The In Vivo Pattern of Binding of RAG1 and RAG2 to Antigen Receptor Loci (vol 141, pg 419, 2010)

Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, Box 208011, New Haven, CT 06520-8011, USA.
Cell (Impact Factor: 32.24). 04/2010; 141(3):419-31. DOI: 10.1016/j.cell.2010.03.010
Source: PubMed

ABSTRACT The critical initial step in V(D)J recombination, binding of RAG1 and RAG2 to recombination signal sequences flanking antigen receptor V, D, and J gene segments, has not previously been characterized in vivo. Here, we demonstrate that RAG protein binding occurs in a highly focal manner to a small region of active chromatin encompassing Ig kappa and Tcr alpha J gene segments and Igh and Tcr beta J and J-proximal D gene segments. Formation of these small RAG-bound regions, which we refer to as recombination centers, occurs in a developmental stage- and lineage-specific manner. Each RAG protein is independently capable of specific binding within recombination centers. While RAG1 binding was detected only at regions containing recombination signal sequences, RAG2 binds at thousands of sites in the genome containing histone 3 trimethylated at lysine 4. We propose that recombination centers coordinate V(D)J recombination by providing discrete sites within which gene segments are captured for recombination.

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    • "Our findings at naturally occurring translocation sites extend recent correlative observations from genome-wide studies that mapped I-SceI-induced translocation junctions in B cells to within or near transcriptionally active regions of the genome (Chiarle et al. 2011; Klein et al. 2011) and in translocation-negative prostate cancer cells, where liganded androgen receptor (AR) binds near the TMPRSS2 translocation gene and increases its transcriptional activity, leading to elevated H3K4me3 levels across the breakpoint region (Lin et al. 2009; Yu et al. 2010). Furthermore, H3K4me3 has been implicated in DSBs generated by endonucleases during class switch and V(D)J recombination processes in lymphocytes (Daniel and Nussenzweig 2012), and both RAG2 and AID target extrachromosomal sites that are highly enriched in H3K4me3 in a sequence-independent manner (Shimazaki et al. 2009; Ji et al. 2010; Stanlie et al. 2010). The increased formation of DSBs by endonucleases in regions of high H3K4 methylation is likely due to chromatin decondensation, resulting in higher chromatin accessibility (Mostoslavsky et al. 2003). "
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    ABSTRACT: Chromosome translocations are well-established hallmarks of cancer cells and often occur at nonrandom sites in the genome. The molecular features that define recurrent chromosome breakpoints are largely unknown. Using a combination of bioinformatics, biochemical analysis, and cell-based assays, we indentify here specific histone modifications as facilitators of chromosome breakage and translocations. We show enrichment of several histone modifications over clinically relevant translocation-prone genome regions. Experimental modulation of histone marks sensitizes genome regions to breakage by endonuclease challenge or irradiation and promotes formation of chromosome translocations of endogenous gene loci. Our results demonstrate that histone modifications predispose genome regions to chromosome breakage and translocations. © 2015 Burman et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & development 06/2015; 29(13). DOI:10.1101/gad.262170.115 · 10.80 Impact Factor
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    • "Green shading indicates whether cross-linking in mE relative to WT alleles is unchanged (darkest green), reduced significantly (lighter green), or reduced to background levels in pro-B cells (white). (F) 3C assays were performed with the E viewpoint (anchor) in DN thymocytes and pro-B cells from RAG-deficient mice, either lacking or expressing a D708A RAG transgene (Ji et al., 2010b). (G) 3C assays were performed with the D1 viewpoint (anchor) in DN thymocytes (WT, E, or E alleles) and pro-B cell controls. "
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    ABSTRACT: Gene regulation relies on dynamic changes in three-dimensional chromatin conformation, which are shaped by composite regulatory and architectural elements. However, mechanisms that govern such conformational switches within chromosomal domains remain unknown. We identify a novel mechanism by which cis-elements promote long-range interactions, inducing conformational changes critical for diversification of the TCRβ antigen receptor locus (Tcrb). Association between distal Vβ gene segments and the highly expressed DβJβ clusters, termed the recombination center (RC), is independent of enhancer function and recruitment of V(D)J recombinase. Instead, we find that tissue-specific folding of Tcrb relies on two distinct architectural elements located upstream of the RC. The first, a CTCF-containing element, directly tethers distal portions of the Vβ array to the RC. The second element is a chromatin barrier that protects the tether from hyperactive RC chromatin. When the second element is removed, active RC chromatin spreads upstream, forcing the tether to serve as a new barrier. Acquisition of barrier function by the CTCF element disrupts contacts between distal Vβ gene segments and significantly alters Tcrb repertoires. Our findings reveal a separation of function for RC-flanking regions, in which anchors for long-range recombination must be cordoned off from hyperactive RC landscapes by chromatin barriers. © 2015 Majumder et al.
    Journal of Experimental Medicine 12/2014; 212(1). DOI:10.1084/jem.20141479 · 12.52 Impact Factor
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    • "Although this means that the catalysis is done by a tetramer, we cannot exclude a noncatalytic role of a RAG octamer. For example, there is the possibility that a RAG octamer exists and serves a role in bringing two distant portions of DNA closer together at two specific RSS sites (regional synapsis), and this could create a high local DNA concentration for RSS recombination (Ji et al., 2010b; Schatz and Ji, 2011). Then, other RAGs, existing as catalytically active RAG tetramers, could carry out nicking and hairpin formation at other RSS sites nearby the synapsed sites. "
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    ABSTRACT: 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.
    Cell Reports 04/2014; 7(2). DOI:10.1016/j.celrep.2014.03.005 · 8.36 Impact Factor
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