RAG Proteins Research Articles

Novel ubiquitination-like modifications that regulate recombination of immunoglobulin loci gene segments leading to antibody diversification (IRM8P.711)

Abstract Programmed DNA mutagenesis events in the immunglobulin (Ig) loci of B cells utilize the common and conserved mechanism of ubiquitination of proteins prior to proteasomal degradation to generate the required antigen-receptor diversity. Recombinase proteins RAG1 and RAG2, required for V(D)J recombination, and activation-induced cytidine deaminase (AID), an essential mutator protein for catalyzing somatic hypermutation (SHM) and class switch recombination (CSR), are regulated by various ubiquitination events affecting protein stability and activity. AID associates with transcribing RNA polII and non-coding RNA processing complex RNA exosome to introduce DNA lesions in the IgH locus that are required for CSR and SHM. Failure in regulating AID’s DNA mutagenic activity is considered as a primary cause for B cell lymphomagenesis, especially Diffuse Large B cell Lymphoma (DLBCL). Here, we present our recent observations that demonstrate that the AID/RNA exosome/RNA polymerase II complex is regulated by ubiquitination and neddylation modifications. Using proteomics based experiments we have identified AID associated protein factors that promote ubiquitin-like modification of AID/RNA polII/RNA exosome complex. Using various biochemical assays, knockdown experiments and genetically mutated mouse models, we demonstrate the ubiquitin-like modification of AID-protein complex is important for regulating AID activity during CSR and overall maintenance of B cell genomic integrity.

Modeling of the RAG Reaction Mechanism

In vertebrate V(D)J recombination, it remains unclear how the RAG complex coordinates its catalytic steps with binding to two distant recombination sites. Here, we test the ability of the plausible reaction schemes to fit observed time courses for RAG nicking and DNA hairpin formation. The reaction schemes with the best fitting capability (1) strongly favor a RAG tetrameric complex over a RAG octameric complex; (2) indicate that once a RAG complex brings two recombination signal sequence (RSS) sites into synapsis, the synaptic complex rarely disassembles; (3) predict that the binding of both RSS sites (synapsis) occurs before catalysis (nicking); and (4) show that the RAG binding properties permit strong distinction between RSS sites within active chromatin versus nonspecific DNA or RSS sites within inactive chromatin. The results provide general insights for synapsis by nuclear proteins as well as more specific testable predictions for the RAG proteins.

Clinical Spectrum of SCID: The Key is in the Thymus?

Clinical Spectrum of SCID: The Key is in the Thymus?

Ubiquitination Events That Regulate Recombination of Immunoglobulin Loci Gene Segments

Programed DNA mutagenesis events in the immunoglobulin (Ig) loci of developing B cells utilize the common and conserved mechanism of protein ubiquitination for subsequent proteasomal degradation to generate the required antigen-receptor diversity. Recombinase proteins RAG1 and RAG2, necessary for V(D)J recombination, and activation-induced cytidine deaminase, an essential mutator protein for catalyzing class switch recombination and somatic hypermutation, are regulated by various ubiquitination events that affect protein stability and activity. Programed DNA breaks in the Ig loci can be identified by various components of DNA repair pathways, also regulated by protein ubiquitination. Errors in the ubiquitination pathways for any of the DNA double-strand break repair proteins can lead to inefficient recombination and repair events, resulting in a compromised adaptive immune system or development of cancer.

Noncore RAG1 Regions Promote Vβ Rearrangements and αβ T Cell Development by Overcoming Inherent Inefficiency of Vβ Recombination Signal Sequences

The RAG proteins are comprised of core endonuclease domains and noncore regions that modulate endonuclease activity. Mutation or deletion of noncore RAG regions in humans causes immunodeficiency and altered TCR repertoire, and mice expressing core but not full-length Rag1 (Rag1(C/C)) or Rag2 (Rag2(C/C)) exhibit lymphopenia, reflecting impaired V(D)J recombination and lymphocyte development. Rag1(C/C) mice display reduced D-to-J and V-to-DJ rearrangements of TCRβ and IgH loci, whereas Rag2(C/C) mice show decreased V-to-DJ rearrangements and altered Vβ/VH repertoire. Because Vβs/VHs only recombine to DJ complexes, the Rag1(C/C) phenotype could reflect roles for noncore RAG1 regions in promoting recombination during only the D-to-J step or during both steps. In this study, we demonstrate that a preassembled TCRβ gene, but not a preassembled DβJβ complex or the prosurvival BCL2 protein, completely rescues αβ T cell development in Rag1(C/C) mice. We find that Rag1(C/C) mice exhibit altered Vβ utilization in Vβ-to-DJβ rearrangements, increased usage of 3'Jα gene segments in Vα-to-Jα rearrangements, and abnormal changes in Vβ repertoire during αβ TCR selection. Inefficient Vβ/VH recombination signal sequences (RSSs) have been hypothesized to cause impaired V-to-DJ recombination on the background of a defective recombinase as in core-Rag mice. We show that replacement of the Vβ14 RSS with a more efficient RSS increases Vβ14 recombination and rescues αβ T cell development in Rag1(C/C) mice. Our data indicate that noncore RAG1 regions establish a diverse TCR repertoire by overcoming Vβ RSS inefficiency to promote Vβ recombination and αβ T cell development, and by modulating TCRβ and TCRα gene segment utilization.

Targeting glutamine uptake in AML.

Cancer cells require nutrients and energy to adapt to increased biosynthetic activity and depend on mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. Whereas they exhibit a pronounced Warburg effect, their TCA cycle remains intact and becomes more dependent on glutamine metabolism through glutaminolysis[1]. Besides this role, intracellular glutamine is also essential for mTORC1 activation by leucine[2]. Many upstream signals regulate mTORC1 activation. Among them, a major process is the availability of leucine, which is required to activate the Rag (for Ras-related GTPases) proteins that enable the proper localization of mTORC1 at the lysosome surface close to its activator Rheb[3]. Leucine uptake into the cells is regulated by the bidirectional transporter SLC7A5/3A2, in exchange for glutamine. The level of leucine thereby depends on the intracellular glutamine concentrations, which is mainly mediated by the high affinity transporter SLC1A5. Thus, the cellular uptake and subsequent rapid efflux of glutamine in the presence of leucine make glutamine availability a limiting step for the activation of mTORC1. MTORC1 positively regulates protein translation through phosphorylation of protein S6 Kinase (P70S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). Protein synthesis is controlled by the translational repressor 4E-BP1 whose phosphorylation at serine 65 is required to initiate the formation of the translation initiation complex.

Suppression of Lysosome Function Induces Autophagy via a Feedback Down-regulation of MTOR Complex 1 (MTORC1) Activity

Autophagy can be activated via MTORC1 down-regulation by amino acid deprivation and by certain chemicals such as rapamycin, torin, and niclosamide. Lysosome is the degrading machine for autophagy but has also been linked to MTORC1 activation through the Rag/RRAG GTPase pathway. This association raises the question of whether lysosome can be involved in the initiation of autophagy. Toward this end, we found that niclosamide, an MTORC1 inhibitor, was able to inhibit lysosome degradation and increase lysosomal permeability. Niclosamide was ineffective in inhibiting MTORC1 in cells expressing constitutively activated Rag proteins, suggesting that its inhibitory effects were targeted to the Rag-MTORC1 signaling system. This places niclosamide in the same category of bafilomycin A1 and concanamycin A, inhibitors of the vacuolar H(+)-ATPase, for its dependence on Rag GTPase in suppression of MTORC1. Surprisingly, classical lysosome inhibitors such as chloroquine, E64D, and pepstatin A were also able to inhibit MTORC1 in a Rag-dependent manner. These lysosome inhibitors were able to activate early autophagy events represented by ATG16L1 and ATG12 puncta formation. Our work established a link between the functional status of the lysosome in general to the Rag-MTORC1 signaling axis and autophagy activation. Thus, the lysosome is not only required for autophagic degradation but also affects autophagy activation. Lysosome inhibitors can have a dual effect in suppressing autophagy degradation and in initiating autophagy.

Finding the GAPs in mTORC1 signalling

A new study identifies the tumour suppressor folliculin as a GTPase-activating protein that regulates the nucleotide status of the RAG proteins, which are important regulators of mTOR complex 1 kinase activity.

Ebf1 and c-Myb Repress Rag Transcription Downstream of Stat5 during Early B Cell Development

The temporal control of RAG (Rag) expression in developing lymphocytes prevents DNA breaks during periods of proliferation that could threaten genomic integrity. In developing B cells, the IL-7R and precursor B cell Ag receptor (pre-BCR) synergize to induce proliferation and the repression of Rag at the protein and mRNA levels for a brief period following successful Ig H chain gene rearrangement. Whereas the mechanism of RAG2 protein downregulation is well defined, little is known about the pathways and transcription factors that mediate transcriptional repression of Rag. Using Abelson murine leukemia virus-transformed B cells to model this stage of development, we identified early B cell factor 1 (Ebf1) as a strong repressor of Rag transcription. Short hairpin RNA-mediated knockdown of either Ebf1 or its downstream target c-Myb was sufficient to induce Rag transcription in these highly proliferative cells. Ebf1 and c-Myb antagonize Rag transcription by negatively regulating the binding of Foxo1 to the Rag locus. Ebf1 accomplishes this through both direct negative regulation of Foxo1 expression and direct positive regulation of Gfi1b expression. Ebf1 expression is driven by the IL-7R downstream effector Stat5, providing a link between the negative regulation of Rag transcription by IL-7 and a novel repressive pathway involving Ebf1 and c-Myb.

CD40 interacts directly with RAG1 and RAG2 in autoaggressive T cells and Fas prevents CD40-induced RAG expression

CD4(+) T cells expressing CD40 (Th40 cells) constitute a pathogenic T-cell subset that is necessary and sufficient to transfer autoimmune disease. We have previously demonstrated that CD40 signals peripheral Th40 cells to induce RAG1 and RAG2 expression, proteins necessary for the expression of T-cell receptor (TCR), leading to TCR revision. The dependency of TCR expression in the thymus on RAG proteins has long been known. However, despite numerous publications, there is controversy as to whether TCR expression can be altered in the periphery, post-thymic selective pressures. Therefore, a better understanding of TCR expression in primary peripheral cells is needed. We now show that the CD40 protein itself interacts with RAG1 and RAG2 as well as with Ku70 and translocates to the nucleus in Th40 cells. This indicates that the CD40 molecule is closely involved in the mechanism of TCR expression in the periphery. In addition, Fas signals act as a silencing mechanism for CD40-induced RAGs and prevent CD40 translocation to the nucleus. It will be important to further understand the involvement of CD40 in peripheral TCR expression and how TCR revision impacts auto-antigen recognition in order to effectively target and tolerize autoaggressive T cells in autoimmune disease.

Expanding the spectrum of recombination-activating gene 1 deficiency: A family with early-onset autoimmunity

Expanding the spectrum of recombination-activating gene 1 deficiency: A family with early-onset autoimmunity

Metabolic sensor AMPK directly phosphorylates RAG1 protein and regulates V(D)J recombination

The ability to sense metabolic stress is critical for successful cellular adaptation. In eukaryotes, the AMP-activated protein kinase (AMPK), a highly conserved serine/threonine kinase, functions as a critical metabolic sensor. AMPK is activated by the rising ADP/ATP and AMP/ATP ratios during conditions of energy depletion and also by increasing intracellular Ca(2+). In response to metabolic stress, AMPK maintains energy homeostasis by phosphorylating and regulating proteins that are involved in many physiological processes including glucose and fatty acid metabolism, transcription, cell growth, mitochondrial biogenesis, and autophagy. Evidence is mounting that AMPK also plays a role in a number of pathways unrelated to energy metabolism. Here, we identify the recombination-activating gene 1 protein (RAG1) as a substrate of AMPK. The RAG1/RAG2 complex is a lymphoid-specific endonuclease that catalyzes specific DNA cleavage during V(D)J recombination, which is required for the assembly of the Ig and T-cell receptor genes of the immune system. AMPK directly phosphorylates RAG1 at serine 528, and the phosphorylation enhances the catalytic activity of the RAG complex, resulting in increased cleavage of oligonucleotide substrates in vitro, or increased recombination of an extrachromosomal substrate in a cellular assay. Our results suggest that V(D)J recombination can be regulated by AMPK activation, providing a potential new link between metabolic stress and development of B and T lymphocytes.

Genetic mutation of recombination activating gene 1 in Dahl salt-sensitive rats attenuates hypertension and renal damage.

Hypertension and renal damage in Dahl SS rats are associated with increased infiltrating immune cells in the kidney. To examine the role of infiltrating immune cells in this disease process, a zinc finger nuclease targeting bases 672-706 of recombination-activating gene 1 (Rag1) was injected into the pronucleus of Dahl SS (SS/JrHsdMcwi) strain embryos and implanted in pseudopregnant females. This strategy yielded a rat strain with a 13-base frame-shift mutation in the target region of Rag1 and a deletion of immunoreactive Rag1 protein in the thymus. Flow cytometry demonstrated that the Rag1-null mutant rats have a significant reduction in T and B lymphocytes in the circulation and spleen. Studies were performed on SS and Rag1-null rats fed a 4.0% NaCl diet for 3 wk. The infiltration of T cells into the kidney following high-salt intake was significantly blunted in the Rag1-null rats (1.7 ± 0.6 × 10(5) cells/kidney) compared with the Dahl SS (5.6 ± 0.9 × 10(5) cells/kidney). Accompanying the reduction in infiltration of immune cells in the kidney, mean arterial blood pressure and urinary albumin excretion rate were significantly lower in Rag1-null mutants (158 ± 3 mmHg and 60 ± 16 mg/day, respectively) than in SS rats (180 ± 11 mmHg and 251 ± 37 mg/day). Finally, a histological analysis revealed that the glomerular and tubular damage in the kidneys of the SS rats fed a high-salt diet was also attenuated in the Rag1 mutants. These studies demonstrate the importance of renal infiltration of immune cells in the pathogenesis of hypertension and renal damage in Dahl SS rats.

RAG and HMGB1 create a large bend in the 23RSS in the V(D)J recombination synaptic complexes

During V(D)J recombination, recombination activating gene proteins RAG1 and RAG2 generate DNA double strand breaks within a paired complex (PC) containing two complementary recombination signal sequences (RSSs), the 12RSS and 23RSS, which differ in the length of the spacer separating heptamer and nonamer elements. Despite the central role of the PC in V(D)J recombination, little is understood about its structure. Here, we use fluorescence resonance energy transfer to investigate the architecture of the 23RSS in the PC. Energy transfer was detected in 23RSS substrates in which the donor and acceptor fluorophores flanked the entire RSS, and was optimal under conditions that yield a cleavage-competent PC. The data are most easily explained by a dramatic bend in the 23RSS that reduces the distance between these flanking regions from >160 Å in the linear substrate to <80 Å in the PC. Analysis of multiple fluorescent substrates together with molecular dynamics modeling yielded a model in which the 23RSS adopts a U shape in the PC, with the spacer located centrally within the bend. We propose that this large bend facilitates simultaneous recognition of the heptamer and nonamer, is critical for proper positioning of the active site and contributes to the 12/23 rule.

A New Take on V(D)J Recombination: Transcription Driven Nuclear and Chromatin Reorganization in Rag-Mediated Cleavage

It is nearly 30 years since the Alt lab first put forward the accessibility model, which proposes that cleavage of the various antigen receptor loci is controlled by lineage and stage specific factors that regulate RAG access. Numerous labs have since demonstrated that locus opening is regulated at multiple levels that include sterile transcription, changes in chromatin packaging, and alterations in locus conformation. Here we focus on the interplay between transcription and RAG binding in facilitating targeted cleavage. We discuss the results of recent studies that implicate transcription in regulating nuclear organization and altering the composition of resident nucleosomes to promote regional access to the recombinase machinery. Additionally we include new data that provide insight into the role of the RAG proteins in defining nuclear organization in recombining T cells.

Clinical and laboratory phenotypes of severe combined immunodeficiencies with mutations in RAG1/RAG2 genes

Background. The RAG1 and RAG2 proteins are key players in the V(D)J recombination process leading to the assembly of antigen receptor genes. Defects in RAG1/RAG2 genes are caused to different phenotypes of severe combined immunodeficiencies (SCID). Objective — to determine the clinical and laboratory manifestations in patients with RAG1 / RAG2 mutations from one single center, to identify the phenotype-genotype correlations. Materials and methods. We described 4 children with RAG1 mutations. Diagnosis of SCID was confirmed by criteria’s of European society of immunodeficiencies (ESID). Results. In two patients we observed Omenn syndrome, in 1 — classic T -B -NK + SCID, in 1 — «sof» T +B -NK + SCID. One patient with Omenn syndrome and patient with «soft» SCID had same RAG1 mutations. Conclusions. RAG 1 / RAG2 mutations are caused to severe life-threatening combined immunodeficiency, requiring radical therapy. We found no genotype-phenotype correlations in patients with RAG1 defects.

TRIM28 mediates chromatin modifications at the TCRα enhancer and regulates the development of T and natural killer T cells

T-cell receptor-α (TCRα) rearrangement in CD4(+)CD8(+) double-positive immature thymocytes is a prerequisite for production of αβ T cells and invariant natural killer T cells. This developmental event is regulated by the TCRα enhancer (Eα), which induces chromatin modification and recruitment of the recombination-activating proteins Rag1 and Rag2. However, the molecular mechanism underlying the activation and long-range action of Eα remains incompletely understood. We show here that the chromatin-modifying factor TRIM28 is highly expressed in double-positive thymocytes and persistently phosphorylated at serine 473. TRIM28 binds to Eα and induces histone 3 lysine 4 trimethylation in the Eα and distant regions of the TCRα locus, coupled with recruitment of Rag proteins. T-cell-conditional ablation of TRIM28 impaired TCRα gene rearrangement and compromised the development of αβ T cells and invariant natural killer T cells. These findings establish TRIM28 as a unique regulator of thymocyte development and highlight an epigenetic mechanism involving TRIM28-mediated active chromatin modification in the TCRα locus.

Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat

BackgroundEngineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain.ResultsAfter microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαβ+ as well as CD8+/CD3+/TCRαβ+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat.ConclusionThe Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.

Localized epigenetic changes induced by DH recombination restricts recombinase to DJH junctions.

Immunoglobulin heavy chain (Igh) genes are assembled by sequential rearrangements of diversity (DH) and variable (VH) gene segments. Three critical constraints govern VH recombination. These include timing (VH recombination follows DH recombination), precision (VHs recombine only to DJH junctions) and allele specificity (VH recombination is restricted to DJH recombined alleles). We provide a model for these universal features of VH recombination. Analyses of DJH recombined alleles revealed that DJH junctions were selectively epigenetically marked, became nuclease sensitive and bound RAG proteins, thereby permitting DH-associated recombination signal sequences to initiate the second step of Igh gene assembly. We propose that VH recombination is precise because these changes did not extend to germline DH gene segments located 5′ of the DJH junction.

A Dual Interaction between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase

The first step in V(D)J recombination is the formation of specific DNA double-strand breaks (DSBs) by the RAG1 and RAG2 proteins, which form the RAG recombinase. DSBs activate a complex network of proteins termed the DNA damage response (DDR). A key early event in the DDR is the phosphorylation of histone H2AX around DSBs, which forms a binding site for the tandem BRCA1 C-terminal (tBRCT) domain of MDC1. This event is required for subsequent signal amplification and recruitment of additional DDR proteins to the break site. RAG1 bears a histone H2AX-like motif at its C terminus (R1Ct), making it a putative MDC1-binding protein. In this work we show that the tBRCT domain of MDC1 binds the R1Ct motif of RAG1. Surprisingly, we also observed a second binding interface between the two proteins that involves the Proline-Serine-Threonine rich (PST) repeats of MDC1 and the N-terminal non-core region of RAG1 (R1Nt). The repeats-R1Nt interaction is constitutive, whereas the tBRCT-R1Ct interaction likely requires phosphorylation of the R1Ct motif of RAG1. As the C terminus of RAG1 has been implicated in inhibition of RAG activity, we propose a model in which phosphorylation of the R1Ct motif of RAG1 functions as a self-initiated regulatory signal.