Replication of the transcriptionally inactive and transcriptionally active immunoglobulin heavy chain gene locus

Abstract

The temporal order of replication of sequences within the murine immunoglobulin heavy chain (Igh) gene locus differs in B cells and other cell types. In the murine erythroleukemia (MEL) cell line in which immunoglobulin genes are not expressed, it has been demonstrated that the most distal immunoglobulin heavy chain constant region gene (Igh-C), i.e. Calpha, replicates early in S; other Ch genes, Jh and Dh segments, and the first member of the variable region gene family (Vh81X) replicate successively later in S proportional to their distance from Calpha. These data can best be explained by: (1) a single bidirectional replicon, or (2) multiple bidirectional replicons that fire sequentially. To discriminate between these models, I have used neutral/alkaline (N/A) two-dimensional (2D) gel electrophoresis; to determine the direction of replication fork movement within DNA segments and to reveal the region from which replication forks originate. These studies---the first to monitor replication fork progression throughout a single copy tissue-specific gene locus---have shown that replication forks proceed exclusively in the 3 ' to 5' direction from the region 45 kb 3' of Calpha to the Dh region, and within the Vh81X gene, a total distance of ∼400 kb. In accord with the timing data, the results of N/A 2D gel electrophoresis indicate that the replication of these sequences of the Igh locus occurs by a single replicon. Distal to the putative initiation region (located between Calpha-45 and Calpha-130) is an early replicating region of at least 250 kb. Downstream of this 250 kb region, there is at least one additional replicon. Thus, multiple early activated replicons are located downstream of the 3'RR. Distal to the Vh81X gene is a late replicating region of at least 700 kb, where multiple replicons may be activated. The Igh-C sequences represent a gradual temporal transition between early and late replicated domains. I have demonstrated for the first time, that in mammalian cells the transition between temporal domains can be achieved by a single replication fork, raising the possibility that the transition between other early and late replicating regions is also achieved in this manner.;In cell lines of the B lineage, in which the immunoglobulin heavy chain locus (Igh) is transcriptionally active, replication of the locus is different from the transcriptionally inactive locus (non-B cell lineage). In plasmacytoma cells, sequences 3' of the-locus, the entire Igh-C locus, and the expressed Vh genes all replicate early in S phase. In pre-B cells, early replication also includes several Vh gene families. Moreover, on the molecular level we demonstrated that in two pre B-cell lines (38C-13 and 70Z/3) replication forks progress in both directions through the Igh-C locus. Replication forks originate both downstream of the Calpha gene and also upstream of the Jh segments. Based on replication timing data and the results of N/A 2D gel electrophoresis obtained from two pre-B cell lines and the plasmacytoma cell line S107 we postulate that at least one additional replication initiation region is activated within the Igh-C sequences in pre-B, cells. However, the possibility of the existence of a delocalized initiation zone dispersed through the Igh-C locus in pre-B cells cannot be excluded. Therefore, the activation of one or more additional replicons in the Igh locus is developmentally regulated. The Igh-C locus offers an opportunity to begin examination of the relationship between replicon activation and nuclear organization in mitotic and interphase chromatin.