Early B cell development is characterized by large-scale Igh locus contraction prior to V(D)J recombination to facilitate a highly diverse Ig repertoire. However, an understanding of the molecular architecture that mediates locus contraction remains unclear. We have combined high-resolution chromosome conformation capture (3C) techniques with 3D DNA FISH to identify three conserved topological subdomains. Each of these topological folds encompasses a major VH gene family that become juxtaposed in pro-B cells via megabase-scale chromatin looping. The transcription factor Pax5 organizes the subdomain that spans the VHJ558 gene family. In its absence, the J558 VH genes fail to associate with the proximal VH genes, thereby providing a plausible explanation for reduced VHJ558 gene rearrangements in Pax5-deficient pro-B cells. We propose that Igh locus contraction is the cumulative effect of several independently controlled chromatin subdomains that provide the structural infrastructure to coordinate optimal antigen receptor assembly.

V(D)J joining is mediated by RAG recombinase during early B-lymphocyte development in the bone marrow (BM). Activation-induced deaminase initiates isotype switching in mature B cells of secondary lymphoid structures. Previous studies questioned the strict ontological partitioning of these processes. We show that pro-B cells undergo robust switching to a subset of immunoglobulin H (IgH) isotypes. Chromatin studies reveal that in pro-B cells, the spatial organization of the Igh locus may restrict switching to this subset of isotypes. We demonstrate that in the BM, V(D)J joining and switching are interchangeably inducible, providing an explanation for the hyper-IgE phenotype of Omenn syndrome.

Ab class switching is induced upon B cell activation in vivo by immunization or infection or in vitro by treatment with mitogens, e. g. LPS, and results in the expression of different heavy chain constant region (CH) genes without a change in the Ab variable region. This DNA recombination event allows Abs to alter their biological activity while maintaining their antigenic specificity. Little is known about the molecular mechanism of switch recombination. To attempt to develop an assay for enzymes, DNA binding proteins, and DNA sequences that mediate switch recombination, we have constructed a plasmid DNA substrate that will undergo switch recombination upon stable transfection into the surface IgM+ B cell line (I.29 mu), a cell line capable of undergoing switch recombination of its endogenous genes. We demonstrate that recombination occurs between the two switch regions of the plasmid, as assayed by PCRs across the integrated plasmid switch regions, followed by Southern blot hybridization. Nucleotide sequence analysis of the PCR products confirmed the occurrence of S mu-S alpha recombination in the plasmid. Recombination of the plasmid in I.29 mu cells does not require treatment with inducers of switch recombination, suggesting that recombinase activity is constitutive in I.29 mu cells. Recombination does not require high levels of transcription across the switch regions of the plasmid. Fewer recombination events are detected in four different B and T cell lines that do not undergo switch recombination of their endogenous genes.