RAG Represents a Widespread Threat to the Lymphocyte Genome

Teng, Grace; Maman, Yaakov; Resch, Wolfgang; Kim, Min; Yamane, Arito; Qian, Jason; Kieffer-Kwon, Kyong Rim; Mandal, Malay; Ji, Yanhong; Meffre, Eric; Clark, Marcus R.; Cowell, Lindsay G.; Casellas, Rafael; Schatz, David G.

doi:10.1016/j.cell.2015.07.009

Cited by 112 publications

(166 citation statements)

References 48 publications

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“…5C). This finding is consistent with our recent RAG1 ChIP-seq data that revealed the binding of RAG1 at a wide range of intensi-ties to thousands of active promoters in developing B and T cells (38). We conclude that RAG1 binding is not limited to regions containing RSSs, in contrast to the conclusions of our previous study (25), and that the association of RAG1 with chromatin might be mediated by various types of interactions, including sequence-specific and nonspecific DNA binding and interactions with nucleosomes.…”

Section: Discussionsupporting

confidence: 82%

“…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). We also note that our recent ChIP-seq analysis using mouse thymocytes revealed that RAG1 binding was reduced in intensity and less well focused at regions of high H3K4me3 levels in the absence of RAG2 than in its presence (38). These results (which represent a steady-state analysis) are more in keeping with the models derived from in vitro kinetic studies (44).…”

Section: Discussionsupporting

confidence: 76%

“…Isolated heptamer-like and nonamer-like sequences are abundant in the genome, and multiple such sequences are present in the vicinity of the Pou2af1 and ␥-actin ChIP-qPCR amplicons. Our data reported here as well as our finding that high-scoring nonamer sequences are more prevalent than expected by chance at RAG1 binding sites genome wide (38) suggest that isolated nonamers might contribute to RAG1 binding at locations lacking discernible RSSs.…”

Section: Discussionsupporting

confidence: 50%

“…ChIP-sequencing (ChIP-seq) data from our laboratory revealed the ability of both RAG1 and RAG2 to bind at certain non-antigen-receptor genes, which do not harbor RSSs (25,38). This result was surprising given that RAG1 has RSS binding domains and that the only known function of the RAG proteins is to assemble antigen receptor genes.…”

Section: Experimental System For Determining Rag Binding Dynamics In mentioning

confidence: 52%

“…7C). Finally, we designed an additional substrate with a consensus 23RSS paired with a cryptic RSS (cRSS) comprised of a CA dinucleotide repeated 38 times [p(CA) 38 /23] (Fig. 7A).…”

Section: Rag1 Recruitment Is Mediated Primarily By the Nonamermentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 82%

Section: Discussionsupporting

confidence: 76%

Section: Discussionsupporting

confidence: 50%

Section: Experimental System For Determining Rag Binding Dynamics In mentioning

confidence: 52%

“…7C). Finally, we designed an additional substrate with a consensus 23RSS paired with a cryptic RSS (cRSS) comprised of a CA dinucleotide repeated 38 times [p(CA) 38 /23] (Fig. 7A).…”

Section: Rag1 Recruitment Is Mediated Primarily By the Nonamermentioning

confidence: 99%

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

Shetty

Schatz

2015

Molecular and Cellular Biology

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Broad H3K4me3 domains: Maintaining cellular identity and their implication in super‐enhancer hijacking

et al. 2023

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The human and mouse genomes are complex from a genomic standpoint. Each cell has the same genomic sequence, yet a wide array of cell types exists due to the presence of a plethora of regulatory elements in the non‐coding genome. Recent advances in epigenomic profiling have uncovered non‐coding gene proximal promoters and distal enhancers of transcription genome‐wide. Extension of promoter‐associated H3K4me3 histone mark across the gene body, known as a broad H3K4me3 domain (H3K4me3‐BD), is a signature of constitutive expression of cell‐type‐specific regulation and of tumour suppressor genes in healthy cells. Recently, it has been discovered that the presence of H3K4me3‐BDs over oncogenes is a cancer‐specific feature associated with their dysregulated gene expression and tumourigenesis. Moreover, it has been shown that the hijacking of clusters of enhancers, known as super‐enhancers (SE), by proto‐oncogenes results in the presence of H3K4me3‐BDs over the gene body. Therefore, H3K4me3‐BDs and SE crosstalk in healthy and cancer cells therefore represents an important mechanism to identify future treatments for patients with SE driven cancers.

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DNA crosslinking and recombination‐activating genes 1/2 (RAG1/2) are required for oncogenic splicing in acute lymphoblastic leukemia

Zhang

Cheng

et al. 2021

Cancer Communications

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Background Abnormal alternative splicing is frequently associated with carcinogenesis. In B‐cell acute lymphoblastic leukemia (B‐ALL), double homeobox 4 fused with immunoglobulin heavy chain (DUX4/IGH) can lead to the aberrant production of E‐26 transformation‐specific family related gene abnormal transcript (ERGalt) and other splicing variants. However, the molecular mechanism underpinning this process remains elusive. Here, we aimed to know how DUX4/IGH triggers abnormal splicing in leukemia. Methods The differential intron retention analysis was conducted to identify novel DUX4/IGH‐driven splicing in B‐ALL patients. X‐ray crystallography, small angle X‐ray scattering (SAXS), and analytical ultracentrifugation were used to investigate how DUX4/IGH recognize double DUX4 responsive element (DRE)‐DRE sites. The ERGalt biogenesis and B‐cell differentiation assays were performed to characterize the DUX4/IGH crosslinking activity. To check whether recombination‐activating gene 1/2 (RAG1/2) was required for DUX4/IGH‐driven splicing, the proximity ligation assay, co‐immunoprecipitation, mammalian two hybrid characterizations, in vitro RAG1/2 cleavage, and shRNA knock‐down assays were performed. Results We reported previously unrecognized intron retention events in C‐type lectin domain family 12, member A abnormal transcript (CLEC12Aalt) and chromosome 6 open reading frame 89 abnormal transcript (C6orf89alt), where also harbored repetitive DRE‐DRE sites. Supportively, X‐ray crystallography and SAXS characterization revealed that DUX4 homeobox domain (HD)1‐HD2 might dimerize into a dumbbell‐shape trans configuration to crosslink two adjacent DRE sites. Impaired DUX4/IGH‐mediated crosslinking abolishes ERGalt, CLEC12Aalt, and C6orf89alt biogenesis, resulting in marked alleviation of its inhibitory effect on B‐cell differentiation. Furthermore, we also observed a rare RAG1/2‐mediated recombination signal sequence‐like DNA edition in DUX4/IGH target genes. Supportively, shRNA knock‐down of RAG1/2 in leukemic Reh cells consistently impaired the biogenesis of ERGalt, CLEC12Aalt, and C6orf89alt. Conclusions All these results suggest that DUX4/IGH‐driven DNA crosslinking is required for RAG1/2 recruitment onto the double tandem DRE‐DRE sites, catalyzing V(D)J‐like recombination and oncogenic splicing in acute lymphoblastic leukemia.

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RAG Represents a Widespread Threat to the Lymphocyte Genome

Cited by 112 publications

References 48 publications

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

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

Broad H3K4me3 domains: Maintaining cellular identity and their implication in super‐enhancer hijacking

DNA crosslinking and recombination‐activating genes 1/2 (RAG1/2) are required for oncogenic splicing in acute lymphoblastic leukemia

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