Recombination centres and the orchestration of V(D)J recombination

Key Points

  • V(D)J recombination, which assembles antigen receptor genes during lymphocyte development, is initiated when the recombination activating gene 1 (RAG1) and RAG2 proteins bind and cleave genomic DNA at recombination signal sequences that lie adjacent to antigen receptor gene segments.

  • Modulation of chromatin structure, histone modifications and transcriptional activity determine the accessibility of recombination signal sequences for binding by RAG1 and RAG2 and thereby help to dictate the developmentally ordered sequence of V(D)J recombination events.

  • V(D)J recombination is also controlled by the association of antigen receptor genes with active or inactive nuclear compartments and by changes in the higher order chromatin architecture (such as looping and contraction) of antigen receptor genes.

  • The RAG proteins associate with a small region of highly active chromatin in each antigen receptor locus, forming recombination centres within which V(D)J recombination might be regulated.

  • The interaction of the RAG2 plant homeodomain (PHD) finger with trimethylated histone H3 lysine 4 (H3K4me3; a modification found in active chromatin) is important for efficient V(D)J recombination and results in the association of RAG2 with many thousands of sites in the genome.

  • The ectopic recruitment and activity of RAG1 and RAG2 at loci that do not encode antigen receptors contributes to genome instability and the development of lymphoid malignancies.

Abstract

The initiation of V(D)J recombination by the recombination activating gene 1 (RAG1) and RAG2 proteins is carefully orchestrated to ensure that antigen receptor gene assembly occurs in the appropriate cell lineage and in the proper developmental order. Here we review recent advances in our understanding of how DNA binding and cleavage by the RAG proteins are regulated by the chromatin structure and architecture of antigen receptor genes. These advances suggest novel mechanisms for both the targeting and the mistargeting of V(D)J recombination, and have implications for how these events contribute to genome instability and lymphoid malignancy.

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Figure 1: The structure of antigen receptor genes.
Figure 2: Mechanism of V(D)J recombination.
Figure 3: The recombination centre model.
Figure 4: The nonamer-first model for RSS recognition.

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Acknowledgements

The authors wish to thank E. Oltz, G. Teng, K. Shetty and J. Banerjee for comments on the manuscript. We apologize that not all of the relevant literature could be cited owing to space constraints.

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Glossary

V(D)J recombination

Somatic rearrangement of variable (V), diversity (D) and joining (J) regions of the genes that encode antigen receptors, leading to repertoire diversity of immunoglobulins and T cell receptors.

Chromosomal translocation

An aberration of chromosome structure in which a portion of one chromosome is broken off and becomes attached to another.

Non-homologous end joining

(NHEJ). A DNA repair process that joins broken DNA ends (double strand breaks) without using homologous DNA as a template. Components of this pathway include the proteins Ku70 (also known as XRCC6), Ku80 (also known as XRCC5), Artemis, X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs).

Chromatin

The combination of DNA, histones and other proteins that comprises eukaryotic chromosomes. The basic repeating unit of chromatin is the nucleosome, which consists of an octamer of histone proteins around which 146 base pairs of DNA is wound.

Allelic exclusion

In theory, every B cell has the potential to produce two immunoglobulin heavy chains and two immunoglobulin light chains. In practice, however, a B cell produces only one immunoglobulin heavy chain and the majority produce only one immunoglobulin light chain. Similarly, most T cells produce only a single T cell receptor β-chain protein. The process by which the production of two different chains is prevented is known as allelic exclusion. Allelic exclusion is accomplished primarily through regulated V(D)J recombination.

Germline transcription

Transcription of unrearranged antigen receptor gene loci that begins before or coincident with their activation. It is not thought to produce functional protein, and the promoter and initiation sites are often lost in the subsequent rearrangement events.

Nucleosome

The fundamental structural unit of eukaryotic chromosomes. It consists of pairs of each of the core histones (H2A, H2B, H3 and H4), thereby creating the histone octamer, and a single molecule of the linker histone H1. The nucleosome spans 146 base pairs of DNA.

Chromatin remodelling complex

An enzymatic complex that remodels the DNA–nucleosome architecture and thus can determine transcriptional activity. The SWI–SNF ATPase is an example of a complex that remodels chromatin.

Antisense transcription

Transcription in the opposite direction and of the opposite strand from that used to generate the normal product of a gene. It is not thought to generate a protein product but instead might alter chromatin structure either directly (via the act of transcription) or indirectly (via the antisense RNA produced).

Heterochromatin

High-density regions in the nucleus that are thought to contain compacted chromatin structures associated with silent genes.

DNase I hypersensitive site

A site of nuclease sensitivity when nuclei from cells are exposed to limiting concentrations of the enzyme DNase I. The digested regions of DNA correspond to sites of open DNA, which might be transcription factor binding sites or areas of altered nucleosome conformation.

Bromodomain

A module of 110 amino acids that is found in several transcriptional regulators. A bromodomain consists of a four-helix bundle with a single binding pocket for Nɛ-acetyl-lysine on histone tails.

Pro-B cell

A cell in the earliest stage of B cell development in the bone marrow. Pro-B cells are characterized by incomplete immunoglobulin heavy chain rearrangements and are defined as CD19+ and cytoplasmic IgM or, sometimes, as B220+CD43+ (by the Hardy classification scheme).

Pericentric heterochromatin

Regions of very densely packed chromatin fibres located near the centromere of each chromosome. These regions are typically inactive and often cluster to form discrete clumps in the nucleus.

Fluorescence in situ hybridization

(FISH). The use of fluorescent probes to visually label specific DNA sequences in the nuclei of cells that are in the interphase or metaphase stages of mitosis.

Chromatin immunoprecipitation

An experimental technique that analyses direct binding of an endogenous transcription factor to chromatin by fixation with formaldehyde followed by immunoprecipitation with a transcription factor-specific antibody. Gene-specific enrichment is then assessed by polymerase chain reaction analysis of the immunoprecipitated DNA.

Recombination centre

A region of an antigen receptor locus that is characterized by strong binding of recombination activating gene 1 (RAG1) and RAG2 and high levels of germline transcription, RNA polymerase II, histone acetylation and trimethylated histone H3 lysine 4 (H3K4me3).

Cryptic RSS

A region of DNA that resembles a true recombination signal sequence (RSS) in some of its functionally important sequence features but does not lie adjacent to an antigen receptor gene segment.

Homologous recombination

Genetic recombination that occurs between regions of DNA with long stretches of homology. This occurs with a low frequency in somatic cells and at a much higher frequency in germ cells.

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Schatz, D., Ji, Y. Recombination centres and the orchestration of V(D)J recombination. Nat Rev Immunol 11, 251–263 (2011). https://doi.org/10.1038/nri2941

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