining pathway (a‐NHEJ), which could contribute genomic instability through increasing the expression of a‐NHEJ‐related MRE11 and RAD50 proteins. As a result, RAG‐expressing BCR‐ABL1 + cells decrease sensitivity to tyrosine kinase inhibitors (TKI) by activating BCR‐ABL1 signaling but independent of the levels of BCR‐ABL1 expression and mutations in the BCR‐ABL1 tyrosine kinase domain. These findings identify a surprising and novel role of RAG in the functional specialization of disease progression in BCR‐ABL1 + leukemia through its endonuclease activity.

The role of chromatin organizer Satb1 in shaping TCR repertoire in adult thymus.

T cells recognize the universe of foreign antigens with a diverse repertoire of T cell receptors generated by V(D)J recombination. Special AT-rich binding protein 1 (Satb1) is a chromatin organizer that plays an essential role in T cell development. Previous study has shown that Satb1 regulates the re-induction of recombinase Rag1 and Rag2 in CD4+CD8+ thymocytes, affecting the secondary rearrangement of the Tcra gene. Here, we detected the repertoires of four TCR genes, Tcrd, Tcrg, Tcrb, and Tcra, in the adult thymus, and explored the role of the Satb1 in shaping the TCR repertoires. We observed a strong bias in the V and J gene usages of the Tcrd and Tcrg repertoires in WT and Satb1-deleted thymocytes. Satb1 deletion had few effects on the V(D)J rearrangement and repertoire of the Tcrg, Tcrd, and Tcrb genes. The Tcra repertoire was severely impaired in Satb1-deleted thymocytes, while the primary rearrangement was relatively normal. We also found the CDR3 length of TCRα chain was significantly longer in Satb1-deleted thymocytes, which can be explained by the strong bias of the proximal Jα usage. Our results showed that Satb1 plays an essential role in shaping TCR repertoires in αβ T cells.

Dynamic 3D Locus Organization and Its Drivers Underpin Immunoglobulin Recombination.

A functional adaptive immune system must generate enormously diverse antigen receptor (AgR) repertoires from a limited number of AgR genes, using a common mechanism, V(D)J recombination. The AgR loci are among the largest in the genome, and individual genes must overcome huge spatial and temporal challenges to co-localize with optimum variability. Our understanding of the complex mechanisms involved has increased enormously, due in part to new technologies for high resolution mapping of AgR structure and dynamic movement, underpinning mechanisms, and resulting repertoires. This review will examine these advances using the paradigm of the mouse immunoglobulin heavy chain (Igh) locus. We will discuss the key regulatory elements implicated in Igh locus structure. Recent next generation repertoire sequencing methods have shown that local chromatin state at V genes contribute to recombination efficiency. Next on the multidimensional scale, we will describe imaging studies that provided the first picture of the large-scale dynamic looping and contraction the Igh locus undergoes during recombination. We will discuss chromosome conformation capture (3C)-based technologies that have provided higher resolution pictures of Igh locus structure, including the different models that have evolved. We will consider the key transcription factors (PAX5, YY1, E2A, Ikaros), and architectural factors, CTCF and cohesin, that regulate these processes. Lastly, we will discuss a plethora of recent exciting mechanistic findings. These include Rag recombinase scanning for convergent RSS sequences within DNA loops; identification of Igh loop extrusion, and its putative role in Rag scanning; the roles of CTCF, cohesin and cohesin loading factor, WAPL therein; a new phase separation model for Igh locus compartmentalization. We will draw these together and conclude with some horizon-scanning and unresolved questions.

END-seq: An Unbiased, High-Resolution, and Genome-Wide Approach to Map DNA Double-Strand Breaks and Resection in Human Cells.

DNA double-strand breaks (DSBs) represent the most toxic form of DNA damage and can arise in either physiological or pathological conditions. If left unrepaired, these DSBs can lead to genome instability which serves as a major driver to tumorigenesis and other pathologies. Consequently, localizing DSBs and understanding the dynamics of break formation and the repair process are of great interest for dissecting underlying mechanisms and in the development of targeted therapies. Here, we describe END-seq, a highly sensitive next-generation sequencing technique for quantitatively mapping DNA double-strand breaks (DSB) at nucleotide resolution across the genome in an unbiased manner. END-seq is based on the direct ligation of a sequencing adapter to the ends of DSBs and provides information about DNA processing (end resection) at DSBs, a critical determinant in the selection of repair pathways. The absence of cell fixation and the use of agarose for embedding cells and exonucleases for blunting the ends of DSBs are key advances that contribute to the technique's increased sensitivity and robustness over previously established methods. Overall, END-seq has provided a major technical advance for mapping DSBs and has also helped inform the biology of complex biological processes including genome organization, replication fork collapse and chromosome fragility, off-target identification of RAG recombinase and gene-editing nucleases, and DNA end resection at sites of DSBs.

Binding and allosteric transmission of histone H3 Lys-4 trimethylation to the recombinase RAG-1 are separable functions of the RAG-2 plant homeodomain finger

V(D)J recombination is initiated by the recombination-activating gene protein (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with gene transcription and with epigenetic marks characteristic of active chromatin, including histone H3 trimethylated at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 induces conformational changes in RAG-1, allosterically stimulating substrate binding and catalysis. To better understand the path of allostery from the RAG-2 PHD finger to RAG-1, here we employed phylogenetic substitution. We observed that a chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the shark Chiloscyllium punctatum binds H3K4me3 but fails to transmit an allosteric signal, indicating that binding of H3K4me3 by RAG-2 is insufficient to support recombination. By substituting residues in the C. punctatum PHD with the corresponding residues in the mouse PHD and testing for rescue of allostery, we demonstrate that H3K4me3 binding and transmission of an allosteric signal to RAG-1 are separable functions of the RAG-2 PHD finger.

Sequence-dependent dynamics of synthetic and endogenous RSSs in V(D)J recombination.

Developing lymphocytes of jawed vertebrates cleave and combine distinct gene segments to assemble antigen–receptor genes. This process called V(D)J recombination that involves the RAG recombinase binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG–RSS interaction. We employ a single-molecule method known as tethered particle motion to track the formation, lifetime and cleavage of individual RAG–12RSS–23RSS paired complexes (PCs) for numerous synthetic and endogenous 12RSSs. We reveal that single-bp changes, including in the 12RSS spacer, can significantly and selectively alter PC formation or the probability of RAG-mediated cleavage in the PC. We find that some rarely used endogenous gene segments can be mapped directly to poor RAG binding on their adjacent 12RSSs. Finally, we find that while abrogating RSS nicking with Ca2+ leads to substantially shorter PC lifetimes, analysis of the complete lifetime distributions of any 12RSS even on this reduced system reveals that the process of exiting the PC involves unidentified molecular details whose involvement in RAG–RSS dynamics are crucial to quantitatively capture kinetics in V(D)J recombination.

Cutting antiparallel DNA strands in a single active site.

SummaryA single enzyme active site that catalyzes multiple reactions is a well-established biochemical theme, but how one nuclease site cleaves both DNA strands of a double helix has not been well understood. In analyzing site-specific DNA cleavage by the mammalian RAG1-RAG2 recombinase, which initiates V(D)J recombination, we find that the active site is reconfigured for the two consecutive reactions, and the DNA double helix adopts drastically different structures. For initial nicking of the DNA, a locally unwound and unpaired DNA duplex forms a zipper via alternating inter-strand base stacking, rather than melting as generally thought. The second strand cleavage and formation of a hairpin-DNA product requires a global scissor-like movement of protein and DNA, delivering the scissile phosphate into the rearranged active site.

How mouse RAG recombinase avoids DNA transposition.

The RAG1-RAG2 recombinase (RAG) cleaves DNA to initiate V(D)J recombination. But RAG also belongs to the RNH-type transposase family. To learn how RAG-catalyzed transposition is inhibited in developing lymphocytes, we determined the structure of a DNA strand-transfer complex of mouse RAG at 3.1 Å resolution. The target DNA is a T form (T for transpositional target), which contains two >80° kinks towards the minor groove, only 3 bp apart. RAG2, a late evolutionary addition in V(D)J recombination, appears to enforce the sharp kinks and additional inter-segment twisting in target DNA and thus attenuate unwanted transposition. In contrast to strand-transfer complexes of genuine transposases, where severe kinks occur at the integration sites of target DNA and thus prevent the reverse reaction, the sharp kink with RAG is 1 bp away from the integration site. As a result, RAG efficiently catalyzes the disintegration reaction that restores the RSS (donor) and target DNA.

Functional requirement of terminal inverted repeats for efficient ProtoRAG activity reveals the early evolution of V(D)J recombination.

The discovery of ProtoRAG in amphioxus indicated that vertebrate RAG recombinases originated from an ancient transposon. However, the sequences of ProtoRAG terminal inverted repeats (TIRs) were obviously dissimilar to the consensus sequence of mouse 12/23RSS and recombination mediated by ProtoRAG or RAG made them incompatible with each other. Thus, it is difficult to determine whether or how 12/23RSS persisted in the vertebrate RAG system that evolved from the TIRs of ancient RAG transposons. Here, we found that the activity of ProtoRAG is highly dependent on its asymmetric 5′TIR and 3′TIR, which are composed of conserved TR1 and TR5 elements and a partially conserved TRsp element of 27/31 bp to separate them. Similar to the requirements for the recombination signal sequences (RSSs) of RAG recombinase, the first CAC in TR1, the three dinucleotides in TR5 and the specific length of the partially conserved TRsp are important for the efficient recombination activity of ProtoRAG. In addition, the homologous sequences flanking the signal sequences facilitate ProtoRAG- but not RAG-mediated recombination. In addition to the diverged TIRs, two differentiated functional domains in BbRAG1L were defined to coordinate with the divergence between TIRs and RSSs. One of these is the CTT* domain, which facilitates the specific TIR recognition of the BbRAGL complex, and the other is NBD*, which is responsible for DNA binding and the protein stabilization of the BbRAGL complex. Thus, our findings reveal that the functional requirement for ProtoRAG TIRs is similar to that for RSS in RAG-mediated recombination, which not only supports the common origin of ProtoRAG TIRs and RSSs from the asymmetric TIRs of ancient RAG transposons, but also reveals the development of RAG and RAG-like machineries during chordate evolution.

Structures of a RAG-like transposase during cut-and-paste transposition.

SummaryTransposons have played a pivotal role in genome evolution1 and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase2, an essential component of the adaptive immune system in jawed vertebrates3. Here we report one crystal and five cryo-electron microscopy structures of a RAG1-like transposase, HzTransib4,5, that capture the entire transposition process from the apo enzyme to the terminal strand transfer complex with transposon ends covalently joined to target DNA, at resolutions of 3.0–4.6 Å. These structures reveal a butterfly-shaped complex that undergoes two cycles of dramatic conformational changes in which the “wings” of the transposase unfurl to bind substrate DNA, close to execute cleavage, open to release the flanking DNA, and close again to capture and attack target DNA. HzTransib possesses unique structural elements that compensate for the absence of a RAG2 partner including a loop that interacts with the transposition target site and an accordion-like C-terminal tail that elongates and contracts to help control the opening and closing of the enzyme and assembly of the active site. Our findings reveal the reaction pathway of a eukaryotic cut-and-paste transposase in unprecedented detail and illuminate some of the earliest steps in the evolution of the RAG recombinase.

Germline DNA Retention in Murine and Human Rearranged T Cell Receptor Gene Coding Joints: Alternative Recombination Signal Sequences and V(D)J Recombinase Errors.

The genes coding for the antigenic T cell receptor (TR) subunits are assembled in thymocytes from discrete V, D, and J genes by a site-specific recombination process. A tight control of this activity is required to prevent potentially detrimental recombination events. V, D, and J genes are flanked by semi-conserved nucleotide motives called recombination signal sequences (RSSs). V(D)J recombination is initiated by the precise introduction of a DNA double-strand break exactly at the border of the genes and their RSSs by the RAG recombinase. RSSs are therefore physically separated from the coding region of the genes before assembly of a rearranged TR gene. During a high throughput profiling of TRB genes in mice, we identified rearranged TRB genes in which part or all of a flanking RSS was retained in V-D or D-J coding joints. In some instances, this retention of germline DNA resulted from the use of an upstream alternative RSS. However, we also identified TRB sequences where retention of germline DNA occurred in the absence of alternative RSS, suggesting that RAG activity was mis-targeted during recombination. Similar events were also identified in human rearranged TRB and TRG genes. The use of alternative RSSs during V(D)J recombination illustrates the complexity of RAG-RSSs interactions during V(D)J recombination. While the frequency of errors resulting from mis-targeted RAG activity is very low, we believe that these RAG errors may be at the origin of oncogenic translocations and are a threat for genetic stability in developing lymphocytes.

Regulation of recombinase RAG-1 expression by female sex steroids in Treg and Th17 lymphocytes. role of oncostatin M

The effect of estradiol (E2), progesterone (P4), and oncostatin M (OSM) on the differentiation of CD4+ T cells to T regulatory (Treg) lymphocytes and T helpers 17 (Th17) was investigated. The possibility of revision of the T cell receptor in these subpopulations by evaluating the expression of RAG-1 recombinase was also studied. E2 at concentrations characteristic of pregnancy trimester I, but no P4 or OSM, increased the Treg level. Combination of sex steroids with OSM increased the percent of CD4+FOXP4+ cells and enhanced RAG-1 expression in these cells, thus promoting the development of immune tolerance during pregnancy. In the study of Th17 such effect of the hormones and OSM was not detected.

RAG2 localization and dynamics in the pre-B cell nucleus.

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 Trp453 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 Thr490 in the noncore region modulates RAG2 localization and dynamics in the pre-B cell nucleus, such as by affecting RAG2 interactions with H3K4me3.

Effect of Estriol, Chorionic Gonadotropin, and Oncostatin M on the Expression of Recombinase RAG-1 in Regulatory T Lymphocyte Subpopulations

We studied the effect of estriol, chorionic gonadotropin, oncostatin M, and hormone-cytokine combinations on the expression of recombinase RAG-1 in T-regulatory (Treg) and T helper 17 (Th17) lymphocytes. It was found that estriol in a concentration corresponding to the first trimester of pregnancy increased the level of Treg (CD4+FoxP3+) cells and suppressed the formation of Th17 (CD4+RORC+) lymphocytes. This effect was nor observed after individual administration of chorionic gonadotropin and oncostatin M, but some combinations of the studied hormones with oncostatin M increased the percentage of CD4+FOXP3+ cells. In the presence of oncostatin M, the studied hormones enhanced the expression of RAG-1 in CD4+FoxP3+ cells, but not in CD4+RORC+ cells, thereby initiating of Treg T-cell receptor (TCR) revision. The mechanisms of hormone cytokine control of induction of the immune tolerance during pregnancy are discussed.

Transposon molecular domestication and the evolution of the RAG recombinase.

Domestication of a transposon to give rise to the RAG1/RAG2 recombinase and V(D)J recombination was a pivotal event in the evolution of the jawed vertebrate adaptive immune system. The evolutionary adaptations that transformed the ancestral RAG transposase into a RAG recombinase with appropriately regulated DNA cleavage and transposition activities are not understood. Here, beginning with cryo-electron microscopy structures of RAG’s evolutionary relative, the ProtoRAG transposase from amphioxus, we identify amino acid residues and domains whose acquisition or loss underpins RAG’s propensity for coupled cleavage, preference for asymmetric DNA substrates, and inability to perform transposition in cells. In particular, we identify two jawed-vertebrate-specific adaptations—arginine 848 in RAG1 and an acidic region in RAG2—that together suppress RAG-mediated transposition more than 1000-fold. Our findings reveal a two-tiered mechanism for suppression of RAG-mediated transposition, illuminate the forces at work during the evolution of V(D)J recombination, and provide insight into the principles governing transposon molecular domestication.