Volume 20, Issue 4, Pages 575-587 (November 2005) Progressive Activation of DNA Replication Initiation in Large Domains of the Immunoglobulin Heavy Chain Locus during B Cell Development  Paolo Norio, Settapong Kosiyatrakul, Qiaoxin Yang, Zeqiang Guan, Nicholas M. Brown, Sharon Thomas, Roy Riblet, Carl L. Schildkraut  Molecular Cell  Volume 20, Issue 4, Pages 575-587 (November 2005) DOI: 10.1016/j.molcel.2005.10.029 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 In Non-B Cells, Initiation Sites Are Not Detected within the Igh D-J-C-3′RR Region (A) Replication timing of the mouse Igh locus. Map of the locus (based on the strain C57BL/6 sequence) with the positions of various genetic elements shown to scale. Red boxes mark the position of the D, J, and C genes. Regulatory elements are indicated by black boxes. Various V region genes families are marked by black bars. Above the map, a schematic summarizes the replication timing of the locus in different cell types (break marks indicate the occurrence of Igh rearrangements in cells of the B lineage). Below the map, blue bars mark the restriction fragments discussed in this study. (B) Diagram for a SMARD experiment (see text for description). (C–L) In non-B cells (ES cells, the T cell line TU5), the SwaI and PacI fragments are replicated by a fork originating downstream of the Igh locus and traveling 5′ throughout the TTR. (C) and (G) show the positions of various genetic elements in these restriction fragments (to scale). The position of the hybridization probes utilized to detect the DNA molecules of interest are indicated by blue bars below the maps. Regions of partial crosshybridization are also indicated by discontinuous blue bars. Aligned to the maps are the images of the RG molecules (D and H), organized from top to bottom by increasing content of IdU (red). Yellow arrowheads mark the position of the red-to-green transitions along the molecules (corresponding to the positions of the replication forks at the end of the first labeling period). In (D) and (H), all forks active in these molecules were traveling leftward. Vertical lines indicate the ends of the molecules (in yellow) and the position of the hybridization signals used to align the molecules (in blue). Molecule 17 in (D) was bent and for reasons of space is not shown, but it was included in the quantitative analysis. The results of SMARD are also presented as replication profiles (E and I), shown below the images of the molecules. The horizontal axes of the diagrams represent arbitrary ∼5 kb intervals of the restriction fragments. The vertical axes show the percentage of RG molecules stained. The intervals more frequently stained in red are those that on average replicate first. (F) and (L) are profiles of replication fork abundance showing the percentage of molecules that contained a replication fork at the time of label switch at each position along the restriction fragments. In these cell lines, all forks move leftward through this region. Molecular Cell 2005 20, 575-587DOI: (10.1016/j.molcel.2005.10.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Pro-B and Pre-B Cells Have High Levels of Initiation Events throughout the C-3′RR Region, in Both Alleles, Regardless of D-J Recombination and Partial Deletion of the 3′RR For each experiment, a schematic of the SwaI fragment shows the positions of various genetic elements (to scale), aligned with the replication profiles (A, D, G, and L), the profiles of replication fork abundance (B, E, H, and M), and the images of the RG molecules (C, F, I, and N). Molecules 25, 34 (in [F]), 13, 21, and 26 (in [N]) are not shown because they are bent. Molecular Cell 2005 20, 575-587DOI: (10.1016/j.molcel.2005.10.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 The Frequency of Initiation Events Declines 5′ of the D Gene Segments in Transformed as Well as in Primary Pro-B Cells Schematics of the PacI fragments show the positions of various genetic elements (to scale), aligned with the corresponding replication profiles (A and D), the profiles of replication fork abundance (B and E), and the images of the RG molecules (C and F). Molecule 11 (in [C]) is not shown because it is bent. The size of the PacI fragment varies between mouse strains due to modifications in the organization of the D genes. Molecular Cell 2005 20, 575-587DOI: (10.1016/j.molcel.2005.10.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 During Late Stages of B Cell Development, Origins Remain Active within the D-J-C-3′RR Region (A–L) In immature- and mature-B cells, initiation events occur throughout the D-J-C-3′RR region, with increased activity near the 3′RR. Similar results were obtained for both the expressed and nonexpressed alleles, regardless of D-J, V-DJ, and class switch rearrangements. Molecules 11, 25, and 28, in (H), are not shown because they are bent. (M and N) In plasmacytoma cells, initiation events occur primarily at the 3′RR as in immature and mature cell lines. Each experiment is presented as in the previous figures. The profiles of replication fork abundance are shown in Figure S3. Molecular Cell 2005 20, 575-587DOI: (10.1016/j.molcel.2005.10.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Model of Origin Activation within the Mouse Igh Locus during B Cell Development Igh alleles during different stages of B cell development showing the position of genes (gray boxes) and regulatory elements (black bars). Deletions due to D-J, V-DJ, and class switch rearrangements are indicated by brackets. The portions of Igh locus containing early origins of replication are marked by black circles labeled “E.” Larger circles indicate the higher frequency of initiation events in the 3′RR region. This model is based on the results presented in this study and on previous information about the replication timing of the Igh locus (Ermakova et al., 1999; Zhou et al., 2002b). Question marks indicate the absence of information about DNA replication. Molecular Cell 2005 20, 575-587DOI: (10.1016/j.molcel.2005.10.029) Copyright © 2005 Elsevier Inc. Terms and Conditions