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...

The murine Igh locus has a 3 regulatory region (3 RR) containing four enhancers (hs3A, hs1,2, hs3B, and hs4) at DNase I-hypersensitive sites. The 3 RR exerts long-range effects on class switch recombination (CSR) to several isotypes through its control of germ line transcription. By measuring levels of acetylated histones H3 and H4 and of dimethylated H3 (K4) with chromatin immunoprecipitation assays, we found that early in B-cell development, chromatin encompassing the enhancers of the 3 RR began to attain stepwise modifications typical of an open conformation. The hs4 enhancer was associated with active chromatin initially in pro-and pre-B cells and then together with hs3A, hs1,2, and hs3B in B and plasma cells. The immunoglobulin heavy chain locus (Igh) (see Fig. 1) is arguably the most complex mammalian locus. B-cell development is marked by the progressive expression of Igh genes (via VDJ recombination, somatic hypermutation, and class switch recombination [CSR]). These regulated processes, some involving substantial DNA rearrangements and deletions, are required for the generation of antibody diversity (reviewed in references 47 and 57). Spanning ϳ3 Mb, the murine Igh locus contains a limited number of enhancers, including the intronic enhancer (E), which has been implicated in V to DJ joining and chain expression (reviewed in reference 47), and a complex 3Ј regulatory region (3Ј RR) located immediately downstream of C␣, the last constant region gene. The human Igh locus contains two 3Ј RRs, each located downstream of one of the duplicated C␣ genes (9, 49, 62).The murine 3Ј RR spans ϳ28 kb downstream of C␣ and comprises four enhancers, each associated with DNase I hypersensitivity (hs3A, hs1,2, hs3B, and hs4) (reviewed in reference 33). hs3A and hs3B are located at the termini of a 25-kb inverted repeat flanking hs1,2 (7, 66), and these three enhancers acquire DNase I hypersensitivity at later stages of B-cell development (66). The hs4 enhancer is the only 3Ј enhancer with activity and DNase I hypersensitivity throughout B-cell development (19, 48), although no role for hs4 or any of the 3Ј enhancers in early B-cell development has been shown.Targeted deletions of the 3Ј RR enhancers have shown that hs3A and hs1,2 are individually dispensable for B-cell development and Igh expression (46), while the combination of hs3B and hs4 is critical for the process of CSR (63). The 3Ј RR has also been proposed to regulate Igh expression in plasma cells (23,40,48,63,70). In accord with earlier suggestions that it functions as a locus control region (43), the 3Ј RR shows synergistic activity and position-independent regulation when tested in transgenic model systems (8,33,59). However, copynumber-dependent expression has not been observed (8). In addition to a function within the Igh locus, the 3Ј RR is likely to be responsible for dysregulation of c-myc expression resulting from certain Igh::c-myc translocations in mouse plasmacytoma cells as well as in human Burkitt's lymphoma and multiple myeloma cells (30,36,43).Th...

We have identified a nuclear factor expressed in pro-B-, pre-B-, and B-cell lines that binds to two sites within the murine immunoglobulin heavy-chain (IgH) 3'at enhancer (3'oE). These sites were defined by oligonucleotide competition in an electrophoretic mobility shift assay (EMSA) and methylation interference footprinting. The 3'aE-binding factor is indistinguishable from NF-HB (B-lineage-specific nuclear factor that binds to the IgH gene) and the B-lineage-specific transcription factor BSAP by several

No abstract

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␣ gene replicate early in S, while heavy-chain variable (Vh) gene sequences replicate late in S. An ϳ500-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.In Saccharomyces cerevisiae, the initiation of DNA replication is sequence specific (27; for a review, see reference 23). In mammalian cells, however, the mechanism underlying this process is still being defined. Although specific origins of replication have been mapped, initiation regions as large as 50 kb have also been described (for reviews, see references 20 and 22), making the potential role of sequence specificity less clear. In mammalian cells, the origins of DNA replication are activated in different intervals of S phase and the timing of the activation of these origins is precisely regulated. The regulation of the replication timing of a specific gene appears to reflect the transcriptional activity of surrounding sequences (for a review, see reference 58). Evidence that shows that the localization of some genes or even of an entire chromosome within the nucleus is nonrandom and is linked both to chromatin structure and replication timing has been presented (9, 16, 41; for a review, see reference 14). Thus, early-and latereplication foci tend to occupy different subnuclear domains in interphase cells (for a review, see reference 28). Recent observations suggest that nuclear structure might ...