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Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching

Abstract

During B-cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons1,2. In mice, six additional sets of constant region exons (CHs) lie 100–200 kilobases downstream in the same transcriptional orientation as V(D)J and Cμ exons2. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH (ref. 2). Activation-induced cytidine deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region2,3; these lesions are converted into DNA double-strand breaks (DSBs) by general DNA repair factors3. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S-region DSB. However, the relative frequency of deletional to inversional CSR junctions has not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved is unknown. To address this question, we adapt high-throughput genome-wide translocation sequencing4 into a highly sensitive DSB end-joining assay and apply it to endogenous AID-initiated S-region DSBs in mouse B cells. We show that CSR is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis Igh organizational features in combination with frequent S-region DSBs initiated by AID. We further implicate ATM-dependent DSB-response factors in enforcing this mechanism and provide an explanation of why CSR is so reliant on the 53BP1 DSB-response factor.

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Figure 1: S-region-dependent orientation-biased joining in CSR-stimulated B cells.
Figure 2: S regions are not sufficient to promote orientation-biased CSR joining.
Figure 3: Orientation-biased joining of AID-initiated endogenous S-region breaks.
Figure 4: Mechanisitic roles of Igh organization and DSBR factors in deletional CSR.

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Gene Expression Omnibus

Data deposits

HTGTS sequencing data has been deposited in the GEO database under the accession number GSE71005.

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Acknowledgements

We thank K. Yu for providing the CH12F3-RMCE (1F7) cell line and exchange cassette plasmid. This work was supported by National Institute of Health grants AI077595 to F.W.A., CA133781 to J.M., AI112602 to D.F.R., and AI037526 and AI072529 to M.C.N. S.V. was supported by NIH training grant T32HL066987. F.W.A. and M.C.N. are investigators of the Howard Hughes Medical Institute. J.H. is supported by a Robertson Foundation/Cancer Research Institute Irvington Fellowship. F.M. is a Lymphoma Research Foundation postdoctoral fellow and was a Cancer Research Institute postdoctoral fellow.

Author information

Authors and Affiliations

Authors

Contributions

J.D., R.P., T.Z., J.P.M. and F.W.A. designed the study; J.D., R.P., T.Z., J.H. and S.V. performed experiments; Y.H. and R.M. designed bioinformatics pipelines; R.M., J.D., R.P. and Z.D. performed computational analyses of sequencing data; J.D., R.P. and F.W.A. wrote the paper. Other authors provided reagents, designed or performed certain experiments, and helped revise the paper.

Corresponding authors

Correspondence to John P. Manis or Frederick W. Alt.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Deletional CSR in in vitro activated B cells by DC-PCR; I-Sce1 DSBs within the Igh constant region locus in activated B cells join with orientation-independence.

a, Schematic representation of DC–PCR assay. b, DC–PCR results from anti-CD40/IL4-activated wild-type and 53BP1−/− B cells. c, Schematic representation of the HTGTS method. d, e, HTGTS libraries analyses of anti-CD40/IL4-stimulated IghI-96k B cells with 3′-broken end (d, red arrow, n = 3) or 5′-broken end (e, blue arrow, n = 3) primers. BE, broken end. f, HTGTS libraries with 5′-broken end primer (blue arrow, n = 3) from ΔSμ2×I/ΔSγ12×I B cells stimulated with anti-CD40/IL4. g, Bar graph depicting deletion:inversion and excision-circle:inversion ratios between two I-SceI sites and between I-SceI and S region in wild-type versus 53BP1−/− backgrounds. For detailed legends and further discussion, refer to the Supplementary Information.

Extended Data Figure 2 Genome-wide translocation junctions lack orientation bias; statistical analyses for experimental replicates orientation-biased joining between I-SceI break in place of Sα and AID-initiated Sμ breaks in CH12F3 cells.

a, b, Circos plots for translocation junctions across the whole genome from 3′-broken end (a, n = 4) or 5′-broken end (b, n = 3) HTGTS with anti-CD40/IL4 stimulated ΔSμ2×I/ΔSγ12×I B cells. c, d, Bar graphs depicting genome-wide percentage of junctions from pooled 3′- and 5′-broken end libraries plotted separately in − or + orientations. Error bars are s.d. e, Joining from ΔSγ12×I 3′-broken end to AID off-target DSBs in Il4ra gene on chromosome 7. f, Bar graph showing the number of junctions (average ± s.d.) recovered from IghI-96k AID−/− 3′-broken end HTGTS libraries (n = 3) at the break site and the upstream 1×I prey break as a percentage of the total number of junctions mapped to the 200 kb Igh constant region. Right panel shows the percentage of junctions mapping at 1×I (average ± s.d.) over the total Igh junctions that are mapped in the deletion (Del) or inversional (Inv) orientation. The numbers above the bar graph (average ± s.d.) denote the ratio of deletional to inversional junctions. g, Percentage of junctions (average ± s.d.) recovered from the IghI-96kAID−/− 5′-broken end HTGTS libraries (n = 3). h, Percentage of junctions (average ± s.d.) recovered from the ΔSμ2×I/ΔSγ12×I 3′-broken end libraries (n = 4). i, Percentage of junctions (average ± s.d.) recovered from the ΔSμ2×I/ΔSγ12×I 5′-broken end libraries (n = 3). j, Percentage of junctions (average ± s.d.) recovered from the wild-type ΔSγ12×I 3′-broken end libraries (n = 3). k, Percentage of junctions (average ± s.d.) recovered from the ΔSα1×I CH12F3 3′-broken end libraries (n = 3) and ΔSα1×I Sμ(INV) CH12F3 cells 3′-broken end libraries (n = 3). l, Bar graphs depicting percentage of trans junctions mapping to Sμ in − and + orientations from libraries of ΔSμ2×I/ΔSγ12×I B cells (n = 3) cloning from ΔSγ12×I 3′-broken ends. m, Bar graphs depicting percentage of trans junctions mapping to Sμ in − and + orientations and to Sε in − and + orientations from libraries of c-myc25×I 5′-broken ends (n = 3). n, o, HTGTS library analyses of ΔSα1×I CH12F3 cells stimulated with anti-CD40, IL4 and TGFβ and nucleofected with I-Sce1 expression plasmid. Cells were harvested on day 3 post-stimulation for 3′-broken ends (n, n = 6) and 5′-broken ends (o, n = 6) libraries. p, 3′- and 5′-broken end libraries are normalized with ‘symmetric junctions’ (see Supplementary Information). q, Bar graph showing percentage of junctions from ΔSα1×I CH12F3 cells (n = 6) from 3′- and 5′-broken end primers. For detailed legends and further discussion refer to the Supplementary Information.

Extended Data Figure 3 Joining between I-SceI break at Sμ and AID-initiated S-region breaks in lipopolysaccharide (LPS)-activated ΔSμ2×I/ΔSγ12×I B cells and clustered I-Sce1 breaks in ΔSμ2×I/ΔSγ128×I B cells in place of Sγ1; inverted Sμ in CH12F3 cells support robust IgA CSR.

a, Diagram of Igh locus organization in ΔSμ2×I/ΔSγ12×I B cells highlighting AID-initiated breaks in Sγ3, Sγ2b and Sγ2a regions upon LPS stimulation and potential outcomes of CSR in the form of deletion (red, −) and inversional joining (blue, +). b, Plots showing enlarged distribution of pooled prey junctions in a 20-kb region flanking Sγ3 and Sγ2b and Sγ2a from HTGTS libraries of ΔSμ2×I/ΔSγ12×I B cells (n = 3) stimulated with LPS and anti-IgD-dextran and infected with I-SceI-expressing retrovirus. c, Bar graph from three independent ΔSμ2×I/ΔSγ12×I 5′-broken end libraries showing the percentage of junctions mapped at different S regions. d, Diagram of Igh locus organization in ΔSμ2×I ΔSγ128×I B cells highlighting joining outcomes of I-Sce1-mediated bait DSBs at ΔSμ2×I to clustered I-SceI DSBs at ΔSγ128×I in the form of deletion (red, −) and inversional joining (blue, +). e, Pooled prey junctions from independent ΔSμ2×I ΔSγ128×I B cell libraries (n = 2, left panel, emulsion-mediated PCR; n = 2, right panel, linear-amplification-mediated HTGTS). f, Southern blot for Sμ inversion on the productive allele of CH12F3 cells with non-productive allele deleted. g, IgA CSR on day 3 for CH12F3 (non-productive ΔSμ-Sα) cells stimulated with anti-CD40, IL4 and TGFβ. h, IgA CSR on CH12F3 (productive allele Sμ(INV), non-productive allele ΔSμ-Sα) cells stimulated with anti-CD40, IL4 and TGFβ. Two independent clones of CH12F3 (Sμ(INV), non-productive ΔSμ-Sα). For detailed legends and further discussion refer to the Supplementary Information.

Extended Data Figure 4 Orientation-biased joining between AID-initiated rSμ and downstream AID-initiated S-region breaks in anti-CD40/IL4-activated and LPS-activated Sμ-truncated B cells.

a, 150-bp rSμ sequence used as HTGTS bait with red arrow indicating rSμ 5′-broken end HTGTS primer; red and blue vertical lines indicate canonical AGCT or other RGYW AID-targeting motifs, respectively. Distribution of rSμ break points in junctions to downstream S regions recovered from anti-CD40/IL4-stimulated rSμ B cells. b, HTGTS analyses of anti-CD40/IL4-activated rSμ B cells, 5′ rSμ AID-initiated broken end (red primer, n = 3) junctions to AID-initiated DSBs in Sγ1 and Sε which includes deletion (− orientation, red) or inversions (+ orientation, blue). c, HTGTS analyses of anti-CD40/IL4-activated rSμ B cells, 3′ rSμ AID-initiated broken end (blue primer, n = 3) junctions to AID-initiated DSBs in Sγ1 and Sε which includes excision circle (+ orientation, blue) or inversions (− orientation, red). d, Bar graph showing the percentage of junctions (average ± s.d.) from anti-CD40/IL4-activated rSμ 5′-broken end libraries mapped to Sγ1 and Sε. e, Bar graph showing the percentage of junctions (average ± s.d.) from anti-CD40/IL4-activated rSμ 3′-broken end libraries mapped to Sγ1 and Sε. f, HTGTS analyses of LPS-activated rSμ B cells, 5′ rSμ (red primer, n = 3) AID-initiated broken end junctions to AID-initiated DSBs in Sγ3, Sγ2b and Sγ2a which include deletional joining (− orientation, red) or inversions (+ orientation, blue). g, HTGTS analyses of LPS-activated rSμ B cells, 3′ rSμ (blue primer, n = 3) AID-initiated broken end junctions to AID-initiated DSBs of above LPS-stimulated cells in Sγ3, Sγ2b and Sγ2a which include excision circle (+ orientation, blue) or inversions (− orientation, red). h, i, Percentage of junction distribution at Sγ3, Sγ2b and Sγ2a in both orientations from both 5′-broken end libraries (h) and 3′-broken end libraries (i) are shown as average ± s.d. from three independent experiments. For detailed legends and further discussion refer to the Supplementary Information.

Extended Data Figure 5 Orientation-biased joining between rSμ and AID-induced Sα DSBs in CSR-activated CH12F3 cells.

a, Diagram outlining potential junction outcomes from 5′ rSμ (red primer) or 3′ rSμ (blue primer) AID-initiated broken end junctions to AID-initiated DSBs in Sα upon anti-CD40, IL4 and TGFβ stimulation of ΔSμ CH12F3 cells. b, c, Top panel shows HTGTS libraries analyses of day 2 (b) and day 3 (c) stimulated CH12F3 (non-productive allele ΔSμ-Sα, productive allele ΔSμ) cells cloning from 5′-broken end rSμ (red primer, n = 3), whereas lower panel shows HTGTS libraries cloning from 3′-broken end rSμ (blue primer, n = 3). d, Bar graph shows percentage of junctions (average ± s.d.) for 5′-broken end and 3′-broken end libraries indicated in b and c. e, Bar graph with percentage of junctions (average ± s.d.) from rSμ libraries mapped to prey Sα in the deletion (DEL) or inversion (INV) for 5′-broken end libraries and in excision circle (EC) or inversion orientation for 3′-broken end libraries. For detailed legends and further discussion refer to the Supplementary Information.

Extended Data Figure 6 Level of junctions to downstream S regions in wild-type and DSBR-deficient 5′Sμ HTGTS libraries correlate with CSR levels; 5′Sμ break site undergoes variable degrees of resection from stimulated DSBR-deficient B cells.

a, Table showing IgG1 and IgE CSR levels of splenic B cells from various genotypes (with number of replicates indicated) activated in vitro with anti-CD40 and IL4. FACS was performed on day 4 and values indicate average ± s.d. WT, wild type. b, Left panel shows bar graph for percentage of junctions (average ± s.d.) recovered from wild-type 5′Sμ 5′-broken end libraries mapped to Sμ, Sγ1 and Sε over the total number of junctions identified from the 200-kb Igh constant region. Remaining panels show the similar results from different DSBR-deficient backgrounds using the same 5′-broken end primer. c, d, 5′Sμ 5′-broken end HTGTS libraries analyses from H2AX−/− and RIF1fl/flCD19cre B cells are shown respectively. e, Diagram of potential junction outcomes from 5′Sμ AID-initiated 5′-broken end junctions to AID-initiated DSBs in Sγ1 and Sε. f, Data from HTGTS libraries mapped to the 20-kb region flanking 5′Sμ break site from B cells stimulated with anti-CD40/IL4 in wild-type and DSBR-deficient backgrounds. For detailed legends and further discussion refer to the Supplementary Information.

Extended Data Figure 7 Orientation-biased joining between rSμ and AID-induced Sγ and Sε DSBs in wild-type, ATM-deficient, and 53BP1-deficient B cells.

a, Diagram of potential junction outcomes from 5′ rSμ AID-initiated broken end junctions to AID-initiated DSBs in Sγ1 and Sε as described earlier. bd, Linear plots of pooled junctions across the 200-kb Igh constant region (first panel), the 20-kb region flanking rSμ break site (second panel), the 20-kb region flanking downstream Sγ1 (third panel) and Sε (last panel) from three independent wild-type (b), ATM- (c), or 53BP1- (d) deficient 5′-broken end rSμ libraries. e, Bar graphs showing invertion:deletion (INV/DEL) bias ratios of HTGTS Sγ1 (left panel) and Sε (right panel) junctions in different genotypes, showing average ± s.d. from three independent libraries for each genotype. P values calculated by unpaired two-tailed t-tests. f, Bar graphs showing percentage of long resection of Sγ1 (left) and Sε (right) junctions in different genotypes as average ± s.d. n.s., not significant. P values calculated by unpaired two-tailed t-tests. g, Bar graphs showing the number of junctions (average ± s.d.) recovered from above experiments from 5′-broken end HTGTS libraries for the indicated genotypes (n = 3 for each) at the break site rSμ and downstream Sγ1 and Sε regions as a percentage of the total number of junctions mapped to the 200-kb IgH constant region. For detailed legends and further discussion refer to the Supplementary Information.

Extended Data Figure 8 Orientation-biased joining of I-SceI DSBs at Sγ1 to AID-induced S-region breaks in various DSBR-deficient backgrounds.

a, Schematic illustration of the ΔSγ12×I allele and joining outcomes from 3′-broken end (red arrow) to AID-initiated upstream Sμ and downstream Sε DSBs. b, Linear distribution of junctions between ΔSγ12×I 3′-broken end to AID-induced Sμ/Sε region DSBs in anti-CD40/IL4-stimulated wild-type (b, n = 4), ATM−/− (c, n = 3), H2AX−/− (d, n = 3) and 53BP1−/− (e, n = 4) cells across the 200-kb Igh region (left panels), 10-kb Sμ (middle panels) and Sε (right panels). f, Bar graphs (average ± s.d.) showing the percentage of junctions mapped at ΔSγ12×I (break site), Sμ and Sε over the total number of junctions in the 200-kb Igh constant region in different genotypes. g, Bar graphs (average ± s.d.) showing the percentage of junctions from above various genotypes of ΔSγ12×I 3′-broken end libraries mapped to Sμ and Sε as average ± s.d. h, Bar graph (average ± s.d.) showing comparison of percentages of junctions cloned using ΔSγ12×I 3′-broken end involving resection of Sμ (top panel) and Sε (bottom panel) breaks in indicated backgrounds. For detailed legends and further discussion refer to the Supplementary Information.

Extended Data Figure 9 Inhibition of resection in 53BP1-deficient B cells by an ATM inhibitor (ATMi) does not rescue directional CSR joining to Sγ1.

ad, Linear plots of pooled junctions across the 200-kb Igh constant region (left panels), the 20-kb region flanking downstream Sγ1 (middle panels) and Sε (right panels) from wild type plus DMSO control (a, n = 2), wild type plus ATMi (b, n = 3), 53BP1−/− plus DMSO (c, n = 3) and 53BP1−/− plus ATMi (d, n = 3) libraries are shown as above. e, Bar graph showing the percentage of Sμ, Sγ1 and Sε junctions (average ± s.d.) from wild type plus DMSO 5′Sμ libraries (left) and wild type plus ATMi 5′-broken end libraries (right, n = 3). f, Bar graph showing the percentage of Sμ, Sγ1 and Sε junctions (average ± s.d.) from 53BP1−/− plus DMSO (left) and 53BP1−/− plus ATMi (right) 5′Sμ libraries. For detailed legends and further discussion refer to the Supplementary Information.

Extended Data Table 1 Statistical comparison for orientation bias and resection in Sγ1 and Sε junctions from wild-type and DSBR-deficient B cell libraries

Supplementary information

Supplementary Information

This file contains Supplementary Text, full legends for Extended Data Figures 1-9 and Extended Data Table 1, and 2 Supplementary Tables, which show the DNA oligos and Plotting coordinates used in this study. (PDF 1167 kb)

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Dong, J., Panchakshari, R., Zhang, T. et al. Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching. Nature 525, 134–139 (2015). https://doi.org/10.1038/nature14970

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