CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells.

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.

The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions.

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.

Sequence and characterization of the Ig heavy chain constant and partial variable region of the mouse strain 129S1.

Although the entire mouse genome has been sequenced, there remain challenges concerning the elucidation of particular complex and polymorphic genomic loci. In the murine Igh locus, different haplotypes exist in different inbred mouse strains. For example, the Igh(b) haplotype sequence of the Mouse Genome Project strain C57BL/6 differs considerably from the Igh(a) haplotype of BALB/c, which has been widely used in the analyses of Ab responses. We have sequenced and annotated the 3' half of the Igh(a) locus of 129S1/SvImJ, covering the C(H) region and approximately half of the V(H) region. This sequence comprises 128 V(H) genes, of which 49 are judged to be functional. The comparison of the Igh(a) sequence with the homologous Igh(b) region from C57BL/6 revealed two major expansions in the germline repertoire of Igh(a). In addition, we found smaller haplotype-specific differences like the duplication of five V(H) genes in the Igh(a) locus. We generated a V(H) allele table by comparing the individual V(H) genes of both haplotypes. Surprisingly, the number and position of D(H) genes in the 129S1 strain differs not only from the sequence of C57BL/6 but also from the map published for BALB/c. Taken together, the contiguous genomic sequence of the 3' part of the Igh(a) locus allows a detailed view of the recent evolution of this highly dynamic locus in the mouse.

Identification of a candidate regulatory element within the 5' flanking region of the mouse Igh locus defined by pro-B cell-specific hypersensitivity associated with binding of PU.1, Pax5, and E2A.

The Igh locus is controlled by cis-acting elements, including Emu and the 3' IgH regulatory region which flank the C region genes within the well-studied 3' part of the locus. Although the presence of additional control elements has been postulated to regulate rearrangements of the VH gene array that extends to the 5' end of the locus, the 5' border of Igh and its flanking region have not been characterized. To facilitate the analysis of this unexplored region and to identify potential novel control elements, we physically mapped the most D-distal VH segments and scanned 46 kb of the immediate 5' flanking region for DNase I hypersensitive sites. Our studies revealed a cluster of hypersensitive sites 30 kb upstream of the most 5' VH gene. Detection of one site, HS1, is restricted to pro-B cell lines and HS1 is accessible to restriction enzyme digestion exclusively in normal pro-B cells, the stage defined by actively rearranging Igh-V loci. Sequence motifs within HS1 for PU.1, Pax5, and E2A bind these proteins in vitro and these factors are recruited to HS1 sequence only in pro-B cells. Transient transfection assays indicate that the Pax5 binding site is required for the repression of transcriptional activity of HS1-containing constructs. Thus, our characterization of the region 5' of the VH gene cluster demonstrated the presence of a single cluster of DNase I hypersensitive sites within the 5' flanking region, and identified a candidate Igh regulatory region defined by pro-B cell-specific hypersensitivity and interaction with factors implicated in regulating VDJ recombination.

Progressive activation of DNA replication initiation in large domains of the immunoglobulin heavy chain locus during B cell development.

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).

Sites that direct nuclear compartmentalization are near the 5' end of the mouse immunoglobulin heavy-chain locus.

VDJ rearrangement in the mouse immunoglobulin heavy chain (Igh) locus involves a combination of events, including a large change in its nuclear compartmentalization. Prior to rearrangement, Igh moves from its default peripheral location near the nuclear envelope to an interior compartment, and after rearrangement it returns to the periphery. To identify any sites in Igh responsible for its association with the periphery, we systematically analyzed the nuclear positions of the Igh locus in mouse non-B- and B-cell lines and, importantly, in primary splenic lipopolysaccharide-stimulated B cells and plasmablasts. We found that a broad approximately 1-Mb region in the 5' half of the variable-gene region heavy-chain (Vh) locus regularly colocalizes with the nuclear lamina. The 3' half of the Vh gene region is less frequently colocalized with the periphery, while sequences flanking the Vh gene region are infrequently so. Importantly, in plasmacytomas, VDJ rearrangements that delete most of the Vh locus, including part of the 5' half of the Vh gene region, result in loss of peripheral compartmentalization, while deletion of only the proximal half of the Vh gene region does not. In addition, when Igh-Myc translocations move the Vh genes to a new chromosome, the distal Vh gene region is still associated with the nuclear periphery. Thus, the Igh region that interacts with the nuclear periphery is localized but is likely comprised of multiple sites that are distributed over approximately 1 Mb in the 5' half of the Vh gene region. This 5' Vh gene region that produces peripheral compartmentalization is the same region that is distinguished by requirements for interleukin-7, Pax5, and Ezh2 for rearrangement of the Vh genes.

Visualization of looping involving the immunoglobulin heavy-chain locus in developing B cells.

The immunoglobulin heavy-chain (IgH) locus undergoes large-scale contraction in B cells poised to undergo IgH V(D)J recombination. We considered the possibility that looping of distinct IgH V regions plays a role in promoting long-range interactions. Here, we simultaneously visualize three subregions of the IgH locus, using three-dimensional fluorescence in situ hybridization. Looping within the IgH locus was observed in both B- and T-lineage cells. However, monoallelic looping of IgH V regions into close proximity of the IgH DJ cluster was detected in developing B cells with significantly higher frequency when compared with hematopoietic progenitor or CD8+ T-lineage cells. Looping of a subset of IgH V regions, albeit at lower frequency, was also observed in RAG-deficient pro-B cells. Based on these observations, we propose that Ig loci are repositioned by a looping mechanism prior to IgH V(D)J rearrangement to facilitate the joining of Ig variable, diversity, and joining segments.

The Sr1 gene that controls diversity of the anti-inulin antibody response maps to mouse chromosome 14.

Previous studies demonstrated that the diversity of the antibody response of mice to the inulin (In) determinant of bacterial levan is regulated by the gene Spectrotype Regulation 1 ( Sr1). BALB/c mice produce a monoclonal anti-In response as shown by isoelectric focusing analysis. In contrast, the anti-In antibody response of (BALB/cxC57BL/6)F1 mice is significantly more heterogeneous. We performed a backcross and a genome-wide scan with microsatellite markers and found that Sr1 is tightly linked to D14Mit121 on chromosome (Chr) 14. This location for Sr1 was supported by analysis of CXB Recombinant Inbred strains. We further confirmed this by finding that the Chr 14 congenic mouse strain B6.C-H8 lacks the C57BL/6 allele of the Sr1 gene, indicating that Sr1 is located in the segment of Chr 14 replaced with BALB/c donor DNA. These data place Sr1 near to or coincident with the Tcra/Tcrd T-cell receptor gene complex and suggest a role for T cells in diversifying the anti-In response.

The origin of a developmentally regulated Igh replicon is located near the border of regulatory domains for Igh replication and expression.

The 3' Ig heavy chain locus (Igh) regulatory region is the most downstream known element of the murine Igh gene cluster. We report here that the nearest non-Igh genes-Crip, Crp2, and Mta1-are located approximately 70 kb further downstream and are beyond the end of the domain of Igh transcriptional regulation. We have localized an origin of replication in MEL cells to a 3-kb segment located between the 3' Igh regulatory region and Crip. Sequences downstream of this origin are replicated by forks that move in both directions. Sequences upstream of this origin (Igh-C, -D, and -J) are replicated in a single direction through a 500-kb segment in which no active bidirectional origins can be detected. We propose that this origin may lie at or near the end of the Igh regulation domain.

Replication and subnuclear location dynamics of the immunoglobulin heavy-chain locus in B-lineage cells.

The murine immunoglobulin heavy-chain (Igh) locus provides an important model for understanding the replication of tissue-specific gene loci in mammalian cells. We have observed two DNA replication programs with dramatically different temporal replication patterns for the Igh locus in B-lineage cells. In pro- and pre-B-cell lines and in ex vivo-expanded pro-B cells, the entire locus is replicated early in S phase. In three cell lines that exhibit the early-replication pattern, we found that replication forks progress in both directions through the constant-region genes, which is consistent with the activation of multiple initiation sites. In contrast, in plasma cell lines, replication of the Igh locus occurs through a triphasic pattern similar to that previously detected in MEL cells. Sequences downstream of the Igh-C alpha gene replicate early in S, while heavy-chain variable (Vh) gene sequences replicate late in S. An approximately 500-kb transition region connecting sequences that replicate early and late is replicated progressively later in S. The formation of the transition region in different cell lines is independent of the sequences encompassed. In B-cell lines that exhibit a triphasic-replication pattern, replication forks progress in one direction through the examined constant-region genes. Timing data and the direction of replication fork movement indicate that replication of the transition region occurs by a single replication fork, as previously described for MEL cells. Associated with the contrasting replication programs are differences in the subnuclear locations of Igh loci. When the entire locus is replicated early in S, the Igh locus is located away from the nuclear periphery, but when Vh gene sequences replicate late and there is a temporal-transition region, the entire Igh locus is located near the nuclear periphery.

A three-megabase yeast artificial chromosome contig spanning the C57BL mouse Igh locus.

The mouse Ig H chain (Igh) complex locus is composed of >100 gene segments encoding the variable, diversity, joining, and constant portions of the Ab H chain protein. To advance the characterization of this locus and to identify all the V(H) genes, we have isolated the entire region from C57BL/6 and C57BL/10 as a yeast artificial chromosome contig. The mouse Igh locus extends approximately three megabases and contains at least 134 V(H) genes classified in 15 partially interspersed families. Two non-Igh pseudogenes (Odc-rs8 and Rpl32-rs14) were localized in the distal part of the locus. This physical yeast artificial chromosome map will provide important structure and guidance for the sequencing of this large, complex, and highly repetitive locus.

Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development.

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.