Hao et al. report that a distant anti-silencer element interacts with the Rag1 and Rag2 gene promoters in double-positive thymocytes and that SATB1 is a regulator of Rag locus organization in these cells.

The sequence specificity of the recombination activating gene (RAG) complex during V(D)J recombination has been well studied. RAGs can also act as structure-specific nuclease; however, little is known about the mechanism of its action. Here, we show that in addition to DNA structure, sequence dictates the pattern and efficiency of RAG cleavage on altered DNA structures. Cytosine nucleotides are preferentially nicked by RAGs when present at single-stranded regions of heteroduplex DNA. Although unpaired thymine nucleotides are also nicked, the efficiency is many fold weaker. Induction of single-or doublestrand breaks by RAGs depends on the position of cytosines and whether it is present on one or both of the strands. Interestingly, RAGs are unable to induce breaks when adenine or guanine nucleotides are present at single-strand regions. The nucleotide present immediately next to the bubble sequence could also affect RAG cleavage. Hence, we propose "C (d) C (S) C (S) " (d, double-stranded; s, single-stranded) as a consensus sequence for RAG-induced breaks at single-/double-strand DNA transitions. Such a consensus sequence motif is useful for explaining RAG cleavage on other types of DNA structures described in the literature. Therefore, the mechanism of RAG cleavage described here could explain facets of chromosomal rearrangements specific to lymphoid tissues leading to genomic instability. The recombination activating gene (RAG)3 complex, consisting of RAG1 and RAG2, is the nuclease responsible for V(D)J recombination, a physiological process by which immunoglobulin and T-cell receptor diversity is generated. RAGs are normally expressed in B-cells and T-cells (1). During V(D)J recombination, the variable (V), diversity (D), and joining (J) subexons are rearranged. Specific sequences present at the ends of the subexon, called recombination signal sequences (RSS) are recognized by RAGs. Each RSS consists of a conserved heptamer and nonamer, separated by a nonconserved spacer, the length of which designates RSS as a 12-signal or 23-signal. Normally during V(D)J recombination, a 12-signal pairs with a 23-signal with the help of proteins like HMGB1 (high mobility group box 1). The nick induced by RAGs during V(D)J recombination is consistently 5Ј of the heptamers (2-14). The nicked strand is then converted to a hairpin in each V, D, and J coding end by a transesterification reaction, leaving each of the signal ends blunt (15). The hairpins are then opened by the ArtemisDNAPKcs complex (16). After cleavage, the RAG complex remains tightly bound to the two signal ends and less tightly bound to the coding end, in a postcleavage complex (17,18). Finally, the complete exon coding for antibody or TCR (T-cell receptor) is generated by joining of the broken subexons by nonhomologous DNA endjoining (NHEJ) (19,20).In the recent past, studies have shown that cryptic RSS sites present elsewhere in the genome can also act as off-target sites for RAG misrecognition, leading to chromosomal translocations in lymphoid cancers such as l...

Expression of Rag1 and Rag2 is tightly regulated in developing T cells to mediate TCR gene assembly. Here we have investigated the molecular mechanisms governing the assembly and disassembly of a transcriptionally active RAG locus chromatin hub in CD4+CD8+ thymocytes. Rag1 and Rag2 gene expression in CD4+CD8+ thymocytes depends on Rag1 and Rag2 promoter activation by a distant antisilencer element (ASE). We identify GATA3 and E2A as critical regulators of the ASE, and Runx1 and E2A as critical regulators of the Rag1 promoter. We reveal hierarchical assembly of a transcriptionally active chromatin hub containing the ASE and RAG promoters, with Rag2 recruitment and expression dependent on assembly of a functional ASE–Rag1 framework. Finally, we show that signal-dependent down-regulation of RAG gene expression in CD4+CD8+ thymocytes depends on Ikaros and occurs with disassembly of the RAG locus chromatin hub. Our results provide important new insights into the molecular mechanisms that orchestrate RAG gene expression in developing T cells.

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