Abstract
Activation-induced cytidine deaminase (AID)-initiated immunoglobulin heavy chain (Igh) class switch recombination (CSR) replaces donor Cμ constant region exons (CHs) with a downstream acceptor CH to generate different functional antibody isotypes. However, mechanisms governing orientation-specific productive CSR remain incompletely understood. Through analysing the characteristics of evolved constant regions and constructing diversified constant regions to recapitulate productive CSR in jawed vertebrates to systematically dissect productive CSR determinants, we find that switch topological configuration (STC), including transcriptional orientation, chromatin distance, and chromatin domain of Igh, determines orientation-specific joining of AID-initiated breaks for productive CSR. Long-distance CHs under co-oriented transcription within Igh domain foster predominantly deletional joining-mediated productive CSR. In contrast, oppositely transcribed and short-distance CHs affect end-joining bias via promoting diffusion-mediated inversional joining for CSR with lower efficiency. Moreover, AID-initiated breaks in different domains, facilitate more diffusion-mediated orientation-unbiased end-joining for non-productive CSR. Our findings uncover chromatin-intrinsic mechanisms safeguarding orientation-specific productive CSR throughout evolution.
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Data availability
CSR-HTGTS-seq, 3C-HTGTS, Pro-seq, and ChIP-seq sequencing data analyzed here have been deposited in the GEO database. The GEO accession number for CSR-HTGTS-seq is GSE299392. The GEO accession number for 3C-HTGTS is GSE299499. The GEO accession number for Pro-seq is GSE299975. The GEO accession number for ChIP-seq is GSE299391. All other data can be found in the Supplementary Data of this paper or in the Source Data. This includes all uncropped gel electrophoresis images and data shown in graphs throughout the manuscript, including the Supplementary Figs. All data are available from the authors upon reasonable request. Source data are provided with this paper.
Code availability
V(D)J-HTGTS-seq, CSR-HTGTS-seq, and 3C-HTGTS data was processed through published pipelines (http://robinmeyers.github.io/transloc_pipeline/). Pro-Seq was aligned to either the mm9 genome or modified genomes with bowtie2 (v.2.4.4) (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml), processed by samtools (v.1.15.1) (https://sourceforge.net/projects/samtools/fles/samtools/1.15.1/), and generated graph files via the RseqQ (v.5.0.1) (https://rseqc.sourceforge.net/) package. ChIP-Seq was aligned to the mm9-derived modified genomes with bowtie2 (v.2.4.4), processed by samtools (v.1.15.1), generated bigwig files via Deeptools (v.3.5.1) (https://github.com/deeptools/deepTools), and generated peak files via MACS2 (v.2.2.7.1) (https://github.com/taoliu/MACS/).
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Acknowledgements
We thank Drs. Frederick W. Alt and Tasuku Honjo for providing reagents. We thank Drs. Xiaoliang Sunney Xie, Zemin Zhang, Fuchou Tang, Guoqiang Li, Jiazhi Hu, and Hsiang-Ying Lee for some technical support. We thank all Zhang lab members for helpful discussions. This work was supported by the National Key R&D Program of China (2022YFA1305000 to X.Z.), the National Natural Science Foundation of China (32270930 to X.Z.), the Beijing Natural Science Foundation (5232007 to X.Z.), and the Beijing Advanced Innovation Center for Genomics at Peking University.
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S.L. (Sha Luo), R.Q., and X.Z. designed the study; S.L. (Sha Luo), R.Q., X.S., S.L. (Shuchan Li), Z.Y., and A.M performed the experiments; H.Z., and L.Y. designed some of the bioinformatics pipelines; S.L. (Sha Luo), R.Q., H.Z., and X.Z. analysed the data; S.L. (Sha Luo), R.Q., H.Z., L.Y., and X.Z. prepared the figures with some input from Y.C. and Y-F.Z.; S.L. (Sha Luo), R.Q., H.Z., L.Y., and X.Z. wrote the manuscript. X.Z. managed and supervised the project.
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Luo, S., Qiao, R., Zha, H. et al. Chromatin-intrinsic mechanisms determine orientation-specific class switch recombination. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70031-z
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DOI: https://doi.org/10.1038/s41467-026-70031-z
