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-Cα gene replicate early in S, while heavy-chain variable (Vh) gene sequences replicate late in S. An ∼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.

J.Z. and O.V.E. contributed equally to this work.

This work was supported by NIH grants GM45751 (C.L.S.), AI13509 (B.K.B.), and AI23548 (R.R.). Support was also provided by Cancer Center Core Grant NIH/NCI P30CA13330.

We thank R. Little and L. Nguyen for their contribution to the early stages of this work, P. Norio for many helpful discussions, and J. Huberman, D. Gilbert, P. Dijkwel, H, Singh, S. Kosak, M. Scharff, A. Sepulveda, F. Garrett, E. Bouhassira, and J. Greally for critical reading of the manuscript. We thank Q. Yang and S. Shenoy for help with FISH. Microscopy was performed at the Analytical Imaging Facility, and the DNA content of separated cell fractions was determined at the fluorescence-activated cell sorter facility of the Albert Einstein College of Medicine.