The immunoglobulin heavy-chain (Igh) locus is organized into distinct regions that contain multiple variable (V(H)), diversity (D(H)), joining (J(H)) and constant (C(H)) coding elements. How the Igh locus is structured in 3D space is unknown. To probe the topography of the Igh locus, spatial distance distributions were determined between 12 genomic markers that span the entire Igh locus. Comparison of the distance distributions to computer simulations of alternative chromatin arrangements predicted that the Igh locus is organized into compartments containing clusters of loops separated by linkers. Trilateration and triple-point angle measurements indicated the mean relative 3D positions of the V(H), D(H), J(H), and C(H) elements, showed compartmentalization and striking conformational changes involving V(H) and D(H)-J(H) elements during early B cell development. In pro-B cells, the entire repertoire of V(H) regions (2 Mbp) appeared to have merged and juxtaposed to the D(H) elements, mechanistically permitting long-range genomic interactions to occur with relatively high frequency.

The E2A gene products, E12 and E47, are critical regulators of B cell development. However, it remains elusive whether E12 and E47 have overlapping and/or distinct functions during B lymphopoiesis. We have generated mice deficient for either E12 or E47 and examined their roles in B cell maturation. We show that E47 is essential for developmental progression at the prepro–B cell stage, whereas E12 is dispensable for early B cell development, commitment, and maintenance. In contrast, both E12 and E47 play critical roles in pre–B and immature B cells to promote immunoglobulin λ (Igλ) germline transcription as well as Igλ VJ gene rearrangement. Furthermore, we show that E12 as well as E47 is required to promote receptor editing upon exposure to self-antigen. We demonstrate that increasing levels of E12 and E47 act to induce Igλ germline transcription, promote trimethylated lysine 4 on histone 3 (H3) as well as H3 acetylation across the Jλ region, and activate Igλ VJ gene rearrangement. We propose that in the pre–B and immature B cell compartments, gradients of E12 and E47 activities are established to mechanistically regulate the sequential rearrangement of the Ig light chain genes.

The adaptive immune system generates a specific response to a vast spectrum of antigens. This remarkable property is achieved by lymphocytes that each express single and unique antigen receptors. During lymphocyte development, antigen receptor coding elements are assembled from widely dispersed gene segments. The assembly of antigen receptors is controlled at multiple levels, including epigenetic marking, nuclear location, and chromatin topology. Here we review recently uncovered mechanisms that underpin long-range genomic interactions and the generation of antigen receptor diversity.

The adaptive immune system generates a specific response to a vast spectrum of antigens. This remarkable property is achieved by lymphocytes that each express single and unique antigen receptors. During lymphocyte development, antigen receptor coding elements are assembled from widely dispersed gene segments. The assembly of antigen receptors is controlled at multiple levels, including epigenetic marking, nuclear location, and chromatin topology. Here we review recently uncovered mechanisms that underpin long-range genomic interactions and the generation of antigen receptor diversity.

RAG1 and RAG2 initiate V(D)J recombination by introducing DNA double strand breaks between each selected gene segment and its bordering recombination signal sequence (RSS) in a two-step mechanism in which the DNA is first nicked, followed by hairpin formation. The RSS consists of a conserved nonamer and heptamer sequence, in which the latter borders the site of DNA cleavage. A region within RAG1, referred to as the central domain (residues 528 -760 of 1040 in the full-length protein), has been shown previously to bind specifically to the double-stranded (ds) RSS heptamer, but with both weak specificity and affinity. However, additional investigations into the RAG1-RSS heptamer interaction are required because the DNA substrate forms intermediate conformations during the V(D)J recombination reaction. These include the nicked and hairpin products, as well as likely base unpairing to produce single-stranded (ss) DNA near the cleavage site. Here, it was determined that although the central domain showed substantially higher binding affinity for ss and nicked versus ds substrate, the interaction with ss RSS was particularly robust. In addition, the central domain bound with greater sequence specificity to the ss RSS heptamer than to the ds form. This study provides important insight into the V(D)J recombination reaction, specifically that significant interaction of the RSS heptamer with RAG1 occurs only after the induction of conformational changes at the RSS heptamer.V(D)J recombination leads to the formation of functional immunoglobulin and T cell receptor genes in developing B and T cells, respectively. Through DNA rearrangement of the immunoglobulin and T cell receptor genetic loci, the genes are assembled by combining selected gene segments termed variable (V), joining (J), and at some loci, diversity (D). The variability of the assembly process in each developing lymphocyte yields an immune system that contains a repertoire of antigen receptors with an array of binding specificities (1, 2).The first phase of V(D)J recombination consists of site-specific DNA cleavage steps adjacent to selected gene segments and requires the lymphoid-specific recombination activating proteins, RAG1 and RAG2 (1, 2). Together the RAG proteins are directed to potential DNA cleavage sites by recognition of the recombination signal sequence (RSS), 1 which flanks each gene segment. The RSS contains a conserved heptamer and nonamer sequence separated by either 12 (12-RSS) or 23 (23-RSS) base pairs of poorly conserved DNA. Successful assembly of two gene segments requires that the segments are adjoined to dissimilar RSSs, a requirement referred to as the 12/23 rule. The RAG proteins cleave at each selected RSS to first nick the double-stranded DNA between the RSS heptamer and the bordering antigen receptor gene segment. The result is a 3Ј-OH group that executes a nucleophilic attack on the opposite strand, generating a covalently closed hairpin at the coding end and a signal end that is blunt-ended at the 5Ј-end of the RSS heptamer. The ...