890 | NOVEMBER 2003 | VOLUME 3 www.nature.com/reviews/immunol REVIEWS V(D)J recombination: levels of regulation V(D)J recombination has three types of regulation — lineage specificity, order within a lineage and allelic exclusion. Transcriptional regulation limits the expres- sion of RAG proteins to the progenitor stages of B- and T-cell development, accounting for the lymphoid lineage specificity of recombination per se. However, immunoglobulin genes fully rearrange in B cells only and TCR genes rearrange in T cells only, so additional regulatory mechanisms must exist to enforce B- versus T-cell lineage specificity. Moreover, in a given lineage, rearrangement is ordered; with immunoglobulin heavy-chain (IgH) or TCRβ locus D-to-J rearrangement preceding V-to-DJ rearrangement, followed most often by rearrangement of immunoglobulin light chain (IgL) or TCRα chains (FIGS 1 and 2). Finally, recombination at most loci is regulated such than an individual B or T cell generates only one functional allele at each locus. This phenomenon — allelic exclusion — results in lymphocytes expressing only a single receptor for antigen 5 . As noted earlier, all rearranging gene segments are flanked by conserved RSSs and all V(D)J recombination events seem to require the same trans-acting factors — the lymphoid-specific RAG1 and RAG2 proteins and It has been nearly 25 years since Tonegawa and col- leagues 1 shattered one of the basic assumptions of molecular biology, the inviolate structure of the genome, and in so doing solved a fundamental puzzle in immunology — the generation of antigen-receptor diversity. The discovery that a complete immunoglob- ulin gene is generated from component gene segments through a series of site-specific DNA recombination reactions known as V(D)J recombination was followed over the ensuing decades by the elucidation of the structures of the seven rearranging antigen-receptor loci (immunoglobulin μ, κ and λ; and T-cell receptor (TCR) α, β, γ and δ), the identification of cis-acting DNA sequences essential for recombination — the RECOMBINATION SIGNAL SEQUENCES (RSSs) — the discovery of two lymphocyte-restricted genes, RAG1 and RAG2, which encode the lymphocyte-specific components of the recombinase, and a thorough description of the recombination-reaction pathway 2–4 (BOX 1). What remains to be understood, however, are the mecha- nisms involved in the regulation of this remarkable site-specific DNA recombination reaction. This review focuses on the regulation of V(D)J recombina- tion from the perspective of both the recombinase and its substrates. REGULATING ANTIGEN-RECEPTOR GENE ASSEMBLY Mark S. Schlissel The genes encoding antigen receptors are unique because of their high diversity and their assembly in developing lymphocytes from gene segments through a series of site-specific DNA recombination reactions known as V(D)J rearrangement. This review focuses on our understanding of how recombination of immunoglobulin and T-cell receptor gene segments is tightly regulated despite being catalysed by a common lymphoid recombinase, which recognizes a widely distributed conserved recombination signal sequence. Probable mechanisms involve precise expression of the lymphoid-restricted recombination-activating genes RAG1 and RAG2, and developmentally regulated epigenetic alterations in template accessibility, which are targeted by transcriptional regulatory elements and involve chromatin-modifying enzymes. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA. e-mail:mss@uclink4. berkeley.edu doi:10.1038/nri1225 RECOMBINATION SIGNAL SEQUENCES (RSSs). Short, conserved DNA sequences that flank all rearranging gene segments and serve as the recognition elements for the recombinase machinery.