V(D)J Recombination: RAG Recombination Centres

ReseaRch highlights

V(D)J RECOmbINAtION RAG recombination centres

The recombination of variable (V), The authors generated mutant In pro‑B cells, RAG protein diversity (D) and joining (J) gene seg‑ mice in which the RAG proteins binding at the Igh locus occurred ments that form immuno globulins could interact with and bind to DNA at the Jh and proximal DQ52 gene and T cell receptors is essential for normally without initiating V(D)J segments but not at other D or V the generation of a highly diverse recombination and assessed RAG pro‑ gene segments. Again, RAG binding repertoire of antigen receptors. tein binding, as well as the chromatin strongly correlated with high levels of V(D)J recombination is initiated by activation status, in purified lympho‑ active chromatin. However, although binding of recombination‑activating cyte populations using chromatin RAG2 could bind in the absence of gene 1 (RAG1) and RAG2 to immunoprecipitation. RAG1, RAG1 required the presence recombination signal sequences At the Igκ locus of pre‑B cells, of RAG2 for binding. Finally, RAG (RSSs) that flank these gene seg‑ the RAG proteins associated with protein binding (both together ments. Schatz and colleagues have the Jκ but not Vκ gene segments. and separately) occurred at Dβ–Jβ now directly assessed RAG protein The binding region contained highly clusters but not at other regions of binding at antigen receptor loci active chromatin, as determined by the Tcrβ locus in pro‑T cells. Of note, in vivo and found that binding high levels of histone 3 trimethyl‑ RAG binding at these sites in the Igh occurs at small regions of active ation at lysine 4 (H3K4me3) and and Tcrβ loci persisted in pre‑B and chromatin encompassing J (and H3 acetyl ation, as well as RNA pre‑T cells, respectively. where present J‑proximal D) polymerase II occupancy. Binding Further analyses showed that segments of Igκ, Igh, Tcrα and of RAG1 and RAG2 could occur the RSSs were required for strong Tcrβ, which they have termed independently of each other, sug‑ binding of RAG1 and RAG2 to the recombination centres. RAG1 and gesting the possibility that RAG1 recombination centres, and mutations RAG2 can bind independently at and RAG2 may be differentially in RAG1 at three known RSS‑binding these sites, and the formation of recruited into the recombination residues decreased RAG1 recruitment the recombination centres is centre. Rearrangement of the Igκ to the RSSs. RAG2 could also bind tightly regulated during locus is upregulated during pro‑B to numerous sites with high levels of lymphocyte to pre‑B cell transition, and binding H3K4me3 that lacked RSSs. Mutation development. of the RAG proteins at the Jκ gene of the plant homeodomain (PHD) of segments and the level of active RAG2, which abrogates H3K4me3 chromatin at these sites binding, decreased its association was highly increased in with numerous H3K4me3‑containing pre‑B cells compared genes, suggesting that RAG2 binds with pro‑B cells. broadly to the genome, whereas Similarly, RAG protein RAG1 binding is restricted to RSSs. binding (both together and The lineage‑ and developmental separately) at the Tcrα locus stage‑specific binding of RAG of pre‑T cells occurred at the proteins to specific recombination Jα cluster in a region of active centres supports the suggestion that chromatin. By contrast, the these sites coordinate V(D)J recom‑ recombination centre had bination but could also contribute to low levels of H3K4me3 in the aberrant recombination events pro‑T cells, in which recom‑ associated with lymphomas. bination does not occur, Olive Leavy and RAG proteins did not bind to any of the Tcrα ORIGINAL RESEARCH PAPER Yanhong, J. et al. gene segments analysed The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci. Cell 141, 419–431 (2010) in these cells.