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

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 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.

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, as is the case in MEL cells. The Igh locus is approximately 3 Mb in size and contains more than 100 Vh genes organized in 15 families, 12 Dh segments, 4 Jh segments, and 8 Ch genes. Only Vh gene families that were examined in the present study are indicated. The 3′ regulatory region (3′ RR) and the array of Ch, Dh, and Jh gene segments occupy ∼400 kb and are collectively referred to as the Igh-C locus. The Eμ enhancer is located between the Jh segments and Cμ gene (26, 29). Downstream of the Cα gene, four enhancers, represented by shaded rectangles, have been identified. They comprise the 3′ regulatory region (54). During the pre-B-cell stage of development, the locus undergoes VDJ rearrangement. As the result of VDJ rearrangement, one variable-region gene, one D segment, and one J segment are brought together, and the intervening sequences are deleted. Deleted regions are indicated by dashed lines. The two allelic chromosomes with different rearrangements in the 70Z/3 cell line are shown. Only the C57BL/6-derived chromosome is productively rearranged, resulting in the transcription of a Vh J558-Cμ gene (51, 53). This allele has a 34-kb deletion that removes Cα and two of the 3′ Igh enhancers (53). The DBA-derived chromosome has a DQ52-to-Jh2 rearrangement, which is transcribed as a sterile (Sμ) transcript (51). The 38C-13 B-lymphoma-cell line represents a B-cell line with a continuing Vκ rearrangement phenotype. The productively rearranged Vh gene of 38C-13 belongs to the S107 family (51). The S107 plasmacytoma cell line represents terminally differentiated B cells. Isotype switching to the Cα gene has occurred in the S107 cell line. The productively rearranged chromosome expresses IgA, and the Vh family used in this cell line is S107 V1. The positions of the BACs used in this study are shown by black rectangles under the maps.

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), and the results are shown as filled circles. The C value is the haploid DNA content of the cells at a particular time in S phase. At the G₁/S boundary, the C value is 2.0; after DNA replication is completed, the C value is 4.0. All the timing data were obtained by the BrdU S-phase fractionation technique. BrdU-labeled DNA was prepared from cells at four different intervals of S phase (early, early middle, late middle, and late) and separated by centrifugal elutriation. The graphs shown represent the average times of replication of the two alleles. According to our timing data from the BrdU S-phase fractionation, the maximum differences in the replication times of the two alleles are not greater than 1.5 h for sequences replicating early and late in S phase and not greater than 2.5 h for sequences within the transition region. These estimates are based on the hybridization signals detected in BrdU-labeled DNA replicated in the four intervals of S phase. (A) Three temporal domains were detected in MEL cells. The early-replication (downstream of Cα) and late-replication regions (Vh genes) are connected by sequences (∼400 kb) that are replicated sequentially throughout S (11, 25, 36, 47). The times of replication of the Vh11, Vh15, Vh3609P, and J558 genes were obtained in this study. (B) Replication of the Igh locus was completed within the first hour of S phase in two pre-B-cell lines. The data presented here were obtained from the 22D6 cell line, but similar results were obtained for the 300-19P cell line. (C and D) The time in S when replication of the murine Igh locus occurs in the WEHI-231 B-cell line (C) and the S107 plasma B-cell line (D) resembles that in the MEL non-B-cell line, whose results are shown in panel A. Three temporal domains were observed. VDJ rearrangement and isotype switching result in deletion of the internal portion of the Igh locus. The lengths of the transition regions are similar to that in MEL cells. Also shown in panel C is the autoradiogram after hybridization (with a Vh15 probe) to DNA transfer of BrdU-labeled WEHI-231-cell DNA replicated during four intervals of S (E, early; EM, early middle; LM, late middle; L, late).

FIG. 3.

(A) Schematic representation of replication forks progressing in both directions through a segment, as detected by the N/A 2D gel electrophoresis technique. We considered two instances in which replication forks progress in both directions through a segment, as detected by either a 5′- or 3′-end probe: (i) replication forks progress towards each other within the examined segment and (ii) replication forks progress in one direction in some cells (one population) and in the opposite direction in other cells (the other population) within the examined segment. These two instances can be discriminated by N/N 2D gel electrophoresis. In the first case, a random or specific termination pattern is expected, while in the second case, no termination is observed. Since we did not detect termination within the segment by N/N 2D gel electrophoresis (Fig. 3B), only the second instance is shown in the diagram. Two identical segments in two different populations of cells are shown. The positions of the probes (filled rectangles) are shown below the segments. The signals that appeared on the film after hybridization to the specific probes are schematically shown below the specific probes and are labeled a to e as follows: a is the nonreplicated linear segments specifically detected by either the 3′- or 5′-end probe (called the 1N spot), b is the full-length parental strands from which the nascent strands are released in the second dimension at alkaline pH, c is the newly replicated nascent strands of multiple (both large and small) sizes, d is the cross-hybridization signals to the nonreplicating linear molecules, and e is the single-stranded molecules that result from breaks in the linear nonreplicating molecules (from the 1N spot). In the segment on the right, replication forks progress in the 3′-to-5′ direction. In this case, the probe at the 3′ end of the segment detects nascent strands of multiple sizes, while the probe at the 5′ end detects only large nascent strands. In the segment on the left, replication forks move from the 5′ end to the 3′ end of the segment. The probe at the 5′ end detects nascent strands of different sizes, and the 3′-end probe detects only large nascent strands. Since both of these populations of cells are present, both the 3′- and 5′-end probes detect nascent strands of multiple sizes (from different cells). The long diagonal nascent strand arcs detected by these probes are shown at the bottom of the figure. (B) Replication forks progress in both directions through examined segments of the Igh-C region in the early-replicating Igh locus. The examined restriction fragments and the positions of the probes (filled rectangles) are indicated. Both the 5′- and 3′-end probes detected nascent strands of all sizes, demonstrating that replication forks enter the segment from upstream and from downstream. Horizontal arrows below (70Z/3) or above (38C-13) the autoradiograms indicate the direction of replication fork movement. In two regions in the 70Z/3 cell line (μ region and 3′ regulatory region [RR]), the direction of replication fork progression on two allelic chromosomes was examined. Two separate arcs are visible for each allele since the probe detects two different restriction fragments (one from each allele). The difference in the sizes of the segments on the two allelic chromosomes for the 3′ regulatory region is much greater than that for the μ region. Thus, the two nascent strand arcs for the 3′ regulatory region are far apart while the two nascent strand arcs for the μ region are close to each other. Nascent strands of all sizes were detected in all segments after Southern hybridization to either the 5′- or 3′-end probe, indicating that replication forks progress in both directions on both alleles. Twelve fragments were examined by N/N 2D gel electrophoresis, which detects the shapes of the replicated molecules (examples are shown). Only a Y-shaped arc was detected in all segments examined, suggesting that replication forks progress through the fragments in either direction. Abbreviations: E, EcoRI; B, BamHI; H, HindIII; X, XbaI; RFLP, restriction fragment length polymorphism. (C) Schematic representation of replication forks progressing in both directions on two allelic chromosomes detected by N/A 2D gel electrophoresis. This scheme represents the ideal pattern of the autoradiogram one would detect if the replication forks move in both directions through the two allelic chromosomes, which can be distinguished by an RFLP. The two separated nascent strand arcs are indicated by arrows. For the Cμ segment, the smallest nascent strands originating from both chromosomes were not easily detectable, in contrast to what is shown in panel B. In the original autoradiogram, nascent strands smaller than 1.5 kb were weakly detected. This may be due to the fact that the intensities of the signals of the nascent strands from a single allele were weaker (by a factor of 2) than those of strands derived from both alleles simultaneously. In the 3′ regulatory region (B), nascent strands of all sizes originating from the DBA allele were easily detected with the 3′-end probe. However, on the C57BL allele, the smallest nascent strands (up to 800 bp) were not apparent on the autoradiogram since the conditions of N/A 2D gel electrophoresis were not ideal for both segments. Since these two segments (2.5 and 4.0 kb) differ in size by a factor of almost 2, the gel could not be run in a range optimal for both. The 5′-end probe for the HindIII/XbaI segment in the 3′ regulatory region detected nascent strands of all sizes from both alleles, which were apparent on the autoradiogram although they appear very weak in the gels shown in panel B. Since these allelic segments differ in size by only one-third, they can be distinguished by use of the same gel conditions.

FIG. 4.

VDJ rearrangement is not essential for activation of B-cell-specific origins. In the 63-12 cell line derived from RAG2^−/− mice, the Igh-C locus is unrearranged, but germ line transcription through Vh genes is activated (4). The restriction fragments examined by N/A 2D gel electrophoresis are indicated by lines below the map. The arrows indicate the probes used. As shown on the autoradiogram, probes from the 5′ and 3′ ends detected nascent strands of all sizes, indicating that replication forks move in both directions through Igh-C. The autoradiograms for Cγ2b and the 3′ regulatory region (RR) were obtained with probes located at only one end of the segments. The 5′-end probe for the Cγ2b region did not detect replication intermediates, although several different membranes were hybridized multiple times. These membranes produced a very strong signal derived from replication intermediates when they were hybridized to other probes. In the 3′ regulatory region, the unique 3′-end probe for the HindIII fragment contained repeated sequences and thus could not be used to examine the direction of replication fork movement. E, EcoRI; H, HindIII.

FIG. 5.

Replication forks progress in one direction through the constant region in the WEHI-231 immature-B-cell line. The positions of the examined segments are shown under the map of the Igh locus. Only 3′-end probes detected nascent strands of all sizes, indicating that replication forks progress from 3′ to 5′ in two regions (Cγ2b, Cα) in WEHI-231. Unique probes were not available to analyze both ends of the two segments containing the γ2b gene. Instead, we used a probe at the 3′ end of one segment and at the 5′ end of the adjacent segment. Probe 7 detected only large nascent strands in the WEHI-231 cell line but detected nascent strands of all sizes in the 70Z/3 cell line (Fig. 3B). The vertical lines to the left of the 1N spot observed for the 6.8-kb EcoRI fragment in WEHI-231 indicate linear, nonreplicating molecules resulting from partial digestion, which can be distinguished from nascent strands that form diagonal arcs extending from the horizontal parental-strand signals (see the diagram in Fig. 3A). The numbered probes are indicated by filled rectangles, and the horizontal arrows indicate the direction of replication fork movement. RR, regulatory region; E, EcoRI; B, BamHI.

FIG. 6.

The position of the Igh locus within the nuclei of B-cell lines is coordinated with the replication program of the Igh locus. (A) Interphase nuclei were hybridized to BAC 34H6 (containing the constant region) (red signals) and BAC 526A21 (containing J558 genes; Igh-V) (green signals). The nuclei were counterstained with DAPI (blue). In the 70Z/3 cell line, only the nonproductively rearranged allele was detected by BAC 526A21. BAC 199M11 (red signals) was used to examine the S107 cell line, because all the constant-region genes, except Cα, were deleted during isotype switching. The green arrow indicates the nontranslocated Igh locus in the S107 cell line. (B and C) Analyses of the subnuclear locations of Igh-C (B) and Igh-V (C) in B-lineage cell lines that showed two different replication programs. Each bar represents the frequency of the signal detected at a particular distance relative to the nuclear center and nuclear edge. The relative position of the signal in each nucleus was normalized to the radius of that nucleus. A value of 0 represents a location at the center of the nucleus, and a value of 1.0 represents a location at the edge of the nucleus. For example, bars between 0.4 and 0.5 show the proportion of the signals with relative positions between 0.4 and 0.5. The distributions of the Igh-V and Igh-C region genes are significantly different in different B-cell lines. In cell lines in which the entire Igh locus replicates early in S, both Igh-C and Igh-V signals are predominantly located away from the nuclear periphery. In B-cell lines that have triphasic-replication patterns (WEHI-231 and S107), Igh signals are preferentially associated with the nuclear periphery. (D) The top portion of the figure indicates the times in S when the different temporal domains of the Igh locus replicate. This is a diagram of the data shown in Fig. 2 (with the same values on the x and y axes), but the directions of replication fork movement and the replication initiation sites are also indicated. The replication initiation regions are indicated by pairs of concentric ellipses (at arbitrary sites). In the early-replication program, the entire Igh locus is replicated within the first hour of S phase and replication forks proceed in both directions through the constant-region genes (blue) and the downstream region (green). The locus is situated away from the nuclear periphery (diagram in the circle in the lower left portion of the figure). Our N/A 2D gel electrophoresis data are consistent with the idea that one or more B-cell-specific origins are activated in the region that serves as a temporal-transition region in MEL cells. In the triphasic-replication program, initiation at origins located in the Vh region (red) is delayed until the end of S. The origins located downstream (green) of the Igh locus are still activated early in these cell lines, and the leftward-moving replication fork from this region progresses toward the late-activation origins (red) in the Vh region, generating the temporal-transition region (∼500 kb in length) (the blue lines indicate constant-region genes, and the red lines indicate Vh region genes). Different sequences encompass the transition region in these two cell lines (the sequences of these transition regions are also different from those in the MEL cell line); therefore, the transition appears to be at least partially sequence independent. In both cell lines, any bidirectional initiation sites within the sequences of the transition regions, including Igh-C in WEHI-231 and the Vh region in WEHI-231 and S107, are silent. The locus is situated near the nuclear periphery (diagram in the circle in the lower right portion of the figure). We suggest that the temporal organization of the replication of the Igh locus is accomplished by (i) either the activation (early-replication program) or silencing (triphasic-replication program) of any initiation site(s) located within the transition region and (ii) the regulation of the firing times of initiation sites located within the Vh region. The silencing or activation of the initiation site(s) within the transition region and the regulation of the firing time of the Vh origins may be accomplished as a result of an alteration of the subnuclear position of the Igh locus.