Volume 147, Issue 1, Pages 95-106 (September 2011) Translocation-Capture Sequencing Reveals the Extent and Nature of Chromosomal Rearrangements in B Lymphocytes  Isaac A. Klein, Wolfgang Resch, Mila Jankovic, Thiago Oliveira, Arito Yamane, Hirotaka Nakahashi, Michela Di Virgilio, Anne Bothmer, Andre Nussenzweig, Davide F. Robbiani, Rafael Casellas, Michel C. Nussenzweig  Cell  Volume 147, Issue 1, Pages 95-106 (September 2011) DOI: 10.1016/j.cell.2011.07.048 Copyright © 2011 Elsevier Inc. Terms and Conditions

Cell 2011 147, 95-106DOI: (10.1016/j.cell.2011.07.048) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 TC-Seq Schematic IgHI or MycI primary B cells are infected with retroviruses encoding I-SceI with or without AID. Genomic DNA is fragmented, blunted, A-tailed, ligated to T-tailed asymmetric linkers and native loci are eliminated by I-SceI digestion. Rearrangements are amplified by semi-nested ligation-mediated PCR followed by linker cleavage and paired-end deep sequencing. Cell 2011 147, 95-106DOI: (10.1016/j.cell.2011.07.048) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Rearrangements to a DSB Site Documented by TC-Seq (A) Genome-wide view of rearrangements to MycI in AID−/− B cells. (B) Rearrangements per mappable megabase to each chromosome in MycIAID−/− B cells. (C) Profile of rearrangements around the I-SceI site in 5 kb intervals. Cell 2011 147, 95-106DOI: (10.1016/j.cell.2011.07.048) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Rearrangements to MycI Occur Near the TSSs of Actively Transcribed Genes (A) Rearrangements, excluding the 1 Mb around I-SceI, were categorized as genic or intergenic. (B) Composite density profile of genomic rearrangements from MycI to genes (red line) and intergenic regions (blue line). TSS = transcription start site, TTS = transcription termination site. (C) Relative frequency (fe) of rearrangements in genes that are either silent or display trace, low, medium or high levels of transcription in activated B cells as determined by RNA-Seq (Figure S1). Dashed line indicates the expected rearrangement frequency based on a random model. p < 0.001 for all (permutation test). (D) Relative frequency of rearrangements in PolII- and activating histone mark-associated gene groups (Yamane et al., 2011). Dashed line indicates expected frequency based on a random model. p < 0.001 for all samples (permutation test). Also see Figure S1. Cell 2011 147, 95-106DOI: (10.1016/j.cell.2011.07.048) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Rearrangements to IgHI or MycI in Primary B Cells Expressing AID (A and B) (A) Rearrangements per kb to I-SceI sites (indicated with an asterisk) in IgH or (B) c-myc in AID−/− (top panel) or AIDRV cells (bottom panel). White boxes in the schematics below each graph represent Ig switch domains while black boxes depict constant regions. (C) Translocations per 100 bp from IgHI to c-myc in AID−/− (top panel) or AIDRV cells (bottom panel). Green arrows indicate c-myc/IgH translocation breakpoints sequenced from primary B cells (Robbiani et al., 2008). (D) Relative frequency of rearrangements in transcription-level gene groups (Figure S1), dashed line indicates expected frequency based on a random model. Asterisks highlight values with a p < 0.001 (permutation test). (E) Relative frequency of rearrangements in PolII-associated or activating histone mark-associated gene groups (Yamane et al., 2011). Dashed line indicates the expected frequency based on a random model. p < 0.001 for all samples (permutation test). (F) Graph comparing the number of translocations per mappable megabase to each chromosome from IgHI (y axis) or MycI (x axis). (G) Ratio of IgHI captured to MycI captured events in 500 kb bins moving away from the I-SceI capture site (both directions combined). Dotted line represents the average transchromosomal joining rate computed on all chromosomes other than 12 or 15. Gray areas show 2 standard deviations around the mean. Also see Figure S2. Cell 2011 147, 95-106DOI: (10.1016/j.cell.2011.07.048) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 AID-Dependent Rearrangement Hot Spots (A) Screenshots of translocations per 100 bp present at Il4i1 and Pax5 genes in all samples. (B) Overlap of AID-dependent hot-spot-bearing genes in IgHIAIDRV and IgHIAIDWT experiments. (C) Overlap of AID-dependent hot-spot-bearing genes in IgHIAIDRV, MycIAID−/− and MycIAIDRV experiments. (D) Empirical cumulative distribution showing transcript abundance in genes displaying (red) or lacking (black) rearrangement hot spots. Filled-in gray slice represents ∼2000 highly transcribed unrearranged genes. (E) Total rearrangements as a function of gene expression (RNA-Seq) (Yamane et al., 2011) in genes bearing rearrangement hot spots. Also see Figure S3. Cell 2011 147, 95-106DOI: (10.1016/j.cell.2011.07.048) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Characterization of AID-Dependent Hot Spots (A) Composite density graph showing the distribution of rearrangements in genes associated with AID-dependent hotspots relative to the TSS. (B) Spt5 and AID recruitment at genomic sites associated with translocation hotspots (Pavri et al., 2010; Yamane et al., 2011). (C) Somatic hypermutation frequency versus number of rearrangements in genes bearing AID-dependent translocation hotspots. (D) Distribution of AID, Spt5. and translocations in Rohema and Hist1 genes. Also see Figure S4. Cell 2011 147, 95-106DOI: (10.1016/j.cell.2011.07.048) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure S1 Gene Expression Groups in LPS+IL-4 Activated B Cells, Related to Figure 3 Genes were divided into silent (0 FPKM [fragments per kilobase of exon per million fragments mapped]) and expressed genes based on mRNA-Seq (Yamane et al., 2011). The expressed group showed a bimodal distribution and was subdivided into trace expressed genes (≤4.04 FPKM) and more highly expressed genes using a normal mixture model implemented in the mclust R package. The higher group was divided again into tertiles (low > 4.04 FPKM; medium > 23.5 FPKM; high > 65 FPKM). Cell 2011 147, 95-106DOI: (10.1016/j.cell.2011.07.048) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure S2 Distribution of Chromosomal Rearrangements in the MycI or IgHI B Cell Genomes, Related to Figure 4 (A) Bar graph displaying total rearrangements normalized per mappable megabase per chromosome in MycIAIDRV and (B) IgHIAIDRV B lymphocytes. In both cases cells were transduced with retroviruses (RV) expressing AID. (C) Rearrangements per 5 kb around MycI, I-SceI site indicated with an asterisk. Distribution in the absence (gray line) or presence (red line) of AID. Green arrows indicate plasmacytoma translocation breakpoints. (D) Composite density profile of rearrangements, genic (red) and intergenic (blue) regions in IgHI (upper) or MycI (lower) AIDRV captures. (E) Frequency of intrachromosomal rearrangements as a function of distance. Ratio of IgHI captured to MycI captured events in 500 kb bins moving away from the I-SceI site. Dotted line represents the average transchromosomal joining rate computed on all chromosomes other than 12 or 15. Gray areas show 2 standard deviations around the mean. (F) Modeling intrachromosomal rearrangement bias. Rearrangements to IgHI (red) and MycI (blue) in the combined AIDRV samples were sorted into variable-sized bins of ≥ 20 events. Rearrangement density was calculated per bin to the left and right of c-myc separately as a step plot. When both directions were combined and plotted as a scatter plot on a log-log scale (inset graph, hotspots in gray) from 5 kb to 1 Mb, a roughly linear relationship suggested that the rearrangement density decreased as a function of a power of the distance. Fitting a linear model to the log transformed data excluding hotspots allowed the exponent of this power law to be estimated as approximately −1.3 (R2 = 0.81). Cell 2011 147, 95-106DOI: (10.1016/j.cell.2011.07.048) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure S3 I-SceI Cryptic Site, Related to Figure 5 Screenshot showing a TC-Seq hotspot associated with a cryptic I-SceI site located in chromosome 15 in MycIAID−/− B cells. Rearrangements accumulate within 200 bp of a site that differs by 1 base (T- > G) from the canonical I-SceI recognition sequence. Cell 2011 147, 95-106DOI: (10.1016/j.cell.2011.07.048) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure S4 AID Recruitment in Rearranged Genes and Rearrangements to Repeat-rich Regions, Related to Figure 6 (A) Empirical cumulative distribution function showing AID accumulation at TSSs of genes displaying (red) or lacking (black) AID-dependent TC-Seq hotspots. (B) Screenshots of TC-Seq hotspots lying in repeat-rich regions. Repeat type and span (open rectangle) of the repeat as annotated by the UCSC browser is indicated. Cell 2011 147, 95-106DOI: (10.1016/j.cell.2011.07.048) Copyright © 2011 Elsevier Inc. Terms and Conditions