Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2

Zhang, Yuhang; Shetty, Keerthi S.; Surleac, Marius; Petrescu, Andrei‐Jose; Schatz, David G.

doi:10.1074/jbc.m115.638627

Cited by 18 publications

(14 citation statements)

References 49 publications

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“…Based on our current and prior findings, we conclude that any type of DSB induced anywhere in the genome of a pre-B cell has the potential to signal immediate cessation of Rag1 and Rag2 transcription, leading to rapid downregulation of Rag1 protein expression and thereby V(D)J recombination throughout the nucleus. Considering that immature T cells express an order of magnitude fewer number of Rag1 monomers than Rag2 monomers (59), DSB-induced downregulation of Rag1 protein in immature lymphocytes would be predicted to be a more effective way to reduce RAG activity than downregulation of Rag2 protein. In this context, the retention of Rag2 protein in thymocytes harboring RAG DSBs at TCR loci (60) may result from a long half-life of endogenous Rag2 protein enabling Rag2 to persists following DSB-induced repression of Rag1/Rag2 transcription and resultant loss of Rag1 protein.…”

Section: Discussionmentioning

confidence: 99%

Immature Lymphocytes Inhibit Rag1 and Rag2 Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks

Fisher

Rivera-Reyes

Bloch

et al. 2017

The Journal of Immunology

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Mammalian cells have evolved a common DNA damage response (DDR) that sustains cellular function, maintains genomic integrity, and suppresses malignant transformation. In pre-B cells, DNA double strand breaks (DSBs) induced at Igκ loci by the Rag1/Rag2 (RAG) endonuclease engage this DDR to modulate transcription of genes that regulate lymphocyte-specific processes. We previously reported that RAG DSBs induced at one Igκ allele signal through the ATM kinase to feedback inhibit RAG expression and RAG cleavage of the other Igκ allele. Here, we show that DSBs induced by ionizing radiation, etoposide, or bleomycin suppress Rag1 and Rag2 mRNA levels in primary pre-B cells, pro-B cells, and pro-T cells, indicating that inhibition of Rag1 and Rag2 expression is a prevalent DSB response among immature lymphocytes. DSBs induced in pre-B cells signal rapid transcriptional repression of Rag1 and Rag2, causing downregulation of both Rag1 and Rag2 mRNA but only Rag1 protein. This transcriptional inhibition requires the ATM kinase and the NF-κB essential modulator protein, implicating a role for ATM-mediated activation of canonical NF-κB transcription factors. Finally, we demonstrate that DSBs induced in pre-B cells by etoposide or bleomycin inhibit recombination of Igκ loci and a chromosomally integrated substrate. Our data indicate that immature lymphocytes exploit a common DDR signaling pathway to limit DSBs at multiple genomic locations within developmental stages wherein monoallelic antigen receptor locus recombination is enforced. We discuss the implications of our findings for mechanisms that orchestrate the differentiation of mono-specific lymphocytes while suppressing oncogenic antigen receptor locus translocations.

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Section: Discussionmentioning

confidence: 99%

Immature Lymphocytes Inhibit Rag1 and Rag2 Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks

Fisher

Rivera-Reyes

Bloch

et al. 2017

The Journal of Immunology

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“…Our results and those from many studies of RAG2 −/− mice (e.g., Shinkai et al 1992) argue that RAG1 levels are sufficiently low in developing lymphocytes that RAG1-only recombination is exceedingly rare. A recent study estimated that mouse thymocytes contain, on average, only ∼1000 RAG1 dimers (Zhang et al 2015).…”

Section: Discussionmentioning

confidence: 99%

“…Lysates were prepared, and pull-downs were performed as previously described (Zhang et al 2015). Immunoblots were developed with anti-GST (Cell Signaling) and anti-MBP (New England Biolabs) antibodies.…”

Section: Pcr Assays For Coding and Signal Jointsmentioning

confidence: 99%

Collaboration of RAG2 with RAG1-like proteins during the evolution of V(D)J recombination

2016

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The recombination-activating gene 1 (RAG1) and RAG2 proteins initiate V(D)J recombination, the process that assembles the B-and T-lymphocyte antigen receptor genes of jawed vertebrates. RAG1 and RAG2 are thought to have arisen from a transposable element, but the origins of this element are not understood. We show that two ancestral RAG1 proteins, Transib transposase and purple sea urchin RAG1-like, have a latent ability to initiate V(D)J recombination when coexpressed with RAG2 and that in vitro transposition by Transib transposase is stimulated by RAG2. Conversely, we report low levels of V(D)J recombination by RAG1 in the absence of RAG2. Recombination by RAG1 alone differs from canonical V(D)J recombination in having lost the requirement for asymmetric DNA substrates, implicating RAG2 in the origins of the "12/23 rule," a fundamental regulatory feature of the reaction. We propose that evolution of RAG1/RAG2 began with a Transib transposon whose intrinsic recombination activity was enhanced by capture of an ancestral RAG2, allowing for the development of adaptive immunity. V(D)J recombination is the process by which the antigen receptor genes of jawed vertebrates are assembled. By joining different combinations of variable (V), diversity (D), and joining (J) gene segments, V(D)J recombination can generate a wide range of receptor specificities, providing the molecular basis for adaptive immunity. The proteins at the heart of this reaction are encoded by recombination-activating gene 1 (RAG1) and RAG2 (Schatz et al. 1989;Oettinger et al. 1990). The RAG1 and RAG2 proteins bind to recombination signal sequences (RSSs) that demarcate the gene segments of the antigen receptor loci and target dsDNA cleavage by RAG. Subsequent repair by the nonhomologous end-joining DNA repair pathway joins the gene segments to form coding joints and the cleaved RSSs to form signal joints (Rooney et al. 2004). The RSS consists of three sequence elements: the nonamer, the spacer, and the heptamer. While the nonamer and the heptamer sequences are well conserved, the length of the spacer, either 12 or 23 base pairs, is its defining characteristic (Ramsden et al. 1994). Therefore, two types of RSS exist depending on spacer length, the 12RSS and the 23RSS, and recombination is only efficient when one 12RSS and one 23RSS are engaged by RAG, a restriction known as the 12/23 rule.Because RAG1-deficient and RAG2-deficient mice and humans exhibit identical phenotypes characterized by a complete absence of V(D)J recombination and lymphocyte development (Mombaerts et al. 1992;Shinkai et al. 1992;Notarangelo et al. 1999), the two RAG proteins are each presumed to be essential for V(D)J recombination. Despite this, RAG1 and RAG2 play vastly different roles in the DNA cleavage reaction. RAG1 contains the RNaseH fold catalytic domain and regions that make direct contact with the RSS and is responsible for the enzymatic activity of the RAG complex (Schatz and Swanson 2011;Kim et al. 2015;Ru et al. 2015). In contrast, RAG2 interacts with and enhances...

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“…Our failure to detect cooperation between the RAG proteins in initial binding at J, while largely consistent with data from our previous ChIP-qPCR study (25), was notable in light of a recent in vitro kinetic analysis of RAG-mediated DNA cleavage, which predicted cooperation between RAG1 and RAG2 by virtue of their ability to recognize the RSS (RAG1) and H3K4me3 (RAG2) (44). This discrepancy might relate to differences in interactions with naked DNA in vitro and with chromatin in vivo as well as the need for RAG1 and RAG2 to associate with one another at their relatively low concentrations in vivo (45). This might be relevant for analyses of initial binding using an inducible expression system (as in this study), given that individually expressed RAG1 core and RAG2 core proteins interact with only a modest affinity, with a K D (equilibrium dissociation constant) value of ϳ0.4 M (45).…”

Section: Discussionmentioning

confidence: 99%

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

Shetty

Schatz

2015

Molecular and Cellular Biology

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b 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 J 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. T he adaptive immune system relies on V(D)J recombination to assemble variable (V), diversity (D), and joining (J) antigen receptor gene segments in generating a diverse repertoire of immunoglobulins and T cell receptors. The process is initiated by the recombination-activating gene (RAG) recombinase, a protein complex consisting of the enzymes encoded by RAG1 and RAG2 (1, 2). RAG1 is 1,040 amino acids long, with a core region spanning amino acids 384 to 1008 that contains recombination signal sequence (RSS) and RAG2 binding activity as well as the catalytic residues responsible for DNA cleavage (3-7). RAG2 is 527 amino acids long, with a core region (amino acids 1 to 387) that interacts with and facilitates DNA binding and cleavage by RAG1 but which has no DNA binding activity on its own (3,4,8,9). The RAG2 noncore region contains a plant homeodomain (PHD) finger that plays an important role in chromatin binding through interactions with the histone H3 N-terminal tail when lysine 4 is trimethylated (H3K4me3) (10-12).The site of recombination is specified by the RSS that immediately flanks each V, D, and J gene segment (Fig. 1A). The RSS consists of a well-conserved 7-bp sequence called the heptamer (consensus sequence of 5=-CACAGTG-3=) and an AT-rich 9-bp sequence called the nonamer (consensus sequence of 5=-ACAAA AACC-3=) separated by a poorly conserved spacer whose length is either 12 or 23 bp (13-15) (Fig. 1B). An RSS with a spacer 12 bp long is defined as a 12RSS, while one with a spacer 23 bp long is defined as a 23RSS (13).The consensus RSS is the most efficiently recombined sequence and therefore has been used for most in vitro experiments; however, endogenous RSSs often deviate from the consensus at multiple hept...

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Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2

Cited by 18 publications

References 49 publications

Immature Lymphocytes Inhibit Rag1 and Rag2 Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks

Immature Lymphocytes Inhibit Rag1 and Rag2 Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks

Collaboration of RAG2 with RAG1-like proteins during the evolution of V(D)J recombination

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

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