Immunoglobulin (Ig) loci are selectively activated for transcription and rearrangement during B lymphocyte development. Using fluorescence in situ hybridization, we show that Ig heavy (H) and Igkappa loci are preferentially positioned at the nuclear periphery in hematopoietic progenitors and pro-T cells but are centrally configured in pro-B nuclei. The inactive loci at the periphery do not associate with centromeric heterochromatin. Upon localization away from the nuclear periphery in pro-B cells, the IgH locus appears to undergo large-scale compaction. We suggest that subnuclear positioning represents a novel means of regulating transcription and recombination of IgH and Igkappa loci during lymphocyte development.

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.

Compaction and looping of the ∼2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V H genes to be brought in proximity with D H -J H segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ∼60 sites throughout the V H region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5′ of DFL16 and the 3′ regulatory region, and also the intronic enhancer (Eμ), creating a D H -J H -Eμ-C H domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the D H region and parts of the V H locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.A ntigen receptors are created through the highly regulated lineage-specific process of V(D)J recombination, creating a diverse repertoire of Ig and T-cell receptors. The generation of the mouse Ig heavy chain in pro-B cells begins with D H -to-J H rearrangement on both alleles, followed by V H -to-D H J H rearrangement. In order for the >100 functional murine V H genes spread across ∼2.5 Mb to gain access to the single D-J rearrangement on that allele, the Igh locus undergoes contraction and looping during the pro-B-cell stage of B-cell differentiation (1-5). By measuring spatial distances between 11 small probes spread throughout the Igh locus, Jhunjhunwala et al. (2) demonstrated that distal and proximal V H genes were approximately equidistant from the D genes specifically at the pro-B-cell stage when the V H genes are rearranging. Computational as well as geometrical approaches have suggested that the locus is organized into rosette-like clusters of loops that compact during rearrangement. Several proteins have been reported to influence Igh locus compaction, including Pax5, YY1, and Ikaros (5-7). These proteins and others, such as Ezh2 (8), are also necessary for the rearrangement of distal V H genes but not proximal V H genes. This is most likely a consequence of the lack of locus compaction in the absence of these proteins. How all these proteins funct...

Seven families of Igh-V genes have been defined by Southern blot analysis of genomic DNA from eighteen inbred strains of mice. Each of twenty-four cloned Vh genes hybridized to one of seven nonoverlapping sets of Eco RI restriction fragments. These families contain from 2 to approximately 40 hybridizing fragments. From these data we estimate that the mouse Igh-V locus consists of one hundred Vh genes. Genes within a Vh family share greater than 80% sequence homology while the sequence homology between families is generally less than 70%. There is extensive restriction fragment length polymorphism among the strains analyzed allowing the assignment of complete (Igh-V + Igh-C) Igh haplotypes for eighteen inbred mouse strains.

In mammalian cells, the replication of tissue-specific gene loci is believed to be under developmental control. Here, we provide direct evidence of the existence of developmentally regulated origins of replication in both cell lines and primary cells. By using single-molecule analysis of replicated DNA (SMARD), we identified various groups of coregulated origins that are activated within the Igh locus. These origin clusters can span hundreds of kilobases and are activated sequentially during B cell development, concomitantly with developmentally regulated changes in chromatin structure and transcriptional activity. Finally, we show that the changes in DNA replication initiation that take place during B cell development, within the D-J-C-3'RR region, occur on both alleles (expressed and nonexpressed).