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␣ 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 ...

Transcription of a Productively Rearranged Ig VDJCα Does Not Require the Presence of HS4 in the Igh 3′ Regulatory Region

“…Interestingly, an ϳ1-Mb region covering the distal V H genes is required for Igh locus association with the nuclear periphery (42). Transient loss of this association appears essential to V to DJ rearrangements involving the distal V H genes, but the Igh locus reforms its attachment to the nuclear lamina in B cells and Ig-secreting cells (43)(44)(45)(46). The effect of this association on Ig transcription is not yet clear.…”

The origin of a developmentally regulatedIghreplicon is located near the border of regulatory domains forIghreplication and expression

“…Replication initiates early in S from a region located downstream of the Igh cluster, and proceeds opposite to the transcriptional orientation through 400 kb of Igh-C (constant), -J (joining), and -D (diversity) sequences, concluding in late S phase with the most proximal Vh genes. More distal Vh genes are all replicated late in S. In B and plasma cell lines, the replication pattern is the same as that of the MEL cells, despite the reduction in the length of the locus resulting from VDJ recombination and class switching (12). The temporal transition region between early-and late-replicating domains extends through rearranged Igh-C sequences and the VDJ gene into formerly distant 5Ј unrearranged Vh genes.…”

“…Our studies on Igh gene replication in B cell lines have raised the possibility that regulators of replication of the Igh gene locus are located downstream of the 3Ј regulatory region (9)(10)(11)(12). Two distinct patterns of replication within the Igh gene cluster are apparent (see Fig.…”

“…Importantly, these studies suggest that during B cell development, replication of Igh is coordinated with various steps in Igh gene expression and rearrangement. In contrast, replication of the region immediately 3Ј to the transition region is regulated independently; sequences in this region are replicated early in non-B cells and in B cells at all stages of B cell differentiation (10,12). In the non-B cell line, MEL, the unidirectional fork movement of the transition region replicon was previously detected as far as Ϸ45 kb downstream of C␣, but forks moved in both directions at a point Ϸ100 kb downstream (9,10).…”

“…3a). Here, all Igh sequences replicate early in S phase (11,12), and replication forks move in both directions throughout the Igh-C region (12). Latent origins of replication within the early to late temporal transition region in MEL apparently become active when there is transcription of unrearranged Vh sequences associated with VDJ joining.…”