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

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 late-replication foci tend to occupy different subnuclear domains in interphase cells (for a review, see reference 28). Recent observations suggest that nuclear structure might play an important role in defining when and where DNA replication initiates (for a review, see reference 21). The immunoglobulin heavy-chain (Igh) locus (consisting of, in the 5′-to-3′ direction, 15 heavy-chain variable [Vh] gene families, 12 heavy-chain diversity [Dh] segments, 4 heavy-chain joining [Jh] segments, an intronic enhancer, 8 constant-region [Ch] genes, and the 3′ regulatory region) is an important model system for the study of mammalian DNA replication. The Igh locus shows successive changes in transcription and DNA rearrangements during B-cell development (Fig. ​(Fig.1).1). V(D)J joining occurs at the pro-B-cell stage, preceded by transcriptional activation of Vh genes. Class switching occurs later in B-cell development. These processes result in unique combinations of V, D, J, and C segments encoding and transcribing individual heavy-chain (and light-chain) genes in individual B cells (for a review, see reference 45). B-cell lines representing different stages of B-cell development are available for the study of the replication and nuclear localization of the Igh locus. The cell lines used in the present study are listed in Table ​Table1.1. The mouse Igh locus is very large (approximately 3 Mb) but has been physically mapped and cloned in yeast artificial chromosome and bacterial artificial chromosome (BAC) arrays (13) and is being sequenced. The information derived from these arrays greatly facilitated the present study. FIG. 1. Rearrangements of the Igh locus during B-cell development.The germ line configuration of the Igh locus on murine chromosome 12 is shown (44, 57). In the 63-12 RAG2−/− pro-B-cell line, the entire locus is unrearranged and in germ line configuration, ... TABLE 1. Characteristics of B-lineage cell lines used in this study Previous studies using bromodeoxyuridine (BrdU) S-phase fractionation have shown that the replication timing of the Igh locus in non-B-cell lines, such as the murine erythroleukemia (MEL) cell line, is different from that in pre-B-cell lines (11, 34, 35). In MEL cells, an ∼400-kb sequence forms a transition region between an early-replication domain downstream of the 3′ regulatory region and a late-replication domain containing Vh genes (triphasic-replication pattern) (Fig. ​(Fig.2A).2A). Sequences within this transition region replicate 3′ to 5′ progressively later during S. A previous study using neutral-alkaline two-dimensional (N/A 2D) gel electrophoresis has shown that the transition region is formed by a single replicon through which replication forks progress in the 3′-to-5′ direction (25). In contrast, the entire Igh locus is replicated within the first hour of S in two pre-B-cell lines. Previous studies also demonstrated that part of the Igh-C region replicates early in S in plasma cell lines (34). FIG. 2. Replication timing of the murine Igh locus in B-lineage cell lines. The time in S phase when a particular segment is replicated is expressed in terms of the C value (y axis) and is plotted versus its distance in kilobases from the Cα gene (x axis), ... One explanation for the dramatically different replication programs detected in non-B cells and pre-B cells is that the DNA replication program is linked to the transcriptional status of the Igh locus. In MEL cells, the Igh locus is transcriptionally silent, while in pre-B-cell lines, this locus is actively transcribed. Since the pattern of transcription along the locus changes during B-cell development, we decided to examine the replication of the Igh locus in B-cell lines at different stages of B-cell development. The experiments presented here reveal two distinct programs of replication for the Igh locus in B-lineage cells. In pro- and pre-B-cell lines, including those derived from RAG2−/− mice, the entire Igh locus is replicated early in S phase. The temporal pattern of Igh replication in other cell lines representing late stages of B-cell development (an immature-B-cell line and plasma cell lines) resembles that in non-B cells. The temporal-transition regions in the different cell lines are similar in length but encompass different sequences, reflecting individual DNA rearrangement events associated with VDJ and class switch recombination. The replication timing of the Igh locus in ex vivo-expanded pro-B cells and lipopolysaccharide (LPS)-stimulated spleen B cells is consistent with the data obtained from the cell lines. In this study, we address the potential mechanism for this dramatic difference in replication programs. N/A 2D gel electrophoresis demonstrates that replication forks progress in both directions through the examined Igh-C genes in cell lines in which the entire locus replicates early in S. Our results suggest that the early replication of the Igh locus is achieved through the activation of one or more replication origins. In contrast, in B cells in which the Igh locus replicates through a triphasic pattern, replication forks progress in a single 3′-to-5′ direction (opposite to the transcriptional orientation) through all the constant-region genes we examined. Our observations that the directions of replication fork progression can differ in B-lineage cell lines raise the possibility that changes in Igh replication are involved in processes of Igh rearrangement and expression. We have found that, associated with the changes in replication, the entire Igh locus is located away from the nuclear periphery when it is replicated early in S phase but is located near the nuclear periphery when it is replicated in a triphasic pattern.