Fundamental roles of chromatin loop extrusion in antibody class switching

Abstract

Antibody class switch recombination (CSR) in B lymphocytes replaces immunoglobulin heavy chain locus (Igh) Cμ constant region exons (CHs) with one of six CHs lying 100–200 kb downstream1. Each CH is flanked upstream by an I promoter and long repetitive switch (S) region1. Cytokines and activators induce activation-induced cytidine deaminase (AID)2 and I-promoter transcription, with 3′ IgH regulatory region (3′ IgHRR) enhancers controlling the latter via I-promoter competition for long-range 3′ IgHRR interactions3,4,5,6,7,8. Transcription through donor Sμ and an activated downstream acceptor S-region targets AID-generated deamination lesions at, potentially, any of hundreds of individual S-region deamination motifs9,10,11. General DNA repair pathways convert these lesions to double-stranded breaks (DSBs) and join an Sμ-upstream DSB-end to an acceptor S-region-downstream DSB-end for deletional CSR12. AID-initiated DSBs at targets spread across activated S regions routinely participate in such deletional CSR joining11. Here we report that chromatin loop extrusion underlies the mechanism11 by which IgH organization in cis promotes deletional CSR. In naive B cells, loop extrusion dynamically juxtaposes 3′ IgHRR enhancers with the 200-kb upstream Sμ to generate a CSR centre (CSRC). In CSR-activated primary B cells, I-promoter transcription activates cohesin loading, leading to generation of dynamic subdomains that directionally align a downstream S region with Sμ for deletional CSR. During constitutive Sα CSR in CH12F3 B lymphoma cells, inversional CSR can be activated by insertion of a CTCF-binding element (CBE)-based impediment in the extrusion path. CBE insertion also inactivates upstream S-region CSR and converts adjacent downstream sequences into an ectopic S region by inhibiting and promoting their dynamic alignment with Sμ in the CSRC, respectively. Our findings suggest that, in a CSRC, dynamically impeded cohesin-mediated loop extrusion juxtaposes proper ends of AID-initiated donor and acceptor S-region DSBs for deletional CSR. Such a mechanism might also contribute to pathogenic DSB joining genome-wide.

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Fig. 1: Cytokine-induced target S-region transcription promotes synapsis with Sμ during CSR.
Fig. 2: Constitutive CH12F3 Sα transcription causes dominant Sα CSR and impedes long-range interactions and CSR to upstream S regions.
Fig. 3: Inserting CBEs upstream of Iα activates inversional CH12F3 CSR.
Fig. 4: CBE insertion in Iα-deleted CH12F3Δ cells impedes upstream transcription, looping and CSR and creates ectopic S region.

Data availability

CSR-HTGTS-seq, 3C-HTGTS, GRO-seq and ChIP–seq sequencing data analysed here have been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE130270. Specifically, the GEO accession number for Figs. 1d, e, 2d–f, 3f, g, 4e–g and Extended Data Figs. 2b–e, 5a, b, 6a–c, 7b, c, 8d, 10a–c is GSE130263. The GEO accession number for Figs. 1c, 2c, 4h and Extended Data Figs. 2a, 4a–c, 10d is GSE130266. The GEO accession number for Figs. 2b, 3c, d, 4b–d and Extended Data Figs. 3f, 8b, 9a, b is GSE130265. The GEO accession number for Figs. 1f, g, 3e and Extended Data Figs. 2f, g, 5c, d, 7a is GSE130264.

Code availability

The CSR-HTGTS-seq and 3C-HTGTS pipelines38 used in this study are available at http://robinmeyers.github.io/transloc_pipeline/.

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Acknowledgements

We thank T. Honjo for the CH12F3 cell line and AID−/− C57BL/6 mice, and J. Chaudhuri for stimulating discussions. This work was supported by NIH R01AI077595. F.W.A. is an investigator of the Howard Hughes Medical Institute. Y.Z. is a special fellow of the Leukemia and Lymphoma Society. Z.B. was a Cancer Research Institute Irvington fellow.

Author information

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Authors

Contributions

X.Z. and F.W.A. designed the study; X.Z. performed all experiments; Y.Z., Z.B. and R.C. provided reagents and advice; N.K. designed some of the bioinformatics pipelines; X.Z. and F.W.A analysed and interpreted data; X.Z. and F.W.A. designed figures and wrote the manuscript; Y.Z., Z.B. and R.C. helped polish the manuscript.

Corresponding author

Correspondence to Frederick W. Alt.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Andre Nussenzweig and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Working model for cohesin-mediated chromatin loop extrusion-driven deletional CSR joining.

a, Cohesin (blue rings) loaded at the indicated HS sites within the 3′ IgHRR dynamically extrude 3′ IgHRR chromatin which aligns the HS sites as transient loop anchors (brown oval). be, In resting B cells, cohesin is loaded (blue arrows) at either Iμ–Sμ (red rectangles) or the 3′ IgHRR. While similar models could be drawn for both, we illustrate one in which loading occurs at 3′ IgHRR (brown oval) and downstream extrusion is impeded by 3′ IgHRR–3′ CBEs chromatin to generate a dynamic impediment for extrusion of upstream chromatin that brings iEμ–Iμ–Sμ into proximity with the 3′ IgHRR to generate a CSRC (grey circle). In this process, upstream extrusion is strongly impeded at the V(D)J–iEμ locale. f, g, B cell activation primes a targeted S-region promoter (light green becoming darker green), which is activated for high level transcription (bright green) after extrusion into proximity with the 3′ IgHRR. h, i, Downstream extrusion of cohesin loaded at the activated S region is impeded by activated S-region chromatin allowing extrusion of upstream chromatin to dynamically align targeted S region with Sμ. jp, Activation-induced AID is transcriptionally targeted to Sμ and the activated S region leading to DSBs (lightning bolts) in one or the other and, ultimately, in both. Cohesin-mediated loop extrusion pulls S-region DSB ends into cohesin rings stalling extrusion and aligning them for deletional end joining. DSBs in the Sμ and activated S regions need not occur at the same spatial location or time in this model. See also Supplementary Video 1.

Extended Data Fig. 2 Cytokine–activator-induced S-region transcription promotes dynamic loop formation and S-S synapsis during CSR.

a, Left, additional repeats of GRO-seq profiles shown in Fig. 1c from non-stimulated and anti-CD40–IL-4-stimulated AID−/− mature splenic B cells. Right, magnified view of the GRO-seq profiles on the left to better reveal the transcription level around the iEμ–Cδ locale from non-stimulated and anti-CD40–IL-4-stimulated AID−/− mature splenic B cells. b, c, Additional repeats of 3C-HTGTS profiles shown in Fig. 1d, e from non-stimulated and anti-CD40–IL-4-stimulated AID−/− mature splenic B cells using iEμ–Iμ (b) or 3′ IgHRR(HS4) (c) locale as baits (blue asterisks). Bar graphs on the right of 3C-HTGTS profiling show the relative iEμ–Iμ or 3′ IgHRR(HS4) interaction frequency with Sγ1 and Sε in anti-CD40–IL-4-stimulated mature splenic B cells. Diagrams on the top of the 3C-HTGTS profiling show the digestion and bait strategies used for the 3C-HTGTS experiments. d, e, 3C-HTGTS profiles of interactions within the 3′ IgH locus domain in anti-CD40–IL-4-stimulated AID−/− mature splenic B cells using the first (d) (two biologically independent repeats) and seventh (e) (three biologically independent repeats) 3′ CBE locale as baits (blue asterisks). Diagrams on the right of the 3C-HTGTS profiling show the digestion and bait strategies used for the 3C-HTGTS experiments. f, g, Additional repeats of NIPBL (f) and RAD21 (g) ChIP–seq shown in Fig. 1f, g from non-stimulated and anti-CD40–IL-4-stimulated AID−/− mature splenic B cells. Bar graphs on the right of the ChIP–seq profiling show NIPBL and RAD21 accumulation of indicated regions. All bar graph data (b, c, f, g) are mean ± s.d. from three biologically independent repeats. P values were calculated via an unpaired two-tailed t-test. All other bars and symbols are as indicated in Fig. 1 legend. Source data

Extended Data Fig. 3 Iα deletion promotes CSR to upstream S regions.

a, Illustration of dominant, deletional CSR between Sμ and Sα in CH12F3 cells. b, Illustration of Cas9–gRNA targeting (lightning bolts) used to generate the CH12F3NCΔ line. c, Southern blot confirmation (using BamHI digestion and a JH4 probe) of the CH12F3NCΔ lines (done twice independently with similar results). d, Western blot confirmation of AID expression or lack of expression, respectively, in AID sufficient and deficient (via targeted deletion) CH12F3NCΔ and IαΔ lines following stimulation with anti-CD40–IL-4–TGFβ for 72 h (done twice independently with similar results). e, FACS analysis for surface IgA expression in CH12F3NCΔ AID−/− cells stimulated with anti-CD40–IL-4–TGFβ for 72 h (done three times independently with similar results). f, Three repeats of CSR-HTGTS-seq data shown in Fig. 2b for anti-CD40–IL-4–TGFβ-stimulated CH12F3NCΔ and IαΔ cells (three biologically independent repeats). Junctions are plotted at 2.5-kb bin size. The blue lines indicate deletional joining and the red lines indicate inversional joining. Bar graph shows percentages of junctions located in different regions from CH12F3NCΔ and IαΔ cells. Data are mean ± s.d. from three biologically independent repeats. P values were calculated via an unpaired two-tailed t-test. g, FACS analysis of IgA, IgG3 and IgG2b surface expression in CH12F3NCΔ and IαΔ cells stimulated with anti-CD40–IL-4–TGFβ for 72 h (four biologically independent repeats). Bar graph shows percentages of IgA, IgG3 and IgG2b expression on CH12F3NCΔ and IαΔ cells. Data are mean ± s.d. from four biologically independent repeats. P values were calculated via an unpaired two-tailed t-test. Source data

Extended Data Fig. 4 Iα deletion promotes transcription to upstream S regions.

a, GRO-seq profile of repeat no. 1 (shown immediately below it) with an enlarged scale to allow better comparison of relative transcription levels of different portions of the IgH constant region in CH12F3NCΔ AID−/− and IαΔ AID−/− cells with or without anti-CD40–IL-4–TGFβ stimulation (three biologically independent repeats with similar results). Green asterisks indicate the HS3a, HS1,2 and HS4 sites within 3′ IgHRR. b, Three repeats of the GRO-seq profiles with a smaller scale to better reveal low, but significant transcription of Cγ2b and Cγ2a (upon Iα deletion) in CH12F3NCΔ AID−/− and IαΔ AID−/− cells with or without anti-CD40–IL-4–TGFβ stimulation. c, Higher magnification view of the three repeats of GRO-seq profiles to better reveal induced antisense transcription in the region between Sγ3 to Sε in IαΔ AID−/− versus CH12F3NCΔ AID−/− cells with or without anti-CD40–IL-4–TGFβ stimulation. d, Bar graph shows GRO-seq transcriptional activity (calculated as RPM) of the different indicated S regions in anti-CD40–IL-4–TGFβ-stimulated CH12F3NCΔ AID−/− cells and IαΔ AID−/− cells (three biologically independent repeats). Bar graph panel represents mean ± s.d. from three biologically independent repeats. P values were calculated via unpaired two-tailed t-test. Grey bars highlight the iEμ–Cμ, Iγ3–Cγ3, Iγ2b–Cγ2b, Iγ2a–Cγ2a, Iα–Cα, 3′ IgHRR and 3′ CBEs. Source data

Extended Data Fig. 5 Constitutively transcribed Sα leads to constitutive synapsis of Sα with Sμ in CH12F3 cells.

a, b, Additional repeats for the 3C-HTGTS profiles shown in Fig. 2d, e, from non-stimulated and anti-CD40–IL-4–TGFβ-stimulated CH12F3NCΔ AID−/− cells using iEμ–Iμ (a) or HS4 (b) locale as baits (blue asterisks). Green asterisks indicate the HS3a, HS1,2 and HS4 sites within 3′ IgHRR. Grey bars highlight the iEμ–Cμ, Sα, 3′ IgHRR and 3′ CBEs. c, d, NIPBL (c) (two biologically independent repeats) and RAD21 (d) (three biologically independent repeats) ChIP–seq profiles of non-stimulated and anti-CD40–IL-4–TGFβ-stimulated CH12F3NCΔ AID−/− cells. Green asterisks indicate the Iα, HS3a, HS1,2, HS3b, HS4 and HS7 sites that were implicated by this experiment as targets for cohesin loading. Grey bars highlight the broader regions around Sμ, Sα, the 3′ IgHRR and the 3′ CBEs. e, Loop extrusion-mediated Sμ–Sα synapsis in CH12F3 cells. I, Cohesin is loaded at various Igh locations including transcriptionally activated Iα–Sα. II–IV, For cohesin loaded at Iα locale downstream extrusion is impeded by transcribed Sα allowing upstream extrusion to proceed until being dynamically impeded by transcribed iEμ–Sμ–Cμ locale resulting in Sμ and Sα being brought into proximity without complete alignment. During upstream extrusion, the activated Iα promoter blocks extrusion-mediated activation of upstream I promoters by the 3′ IgHRR via promoter competition. V, VI, Continued loading of cohesin at the Iα locale is impeded for downstream extrusion allowing continued upstream extrusion until reaching the transcribed Sμ locale causing dynamic Sα–Sμ synapsis. VII–X, Activation-induced AID is transcriptionally targeted to Sμ and the activated Sα leading to DSBs (lightning bolts) in one or the other and, ultimately, in both. Cohesin-mediated loop extrusion pulls S-region DSB ends into cohesin rings stalling extrusion and aligning them for deletional end joining. This model could be explained by other variations including cohesin loading at Sμ or the 3′ IgHRR or a process like the one shown in Extended Data Fig. 1.

Extended Data Fig. 6 Iα deletion increases iEμ–Iμ and HS4 interactions across the upstream CH domain.

a, b, Left, additional repeats for the 3C-HTGTS profiles shown in Fig. 2d, e, from anti-CD40–IL-4–TGFβ-stimulated CH12F3NCΔ AID−/− and IαΔ AID−/− cells using iEμ–Iμ (a) or HS4 (b) locale as baits (blue asterisks). Green asterisks indicate 3′ IgH RR HS sites in all panels. Grey bars highlight the iEμ–Cμ, Sγ3, Sγ2b, Sγ2a, Sα, 3′ IgHRR and 3′ CBEs. Right, magnified view of the 3C-HTGTS profiles on the left to better reveal the interaction patterns in the region from Iγ3 to Cε in anti-CD40–IL-4–TGFβ-stimulated CH12F3NCΔ AID−/− and IαΔ AID−/− cells. c, Left, 3C-HTGTS profiles of interactions across the indicated domain of non-stimulated and anti-CD40–IL-4–TGFβ-stimulated IαΔ AID−/− cells using the iEμ–Iμ locale as bait (blue asterisks). Grey bars highlight the iEμ–Cμ, Sγ3, Sγ2b, Sγ2a, Sα, 3′ IgHRR and 3′ CBEs. Right, magnified view of the 3C-HTGTS profiles on the left to better reveal the interaction patterns in the region from Sγ3 to Sε in non-stimulated and anti-CD40–IL-4–TGFβ-stimulated IαΔ AID−/− cells.

Extended Data Fig. 7 CBEs inserted upstream of Iα lead to increased inversional Sα CSR.

a, Three repeats of RAD21 ChIP–seq profiles of CD40–IL-4–TGFβ-stimulated i3CBEs AID−/− cells. b, c, Additional repeats of the 3C-HTGTS profiles shown in Fig. 3f, g, from CD40–IL-4–TGFβ-stimulated CH12F3NCΔ AID−/− and i3CBEs AID−/− cells using CBE insertion (b) or iEμ–Iμ (c) locale as baits (blue asterisks). b, Right, 3C-HTGTS profiling shows the digestion and bait strategies used. d, Model to address increased inversional Sα CSR in CH12F3 cells with CBEs inserted upstream of Iα. I, Cohesin is loaded at various Igh locations including transcriptionally activated Iα–Sα. II–VII, For cohesin loaded at the Iα locale, extrusion past the CBE impediment allows a significant subset of cells to reach step VII to generate CSRC. VIII–X, In these cells, a significant portion of continued upstream extrusion passes by the CBE impediment to yield cells in the population with configurations shown in steps IX and X. XI–XIV, The cells with the configuration shown in IX will have both deletional (XIII) and inversional (XIV) joining mediated by a diffusion-related process in the absence of complete Sμ–Sα synapsis (see main text for more details). Those with the configuration shown in X will join via deletion as described in Extended Data Fig. 5e. This working model could be explained by other variations as indicated for other model figures.

Extended Data Fig. 8 Deletion of 3′ CBEs in i3CBE cells has little effect on the Sα CSR and Sα inversional joining.

a, Representative FACS analyses for IgH class switching from IgM to IgA for CH12F3NCΔ, i3CBEs and i3CBEs 3′ CBEsΔ cells stimulated with anti-CD40–IL-4–TGFβ for 72 h. Bar graph on right shows FACS data from six biologically independent repeats plotted as mean ± s.d. P values were calculated via an unpaired two-tailed t-test. b, CSR-HTGTS-seq of three repeats that use 5′ Sμ bait for analyses of anti-CD40–IL-4–TGFβ-stimulated CH12F3NCΔ, i3CBEs and i3CBEs-3′ CBEsΔ cells. Junctions are plotted at 200-bp bin size. Blue lines indicate deletional joining and red lines indicate inversional joining. c, Schematic of Igh CH locus from iEμ to 33 kb downstream of 3′ CBEs. Top, magnified view illustrates 3′ CBEs deletion in i3CBEs lines to generate i3CBE 3′ CBEΔ lines. d, Three repeats of 3C-HTGTS profiles of anti-CD40–IL-4–TGFβ-stimulated i3CBE AID−/− and i3CBE 3′ CBEΔ-AID−/− cells using the CBE insertion locale as bait (blue asterisks). Source data

Extended Data Fig. 9 CBE insertion in Iα-deleted CH12F3NCΔ cells impedes IgH class-switching and CSR and creates an ectopic S region.

a, Top and middle, three individual repeats of CSR-HTGTS-seq experiments shown in Fig. 4b that use a 5′ Sμ bait to assay anti-CD40 region–IL-4–TGFβ-stimulated IαΔ and IαΔ i3CBE cells. Junctions are plotted at 2.5-kb bin size. Bottom, magnified views of three repeats of data in Fig. 4c showing junctions located in the AID-targeted ectopic S region between Cγ2a and Iε in assays of IαΔ i3CBE cells. Junctions are plotted at 115-bp bin size. Blue lines indicate deletional joining and red lines indicate inversional joining. b, Bar graph shows percentages of junctions located in indicated AID-targeted regions from IαΔ and IαΔ i3CBE cells. Data are mean ± s.d. from three biologically independent repeats. P values were calculated via an unpaired two-tailed t-test based on the three repeats. c, AID-targeting-motif frequency analysis of 2-kb ectopic S-region targeting peak and in comparably sized region just upstream and downstream of the targeting peak. d, FACS analysis of IgG3 and IgG2b surface expression in IαΔ and IαΔ i3CBE cells stimulated with anti-CD40–IL-4–TGFβ for 72 h (6 biologically independent repeats). Bar graph shows percentages of IgG3 and IgG2b production from IαΔ and IαΔ i3CBE cells. Data are mean ± s.d. from six biologically independent repeats. P values were calculated via an unpaired two-tailed t-test. Source data

Extended Data Fig. 10 Repeats of experiments showing that CBE insertion in Iα-deleted CH12F3Δ cells impedes upstream transcription and looping.

ac, Additional repeats of Fig. 4e–h: 3C-HTGTS profiles from anti-CD40–IL-4–TGFβ-stimulated IαΔ AID−/− and IαΔ i3CBE AID−/−cells using the CBEs insertion (a) (three biologically independent repeats), the iEμ–Iμ (b) (three biologically independent repeats) or the 3′ IgHRR(HS4) (c) (three biologically independent repeats) locale as baits (blue asterisks). Bar graphs on the right of the 3C-HTGTS profiles show the relative iEμ–Iμ or 3′ IgHRR(HS4) interaction frequency with eS and the region between Cδ to Sγ2b. In b, c, data are mean ± s.d. from three biologically independent repeats. P values were calculated via paired two-tailed t-test. d, Three repeats of GRO-seq profiles with larger scale from anti-CD40–IL-4–TGFβ-stimulated IαΔ AID−/− and IαΔ i3CBEs AID−/−cells as in Fig. 4h. All bars and other notations as described in the legend of Fig. 4. Source data

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This file contains the Supplementary Discussion, Supplementary Tables 1-4 and Supplementary Figure 1

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Video 1

: Chromatin loop extrusion plays a fundamental mechanistic role in antibody class switch recombination. This video provides the animation of the loop-extrusion mediated IgH class switch recombination based on the model described in Extended Data Figure 1 and legend. The video has also incorporated self-explanatory text that allows it to be understood on its own

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Zhang, X., Zhang, Y., Ba, Z. et al. Fundamental roles of chromatin loop extrusion in antibody class switching. Nature 575, 385–389 (2019). https://doi.org/10.1038/s41586-019-1723-0

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