Regulation of DNA Replication within the Immunoglobulin Heavy-Chain Locus During B Cell Commitment

Agnieszka Demczuk,Carl L. Schildkraut,Paolo Norio,John Bechhoefer,Ingrid Veras,Michel G. Gauthier,Meinrad Busslinger,Settapong Kosiyatrakul,Tom Misteli

doi:10.1371/journal.pbio.1001360

Abstract

The temporal order of replication of mammalian chromosomes appears to be linked to their functional organization, but the process that establishes and modifies this order during cell differentiation remains largely unknown. Here, we studied how the replication of the Igh locus initiates, progresses, and terminates in bone marrow pro-B cells undergoing B cell commitment. We show that many aspects of DNA replication can be quantitatively explained by a mechanism involving the stochastic firing of origins (across the S phase and the Igh locus) and extensive variations in their firing rate (along the locus). The firing rate of origins shows a high degree of coordination across Igh domains that span tens to hundreds of kilobases, a phenomenon not observed in simple eukaryotes. Differences in domain sizes and firing rates determine the temporal order of replication. During B cell commitment, the expression of the B-cell-specific factor Pax5 sharply alters the temporal order of replication by modifying the rate of origin firing within various Igh domains (particularly those containing Pax5 binding sites). We propose that, within the Igh CH-3′RR domain, Pax5 is responsible for both establishing and maintaining high rates of origin firing, mostly by controlling events downstream of the assembly of pre-replication complexes.

Highlights

During the S phase, mammalian chromosomes replicate in a precise temporal order, with the timing of replication typically changing gradually across hundreds of kilobases
Does this mean that the temporal order of replication is determined by multiple mechanisms? Are origin distribution, firing efficiency, and the timing of origin firing regulated independently? Which aspect of origin activation is controlled by cell differentiation? These are some of the questions addressed in this study
We studied the mouse immunoglobulin heavy-chain locus, one of the antibodyencoding portions of the genome, where origins change activity when antibody-producing B cells differentiate in the bone marrow

Summary

Introduction

During the S phase, mammalian chromosomes replicate in a precise temporal order, with the timing of replication typically changing gradually across hundreds of kilobases. Within a 340 kb portion of the Igh locus, changes in replication timing have been linked to modifications in the distribution of active origins and in their firing efficiency (see definitions in Table 1) [2]. Within the beta-globin locus, changes in replication timing can occur without significant changes in origin distribution, or firing efficiency, and have been ascribed to modifications in the timing of origin firing [3,4,5]. Does this mean that the temporal order of replication is determined by multiple mechanisms? Does this mean that the temporal order of replication is determined by multiple mechanisms? Are origin distribution, firing efficiency, and the timing of origin firing regulated independently? Which aspect of origin activation is controlled by cell differentiation? These are some of the questions addressed in this study

Methods

Results

Discussion

Conclusion