A Direct Interaction between the RAG2 C Terminus and the Core Histones Is Required for Efficient V(D)J Recombination

Immunobiology Center, Mount Sinai School of Medicine, New York, New York 10029, USA.
Immunity (Impact Factor: 21.56). 09/2005; 23(2):203-12. DOI: 10.1016/j.immuni.2005.07.004
Source: PubMed


V(D)J recombination is a tightly controlled process of somatic recombination whose regulation is mediated in part by chromatin structure. Here, we report that RAG2 binds directly to the core histone proteins. The interaction with histones is observed in developing lymphocytes and within the RAG1/RAG2 recombinase complex in a manner that is dependent on the RAG2 C terminus. Amino acids within the plant homeo domain (PHD)-like domain as well as a conserved acidic stretch of the RAG2 C terminus that is considered to be a linker region are important for this interaction. Point mutations that disrupt the RAG2-histone association inhibit the efficiency of the V(D)J recombination reaction at the endogenous immunoglobulin locus, with the most dramatic effect in the V to DJ(H) rearrangement.

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Available from: Hediye Erdjument-Bromage, May 22, 2014
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    • "The C terminus also contains a conserved hinge region (Jones and Simkus, 2009) that lies after a predicted b-propeller (Callebaut and Mornon, 1998) and has a high density of acidic amino acids (Oettinger et al., 1990). A small region of the hinge (residues 402–407) interacts with core histones (West et al., 2005), but the significance of this interaction remains unclear. Nothing further has been reported as to the hinge's function (Jones and Simkus, 2009). "
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    ABSTRACT: V(D)J recombination-associated DNA double-strand breaks (DSBs) are normally repaired by the high-fidelity classical nonhomologous end-joining (cNHEJ) machinery. Previous studies implicated the recombination-activating gene (RAG)/DNA postcleavage complex (PCC) in regulating pathway choice by preventing access to inappropriate repair mechanisms such as homologous recombination (HR) and alternative NHEJ (aNHEJ). Here, we report that RAG2's "acidic hinge," previously of unknown function, is critical for several key steps. Mutations that reduce the hinge's negative charge destabilize the PCC, disrupt pathway choice, permit repair of RAG-mediated DSBs by the translocation-prone aNHEJ machinery, and reduce genomic stability in developing lymphocytes. Structural predictions and experimental results support our hypothesis that reduced flexibility of the hinge underlies these outcomes. Furthermore, sequence variants present in the human population reduce the hinge's negative charge, permit aNHEJ, and diminish genomic integrity.
    Cell Reports 08/2013; 4(5). DOI:10.1016/j.celrep.2013.07.041 · 8.36 Impact Factor
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    • "By analogy with other DNA repair reactions, specific chromatin modifications may provide signals for recruitment and assembly of repair complexes (Altaf et al., 2007). Several lines of evidence indicate that RAG proteins themselves , through domains outside the catalytic core regions, play an active role in modulating the chromosomal V(D)J recombination reaction (Noordzij et al., 2000; Simkus et al., 2007; West et al., 2005). Here we show that RAG1 contains a chromatinbinding domain within its noncore N-terminal region that preferentially interacts in vitro and in vivo with histone H3, mediated by the N-terminal histone tail region. "
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    ABSTRACT: The RAG1 and RAG2 proteins are the only lymphoid-specific factors required to perform the first step of V(D)J recombination, DNA cleavage. While the catalytic domain of RAG1, the core region, has been well characterized, the role of the noncore region in modulating chromosomal V(D)J recombination efficiency remains ill defined. Recent studies have highlighted the role of chromatin structure in regulation of V(D)J recombination. Here we show that RAG1 itself, through a RING domain within its N-terminal noncore region, preferentially interacts directly with and promotes monoubiquitylation of histone H3. Mutations affecting the RAG1 RING domain reduce histone H3 monoubiquitylation activity, decrease V(D)J recombination activity in vivo, reduce formation of both signal-joint and coding-joint products on episomal substrates, and decrease efficiency of V(D)J recombination at the endogenous IgH locus in lymphoid cells. The results reveal that RAG1-mediated histone monoubiquitylation activity plays a role in regulating the joining phase of chromosomal V(D)J recombination.
    Molecular cell 01/2010; 37(2):282-93. DOI:10.1016/j.molcel.2009.12.035 · 14.02 Impact Factor
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    • "Due to greater difficulty in purification of full-length RAG proteins, most early biochemical work on RAGs was done with core regions. However, some patients with severe combined immunodeficiency (SCID) and Ommen syndrome are found to carry mutations in the RAG2 PHD finger, indicating the essential role of this domain during V(D)J recombination (Baker et al., 2008; Ramón-Maiques et al., 2007; West et al., 2005). Consistent with this, mutations of residues contacting H3K4me3 impair V(D)J recombination on both plasmid substrates and genomic loci (Liu et al., 2007; Matthews et al., 2007; Ramón-Maiques et al., 2007). "
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    ABSTRACT: The PHD finger of the RAG2 polypeptide of the RAG1/RAG2 complex binds to the histone H3 modification, trimethylated lysine 4 (H3K4me3), and in some manner increases V(D)J recombination. In the absence of biochemical studies of H3K4me3 on purified RAG enzyme activity, the precise role of H3K4me3 remains unclear. Here, we find that H3K4me3 stimulates purified RAG enzymatic activity at both the nicking (2- to 5-fold) and hairpinning (3- to 11-fold) steps of V(D)J recombination. Remarkably, this stimulation can be achieved with free H3K4me3 peptide (in trans), indicating that H3K4me3 functions via two distinct mechanisms. It not only tethers the RAG enzyme complex to a region of DNA, but it also induces a substantial increase in the catalytic turnover number (k(cat)) of the RAG complex. The H3K4me3 catalytic stimulation applies to suboptimal cryptic RSS sites located at H3K4me3 peaks that are critical in the inception of human T cell acute lymphoblastic lymphomas.
    Molecular cell 07/2009; 34(5):535-44. DOI:10.1016/j.molcel.2009.05.011 · 14.02 Impact Factor
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