Chromatin-intrinsic mechanisms determine orientation-specific class switch recombination

Domain: chemistry
Published: 2026-02-28
Editorial outcome: major_revision

Abstract
Activation-induced cytidine deaminase (AID)-initiated immunoglobulin heavy chain (Igh) class switch recombination (CSR) replaces donor C constant region exons (C s) with a downstream acceptor C 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 C s under co-oriented transcription within Igh domain foster predominantly deletional joining-mediated productive CSR. In contrast, oppositely transcribed and short-distance C s 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.

Introduction
The functional efficacy of adaptive immunity is a defining feature of jawed vertebrates that equips them to counteract rapidly evolving pathogens (e.g., viruses, bacteria), which is crucially hinge on two synergistic processes: V(D)J recombination and class switch recombination (CSR)1. During B lymphocyte development, RAG endonuclease cleaves immunoglobulin heavy chain (Igh) V, D, and J gene segments to assemble a V(D)J exon adjacent to the C constant region exons (C s), encoding the default IgM antibody2,3. Upon B lymphocyte activation, Igh CSR replaces C with one of several downstream acceptor C s to change antibody isotypes and functions3,4. Each C unit contains an I-promoter, a long repetitive switch (S) region intron, and several C coding exons5. Chromatin loop extrusion6,7-mediated Igh 3’ regulatory region (3’RR) enhancer scanning activates transcription of acceptor C s and promotes S-S synapsis for recombination5,8-10. Activation-induced cytidine deaminase (AID)11 targets the transcribed donor S region and an acceptor S region to initiated deamination lesions12, which are converted to double-strand breaks (DSBs) and joined by DNA damage repair factors to accomplish CSR4,13-15. The Igh domain boundary termed 3’CBEs downstream of 3’RR, consisted of ten consecutive CTCF-binding elements (CBEs)16, safeguards loop extrusion-mediated transcriptional and CSR activities within Igh domain17,18.

Unlike the orientation-unbiased random joining of most chromosomal DSB ends genome-wide19-21, AID-initiated CSR within Igh domain employs an orientation-specific joining mechanism to generate productive antibody isotypes22. The mouse Igh domain contains six acceptor C s in the same transcriptional orientation as the upstream donor C, indicating that only deletional joining between donor S breaks and acceptor S breaks results in productive CSR10. DNA damage repair factors, such as ATM22-25, H2AX22,25,26, 53BP122,25,27,28, RIF122,25,29,30, Rev731,32, Shieldin33-37, and Ligase 425,38, have been found to promote deletional joining-mediated productive CSR by facilitating DSB tethering, suppressing DSB processing and decreasing microhomology (MH) of CSR junctions in mice. Previous studies implicate the DNA damage repair factors in enforcing the deletional joining mechanism, but do not implicate them in providing a basis for the orientation-specific CSR22.

Chromatin loop extrusion has been proposed to be the underlying mechanism for CSR in Mus musculus and Homo sapiens, which promotes the formation of dynamic CSR centers where acceptor S regions get activated and synapsed with donor S for CSR9,10,39. Moreover, chromatin loop extrusion has also been proposed to explain the predominantly deletional CSR in Mus musculus, supported by the increase of inversional joining from 3% to 19% during S CSR via inserting CBE-mediated chromatin loop extrusion blocks within Igh constant region in murine CH12F3 cells9,10. However, in some birds and reptiles, CSR must proceed by inversional joining to generate productive antibody isotypes due to the opposite transcriptional orientation of donor C and some acceptor C s40,41. The chromatin loop extrusion-mediated predominantly deletional CSR in mice and the structural diversity of constant regions across jawed vertebrates suggest that regulation of orientation-specific joining for productive CSR is more than previously documented9,10,22,41. Thus, detailed mechanisms need to be investigated to comprehensively explain the joining features of productive CSR with different Igh architectures throughout evolution.

In this study, by comprehensively analysing the constant region across a series of species, we revealed that the distinct chromatin features play critical roles in regulating productive CSR. We constructed series of murine CH12F3 cell mutants with distinct Igh configurations and found that switch topological configuration (STC) including transcription orientation, chromatin distance, and chromatin domain, determines orientation-specific joining of AID-initiated breaks for productive CSR.

Results
Intrinsic chromatin features affect end-joining of CSR
To investigate the potential influencing factors promoting productive CSR with different Igh architectures throughout evolution, we conducted a detailed comparative analysis of constant region across multiple species. The sequence feature analysis of S regions indicated the GC content within S regions was increased in evolution (Supplementary Fig. 1a). In addition, RNA-seq analysis showed an adaptive fixation of unidirectional transcription polarity and a progressive elongation of Igh constant region along the evolutionary lineage from birds to mammals (Supplementary Fig. 1b). Moreover, Hi-C analysis indicated an increasing conservation of constant region within a domain across the Osteichthyes-to-Mammalia transition (Supplementary Fig. 1c). We further manipulated the Igh constant region of murine CH12F3 cells to investigate the roles of chromatin configurations in orientation-specific CSR.

CH12F3 cells undergo robust CSR between S and S to generate predominantly deletional joining-mediated productive IgA antibodies upon CD40/IL4/TGF activation22,42. To eliminate the potentially confounding effects of non-productive Igh allele on the assessment of sequencing results, we employed Cas9/gRNA targeting to delete the entire non-productive Igh locus from upstream of the first V to downstream of 3’CBEs in CH12F3NC cells9, thereby generating CH12F3PO cells (Supplementary Fig. 2a, b). We placed the C unit next to the donor C with opposite transcriptional orientation by inverting the cluster of six acceptor C s from upstream of C3 to downstream of C in CH12F3PO cells to generate 3-Inv cells (Fig. 1a, Supplementary Fig. 2c, d). Then we employed high-sensitive CSR-HTGTS-seq to assay the joining features of CSR junctions (Fig. 1b) and found that 3-Inv cells exhibited predominantly deletional S3 CSR and a small but nonnegligible number of S CSR (Fig. 1c). Remarkably, in contrast to the predominantly deletional S CSR in CH12F3PO cells, S CSR underwent orientation-unbiased joining with 46% inversional joining of S-S junctions in 3-Inv cells without perturbing DNA damage repair factors (Fig. 1d, e).

We further used Pro-seq and 3C-HTGTS analyses to check the transcription status and chromatin interaction of Igh locus for revealing the underlying mechanisms. In contrast to the predominantly transcription of downstream C unit in CH12F3PO cells, 3- cluster inversion significantly activated transcription of downstream C3 unit and repressed upstream C transcription in 3-Inv cells (Fig. 1f-h, Supplementary Fig. 2e, f), suggesting that chromatin loop extrusion-mediated 3’RR scanning predominantly activates C3 unit instead of upstream C unit in 3-Inv cells. And the low transcription of C unit is corresponding to the low level of AID-initiated S CSR in 3-Inv cells (Fig. 1c, g). In addition, unlike the dominant interaction between C unit with iE and 3’RR in CH12F3PO cells, C3 unit interacted with iE and 3’RR within CSR center in 3-Inv cells, facilitating S-S3 synapsis-mediated predominant deletional joining of S-S3 junctions (Fig. 1c, f, g, Si, Supplementary Fig. 2e, f). Although the rarity of interactions between donor C and acceptor C unit limited S-S synapsis formation in 3-Inv cells, low-frequent short-range diffusion could promote orientation-unbiased random joining of S-S junctions, accounting for 46% inversional S CSR for generating productive IgA antibodies (Fig. 1d-i, Supplementary Fig. 2e, f). These data suggest that chromatin configuration including transcriptional orientation and chromatin distance of C s affects orientation-specific joining of CSR within Igh domain.

Transcription orientation of C s affects end-joining of CSR
Given that both transcriptional orientation and chromatin distance of C and C units were changed in 3-Inv cells by inverting the entire acceptor C region (Fig. 1a), we further inverted different acceptor C units individually to change the relative transcription orientation between donor C and acceptor C s. We firstly inverted C unit, the farthest downstream acceptor C from donor C in CH12F3PO cells, to generate Inv cells (Fig. 2a, Supplementary Fig. 3a, b). The results showed that Inv cells slightly increased inversional joining of S-S junctions from 4% to 8% (Fig. 2b-d). And this increase of inversional joining in Inv cells had little effect on the MH of S-S junctions (Supplementary Fig. 3c), suggesting a distinct regulation from DNA damage repair factor-deficiency-mediated increase of inversional joining during CSR. Then we took advantages of the activated CSR from upstream acceptor C s upon I deletion in I CH12F3 cells9. We inverted C2a unit in I cells to generate 2aInv cells (Fig. 2e, Supplementary Fig. 3d, e) and found that 2aInv cells significantly increased inversional joining of S-S2a junctions from 12% to 25% without affecting MHs (Fig. 2f-h, Supplementary Fig. 3f). We further inverted C1 unit in I cells to generate 1Inv cells (Fig.2i, Supplementary Fig. 3g, h) and found that 1Inv cells significantly increased inversional joining of S-S1 junctions from 5% to 43% without affecting MHs (Fig. 2j-2l, Supplementary Fig. 3i). We further inverted C unit in 3-Inv cells to generate 3-Inv-Inv cells (Fig. 2m, Supplementary Fig. 3j, k). Notably, 3-Inv-Inv cells significantly decreased inversional joining of S-S junctions from 43% to 18% without affecting MHs (Fig. 2n-p, Supplementary Fig. 3l). All these data indicate that transcription orientation play distinct roles from DNA damage repair factors in regulating orientation-specific joining of CSR with more inversional joining from two opposite transcribed C s.

Chromatin distance of C s affects end-joining of CSR
To further assess the effects of chromatin distance between donor C and acceptor C on orientation-specific joining of CSR, we changed the chromatin distance between C and C3 unit. We firstly inverted C3 unit in I cells to generate 3Inv cells, which have shorter distance between C and C3 (Fig. 3a, Supplementary Fig. 4a, b), and 3Inv cells significantly increased inversional joining of S-S3 junctions from 8% to 20% (Fig. 3b-d). Given 3Inv cells changed both chromatin distance and transcription orientation of C and C3 compared to I cells, we further inverted C3 unit in 3Inv cells to generate 3Inv-3Inv cells (Fig. 3a, Supplementary Fig. 4a, c). We found that 3Inv-3Inv cells decreased inversional S-S3 junctions from 20% to 14% compared to 3Inv cells (Fig. 3c-e), consistent with the conclusion that opposite transcriptional orientation of C s increases inversional joining of CSR (Fig. 2). Notably, compared to I cells, 3Inv-3Inv cells increased inversional S-S3 junctions from 8% to 14% (Fig. 3b, d, e), indicating that chromatin distance contributes to orientation-specific joining of CSR. We also deleted the intergenic region between CS and C3 unit directly to generate 3IRD cells (Fig. 3a, Supplementary Fig. 4d, e), and S3IRD cells significantly increased inversional joining of S-S3 junctions from 8% to 15% (Fig. 3b, d, f), exhibiting similar trend as 3Inv-3Inv cells. And this short-distance-mediated increase of inversional joining was not dependent on changing MH-related DNA damage repair pathways (Supplementary Fig. 4f). Altogether, these data indicate that chromatin distance of C s play roles in regulating orientation-specific joining of CSR with more diffusion-mediated inversional joining from two short-distance C H s.

Inverted I promoter-mediated block activates inversional CSR
To reveal the regulatory mechanism of transcriptional orientation in orientation-specific joining of CSR, we further fused the full S3 and S in CH12F3PO cells and Inv cells to generate I3-FS-C and I3-FS-I cells respectively (Supplementary Fig. 5a-c). In I3-FS-C cells, both S3 and S were transcribed under I3 promoter with the same transcription orientation as donor C, synapsed with donor C and underwent robust deletional S3 CSR and S CSR at similar levels (Fig. 4a, Supplementary Fig. 5d). However, in I3-FS-I cells, both S3 and S were transcribed under inverted I promoter instead of the upstream I3 promoter, suggesting that 3’RR enhancer scans to activate the closer promoter for transcriptional activation (Fig. 4b, Supplementary Fig. 5e). Compared to the robust synapsis between donor S and both S3 and S in I3-FS-C cells (Fig. 4a, Supplementary Fig. 5d), I3-FS-I cells exhibited major interaction between donor S with inverted I promoter and adjacent S, which blocked transcriptional activation of I3 promoter, leading to predominantly S CSR and less frequent S3 CSR (Fig. 4b, Supplementary Fig. 5e). Meanwhile, cohesin significantly accumulated at the inverted I promoter and this inverted I promoter with cohesin accumulation-mediated block effects increased inversional joining of S-S junctions from 8% to 19% and of less frequent S-S3 junctions from 10% to 36% (Fig. 4a, b, Supplementary Fig. 5f).

We also fused the partial S3 and S in CH12F3PO cells and Inv cells to generate I3-PS-C and I3-PS-I cells respectively (Supplementary Fig. 5a, g, and h). In I3-PS-C cells, both S3 and S were transcribed under I3 promoter with the same transcription orientation as donor C, synapsed with donor C and underwent robust S3 CSR and less frequent S CSR (Fig. 4c, Supplementary Fig. 5i). Compared to the robust S CSR in I3-FS-C and S3 CSR in I3-PS-C, the less frequent S CSR in I3-PS-C cells also suggest that AID targets more 5’S region for CSR (Fig. 4a, c). I3-PS-I cells exhibited a similar trend of transcription status, chromatin interaction, cohesin accumulation, and CSR as I3-FS-I cells (Fig. 4b, d, Supplementary Fig. 5e, j). The inverted I promoter in I3-PS-I cells dominated the transcription orientation, chromatin interaction, cohesin accumulation, and CSR with more diffusion-mediated inversional S-S and S-S3 junctions (Fig. 4d, Supplementary Fig. 5j). These results indicate that unidirectional transcription polarity within Igh constant region orchestrates 3’RR-mediated transcriptional activation, cohesin accumulation, and chromatin interaction to regulate loop extrusion- and diffusion-mediated end-joining of CSR.

Chromatin domains of Igh affect end-joining of CSR
To investigate the joining features of AID-initiated breaks located in different chromatin domains, we placed 3’RR and its associated C unit outside Igh domain by inverting the C-3’CBEs region in Inv cells to generate -3CBEInv cells, in which 3’RR and C are located in a different domain from donor C unit (Fig. 5a, Supplementary Fig. 6a, b). Remarkably, -3CBEInv cells significantly increased inversional S-S junctions from 8% to 53% without affecting MHs (Fig. 5b-d, Supplementary Fig. 6c). In contrast to the predominantly S-S synapsis for robust deletional S CSR in Inv cells (Fig. 5e, Supplementary Fig. 6d), C unit in -3CBEInv cells was isolated by the new topologically-associated domain (TAD) formed by the inverted 3’CBEs and downstream CBE, which prevented S-S synapsis even C unit was highly transcribed (Fig. 5f-j, Supplementary Fig. 6e). These data indicate that the donor C and acceptor C located in different TADs undergo diffusion-mediated orientation-unbiased end-joining of CSR.

Given that the above CSR occurs between classical donor S and acceptor S regions, we also wanted to investigate the general joining mechanism between AID-targeted classical S region and off-targets. Within Igh domain, chromatin loop extrusion-mediated 3’RR scanning activates C for CSR in CH12F3PO cells and scans upstream C s to activate their CSR activity upon the elimination of C competition in I cells9. In -3CBEInv cells, 3’RR activates C for CSR in a new TAD and we further deleted C unit in -3CBEInv cells to generate -3CBEInv- cells (Fig. 6a, Supplementary Fig. 7a, b). The data indicated that 3’RR activated an ectopic S (eS) region immediately downstream of the deleted C unit and donor S recombined, without orientation bias, to this eS region in -3CBEInv- cells (Fig. 6b-g). This eS region indicated a significant lower GC content than classical S and S regions (Supplementary Fig. 7c), corresponding to the less AID targeting frequency (Fig. 6b, c). These data indicate that AID-initiated on- and off-targets located in different domains promotes diffusion-mediated orientation-unbiased recombination.

Methods
Experimental procedures
No statistical methods were used to predetermine sample size and experiments were not randomized in this study. Investigators were not blinded to allocation during experiments and outcome assessment.

Cell culture
CH12F3 cells were cultured in medium R15 and stimulated with CD40 (1 μg/ml, eBioscience), IL4 (20 ng/ml, PeproTech), and TGF (0.5 ng/ml, R&D systems) for 72hrs. CSR-HTGTS-seq was performed in AID-proficient CH12F3 cells. Pro-seq, 3C-HTGTS, and ChIP-seq were performed in AID-deficient CH12F3 cells to obviate confounding effects of CSR-related genomic rearrangements.

Generation of mutant CH12F3 cell lines
gRNA design and vector construction: a Cas9/gRNA approach was employed to generate all the various mutant strains used in this study as described previously9. gRNAs were designed using the E-CRISP web tool (v5.4) with default parameters for SpCas9, targeting the Mus musculus genome. Candidate gRNAs were selected based on high predicted on-target efficiency scores (>80) and the absence of predicted off-target sites (with ≤3 mismatches) within protein-coding exons. gRNAs were individually cloned into the BbsI and BsaⅠ site of the p332 vector following the standard protocol. The specific sequences for all gRNAs used are listed in Supplementary data.

Nucleofection and clonal selection: to generate mutant clones, 2 × 10⁶ CH12F3 cells were nucleofected with 2 µg of p332-based Cas9/gRNA plasmids containing two gRNA sequences using the Lonza 4D-Nucleofector™ system (Solution SF, program CA-137). Twenty-four hours post-nucleofection, single cell subcloning was performed by fluorescence-activated cell sorting (FACS) on a BD FACS ARria™ SORP cell sorter to isolate one cell into one well of 96-well plates. Genomic DNA from individual clones was subsequently extracted for PCR-based genotyping and sequencing to confirm the desired genetic modification. Two pairs of genotyping primers were designed on either side of the two break sites for each mutant. Different combinations of these four primers can be used for PCR genotyping to detect WT bands, deletion bands, and inversion bands. The detailed primer pairs for genotyping were described in the corresponding figure legends. Based on the PCR genotyping and sequencing results, we can only detect WT bands for the control cells and positive bands (deletion or inversion) for the mutant clones. Detailed primers for selecting various mutant CH12F3 cell lines are listed in Supplementary data.

CH12F3PO cells were generated by deleting the entire non-productive Igh locus from upstream of the first V to downstream of 3’CBEs in CH12F3NC cells9 and confirmed by PCR genotyping and sequencing. 3-Inv cells were generated by inverting the six acceptor C s region from upstream of C3 to downstream of C in CH12F3PO cells and confirmed by PCR genotyping and sequencing. Inv cells were generated by inverting the C unit in CH12F3PO cells and confirmed by PCR genotyping and sequencing. We generated the I cells by deleting I promoter in CH12F3 cells in the previous study9. aInv cells were generated by inverting the C2a unit in I cells and confirmed by PCR genotyping and sequencing. Inv cells were generated by inverting the C1 unit in I cells and confirmed by PCR genotyping and sequencing. 3-Inv-Inv cells were generated by inverting the C unit in 3-Inv cells and confirmed by PCR genotyping and sequencing. Inv cells were generated by inverting the C3 unit in I cells and confirmed by PCR genotyping and sequencing. 3-Inv-3Inv cells were generated by inverting the C3 unit in 3-Inv cells and confirmed by PCR genotyping and sequencing. 3IRD cells were generated by deleting the intergenic region between C3 and C to position C3 ~17kb away from C in I cells and confirmed by PCR genotyping and sequencing. I3-FS-C cells were generated by fusing the entire S3 with S in CH12F3PO cells and confirmed by PCR genotyping and sequencing. I3-FS-I cells were generated by fusing the entire S3 with S in Inv cells and confirmed by PCR genotyping and sequencing. I3-PS-C cells were generated by fusing the partial S3 with S in CH12F3PO cells and confirmed by PCR genotyping and sequencing. I3-PS-I cells were generated by fusing the partial S3 with S in Inv cells and confirmed by PCR genotyping and sequencing. -3CBEInv cells were generated by inverting the -3’CBEs region in Inv cells and confirmed by PCR genotyping and sequencing. -3CBEInv- cells were generated by deleting the C unit in -3CBEInv cells and confirmed by PCR genotyping and sequencing. The corresponding AID-deficient cells were generated by deleting the Aicda gene from the above cells and confirmed by PCR genotyping and Western blotting. At least two independent clones were obtained for each derivative mutant genotype.

Chromatin feature analyses of Igh locus across species
Reference genome assemblies used in this paper included Danio rerio (GRCz10), Xenopus tropicalis (UCB_Xtro_10.0), Gallus gallus (GRCg7w), and Mus musculus (mm9). Alligator sinensis was derived from Pan, T et al53. Splicing regions in Mus musculus have been well-annotated. For other species, we aligned the constant region sequences obtained from IMGT54. The splicing regions were defined as 4kb upstream of the first exon of the constant regions. Splicing region sequences were extracted by bedtools (v2.31.1) and the GC contents were calculated with seqkit (v2.8.2). Background GC content was derived genome-wide by sliding 4-kb windows.

Reads of RNA-seq from all species53,55-58 were aligned to their respective reference genomes using STAR (v2.7.11a). Strand-specific bigwig files were generated with deeptools (v3.5.3) and strand orientation was defined from the alignment described above.

All Hi-C data53,59-61 were processed with HiC-Pro (v3.1.0) under default settings, except the LIGATION_SITE was species-specific, to generate valid interaction pair files. The output pair files were then converted to .hic files using the hicpro2juicebox utility. Hi-C files were also converted to .cool files using Hicexplorer (v3.7.2). Except for Gallus gallus, all species were analyzed at 25 kb resolution. Gallus gallus was analyzed at 5 kb resolution because the Igh locus is located on the microchromosome. Insulation scores and TADs were calculated with cooltools (v0.7.1) with a window size of 7 bins. All visualizations were generated by pyGenomeTracks (v3.8).

CSR-HTGTS-seq library preparation and data analysis
CSR-HTGTS-seq libraries with 5’S bait were prepared from different CH12F3 mutants stimulated with CD40/IL4/TGF for 72hrs as described previously22. Briefly, 25 g gDNA from CD40/IL4/TGF-stimulated CH12F3 cells was sonicated on Covaris M220 sonicator. The sonicated DNA segments were amplified by LAM-PCR with biotinylated 5’S primer. The amplified biotin-labeled LAM-PCR products were enriched with streptavidin C1 beads (Thermo Fisher Scientific, #65001) for 4hrs at room temperature, followed by adaptor ligation with the following PCR program: 25 ℃ 1hr, 22 ℃ 3hrs, 16 ℃ overnight. The adaptor-ligated products were subjected to nested-PCR with barcode primers and followed by tag-PCR with Illumina platform-matched P5-I5 and P7-I7 primers. 500-1000bp tag-PCR products were selected by separation on 1% TAE gel. CSR-HTGTS-seq libraries were sequenced by paired-end 150bp on a NovaSeq 6000 (Illumina) sequencer.

Libraries were processed via the published pipeline and mapped against the mm9 genome or corresponding modified genome22. Data were analyzed and plotted after removing the duplicates as described22. Each experiment was repeated at least three times from at least two independent clones. To plot the MH pattern, direct joins (MH=0) and junctions with MH were pooled and sorted by length, and the number of junctions with indicated length of MH were counted and calculated as percentage of the total number of junctions mapped to the region of interest. Primers used for CSR-HTGTS-seq are listed in Supplementary Table 1.

3C-HTGTS library preparation and data analysis
3C-HTGTS analyses were performed as previously described on AID-/- CH12F3 cells9 stimulated with CD40/IL4/TGF for 72hrs. Briefly, 10 million cells were crosslinked with 2% formaldehyde for 10 minutes at room temperature and quenched with glycine at a final concentration of 125 mM. Then, the crosslinked cells were lysed in the 3C lysis buffer and nuclei were digested with NlaIII enzyme (NEB, R01I25) at 37 ℃ overnight. The digested samples were ligated by T4 DNA ligase (NEB, M1801E) at room temperature for at least 6hrs. The ligated products were de-crosslinked with Proteinase K (Roche, #03115852001) at 56 ℃ overnight and the 3C templates were purified by phenol/chloroform. The 3C templates were used for making 3C-HTGTS with different baits including the iE-I, 3’RR(HS4) or 3’CBEs locale. Besides the DNA templates and baits, the 3C-HTGTS library preparation procedures were similar as CSR-HTGTS-seq described above.

The 3C-HTGTS libraries were then sequenced by paired end 150 bp sequencing on a NovaSeq 6000 (Illumina) sequencer and data were processed as previously described9. Each experiment was repeated from at least two independent clones. Before plotting the data for comparison, libraries were size-normalized to total junctions of the smallest library in the set of libraries for comparison. For statistical analyses, we counted the number of junctions within the indicated bait-interacting locals for each sample. For bar graph presentations in Fig. 1 and Fig. 5, the junction number from control samples was normalized to represent 100% and relative experimental values listed as a percentage of the control values. Primers used for 3C-HTGTS are listed in Supplementary Table 1.

Pro-seq library preparation and data analysis
Pro-seq libraries were prepared as described previously62 from AID-/- CH12F3 cells stimulated with CD40/IL4/TGF for 72hrs. Briefly, 10 million cells were collected and permeabilized with permeabilization buffer. The permeabilized cells were resuspended in 100 l of storage buffer for nuclear run-on with 2X run-on mix at 37℃ for 5 mins. RNA was extracted by Trizol and followed by hybrolyzation. The RNA was incubated with streptavidin C1 beads (Thermo Fisher Scientific, #65001) and the enriched run-on samples were incubated with hydroxyl repair with T4 PNK (NEB, M0201S) and RppH (NEB, M0356S), followed by ligating the 5’ and 3’ RNA adaptor. RT-PCR was performed from the adaptor-ligated RNA to obtain cDNA. The cDNA was subjected to making Pro-seq libraries by two rounds of PCR with barcode primers. 200-500 bp products from the first round of PCR were subjected to the second round of PCR with the number of PCR cycles determined by test PCR amplification. The second round of PCR products were size-selected by SPRIselect beads (Beckman Coulter, B23318). Pro-seq libraries were sequenced via single end 50 bp sequencing on a HiSeq2500 (Illumina) sequencer or paired end 150bp sequencing on a NovaSeq 6000 (Illumina) sequencer. The processed reads were mapped to the mm9 genome or corresponding modified genome using Bowtie2 software. The read coverage for each sample was normalized to a coverage of 10 million 100nt reads for display. Each experiment was repeated three times from at least two independent clones.

ChIP-seq library preparation and data analysis
ChIP-seq was carried out in AID-deficient cells stimulated with CD40/IL4/TGF for 72 hrs based on a prior protocol63 with some modifications. Briefly, 20 million cells were collected and fixed with 1% formaldehyde for 10 mins at room temperature, and quenched with glycine at a final concentration of 125 mM. Cells were lysed and sonicated with a Covaris M220 sonicator for 5-8 min until the size of most fragments was in the range of 200–700 bp. The fragmented chromatin was incubated with Rad21 antibody (ab992, abcam) overnight, followed by incubating with magnetic protein A (Invitrogen, 10002D) for 4hrs at 4 °C. The enriched chromatin was used for tagmentation, followed by decrosslinking and elution. Purified DNA was subjected to make ChIP-seq libraries. The amplified libraries were purified by SPRIselect beads (Beckman Coulter, B23318) and were sequenced via paired end 150bp sequencing on a NovaSeq 6000 (Illumina) sequencer. The processed reads were mapped to the modified genome using Bowtie2 software. The uniquely aligned reads were retained, while duplicate reads and those located in blacklist regions were excluded prior to further analyses. The read coverage for each sample was normalized by RPKM.

Quantification and statistical analysis
Statistical analyses for CSR-HTGTS-seq, 3C-HTGTS, and Pro-seq between two samples were performed via two-tailed, unpaired or paired Student’s t-test. At least three biological repeats were done for each statistical analysis. p < 0.05 is considered significant. P value shown in the bar graphs in the main and Supplementary Figures. The statistical analyses were also described in the corresponding Figure legends and Supplementary Figure Legends. Unless indicated otherwise, all agarose gel electrophoresis images are representative of at least two independent experiments, with uncropped gels shown in the Source Data file.

Data availability
CSR-HTGTS-seq, 3C-HTGTS, Pro-seq, and ChIP-seq sequencing data analyzed here has been deposited in the GEO database. The GEO accession number for CSR-HTGTS-seq is GSE299392 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE299392]. The GEO accession number for 3C-HTGTS is GSE299499 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE299499]. The GEO accession number for Pro-seq is GSE299975 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE299975]. The GEO accession number for ChIP-seq is GSE299391 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=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 Figures. 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 were aligned to either 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 RseqQ (v.5.0.1) (https://rseqc.sourceforge.net/) package. ChIP-Seq were 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/).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Luo et al examine how transcriptional orientation determines the relative frequencies of inversional vs deletional end-joining of DNA double strand breaks during the completion phase of class switch recombination. It has been known for years that AID-initiated DSBs within the Igh locus underdo orientation-specific end-joining and while both inversional vs deletional end-joining can theoretically occur, end-joining in mice proceed through deletional recombination to mediate productive CSR. However, in some species such as birds and reptiles, CSR proceeds via inversional recombination. The mechanisms that promote orientation-specific end-joining are poorly defined. This manuscript have genetically manipulated the mouse lymphoma line CH12 to examine how transcriptional orientation and distance between the recombining elements dictate orientation of AID-initiated double strand breaks.\nThe authors have generated a huge number of mutated CH12 lines-from inverting the entire region between Ig3 and Ca to inverting each of the constant region segments individually. Then they carry out high-throughput CSR sequencing (HTGTS-CSR) to examine the switch junctions. They find that co-oriented transcription within Igh promote deletional recombination when the recombining elements are further apart, while oppositely transcribed and short-distance recombination promote inversional end-joining, albeit with lower frequency. The manuscript is a tour-de-force of genetic manipulation of the CH12 cell line, with high quality data.\nThe major issue of the manuscript is the dense description of the study, which the authors should fix. Second, the authors should provide a better description of the strategy they used to invert the entire locus-they mention using CRISPR/Cas9 but the methods are missing details of how this was achieved. Third, they make some blanket statements regarding inversional switching occurring via diffusion-dependent mechanism without providing any data to support this idea. Finally, the authors should examine how the DNA repair proteins, especially 53BP1 which has previously been shown to effect orientation, participate in the process. This is especially important since all the studies here have been carried out in a cell line, and while it is difficult to carry out the analysis in primary mouse B cells, using known effectors can provide additional support for the generality of their models.", "reviewer_2": "The studies reported in this the manuscript reveals that chromatin topology including transcriptional orientation, distance between constant regions, and chromatin domain organization is key to orientation-specific productive class switch recombination (CSR) in the immunoglobulin heavy chain locus. As largely deletional oriented CSR end-joining occurs in mouse and human cells based on a well-known cohesin mediated loop extrusion based end-joining mechanisn, the key contribution of this paper is to elucidate the mechanism by which certain other organisms are able to employ an inversional CSR mechanism. To test the potential determinants of such a mechanism, authors modified the the CH locus in the mouse CH12 cell line, which undergoes CSR in culture, to mimic chromatin features of IgH locus in different species that undergo inversional CSR. Analysis of CSR in the modified CH locus CH12 line, including detailed interrogation of transcription, chromatin distance and chromatin topological association, revealed general principals of how intrinsic chromatin features could influence the occurrence of inversional CSR which provides insight into the interesting question of how convergent switch region n orientation in some of vertebrate species can still efficiently promote functional CSR. The study provides significant mechanistic insight into how intrinsic chromatin architecture promote can promote productive CSR of Igh CH units in other species hat are in opposite orientation to those of mice and humans in other species. The study represents a significant advance to the field and as such,should be published , once the authors address the questions and comments below, most of which just require rewriting as opposed to new experiments.\nComments:\n1. The introduction and discussion is not clearly written and misses important work in the field that set the stage for this current interesting new study. The introduction introduces lists a series of papers that the authors suggest have implicated certain DSB response and end-joining factors in playing major role mechanism of deletional-orientation dependent CSR in mice and humans. However, these papers at most implicate these factors in enforcing the deletional mechanism but do not implicate them in providing a basis for the deletional joining mechanism as clearly reported in the main study referenced in this section (Dong, et al, Nature 2015, reference 22 in the manuscript). The authors should carefully review the conclusions of Dong et al., as well as those of Zhang et al. Nature 2019, (also cited, reference 9). Beyond that they should also read carefully the Zhang et al.2022 Nature Reviews in Immunology (reference 10 in manuscript) section that reviews the CSR mechanism and proposes the model that their study now elegantly proves. The suggestion here is that the authors better incorporate the studies on the role of cohesin-mediated chromatin loop extrusion in CSR and CSR center formation and it activity in promoing deletional CSR into their introduction. These revisions will not in any way detract from the important new findings described in this manuscript. It would just put them into a more appropriate context for the readers and further enhance the significance of their important new studies.\n2. Comparing Figure 2n and Figure 3c, when placed adjacent to Cμ in an inverted orientation, Cα shows 43% inversional joining, whereas Cγ3 shows only 20%. In addition, when placed next to Cμ in the normal orientation, Cα and Cγ3 also show similar inversional joining percentages (Figures 2o and 3e–f). C It would be helpful if the authors commented on their interpretation of the reason for this difference.\n4. Some of the alteration of switch regions have significant effects to the absolute CSR levels, could the authors discuss the underlying mechanisms? And could these effects confound any of the CSR orientation analyses? Also, could the authors comment on whether or not transcription levels might affect the CSR orientation?\n5. Figure 1: The authors state that, because the acceptor Ca unit in chicken is located closer to the donor Cμ and has an opposite transcription orientation, they inverted the CH region in mouse CH12F3 cells. However, Ca is not transcribed after inversion. Therefore, the statement that “ 3- Inv cells significantly increased inversional joining-mediated productive CSR, which can explain how IgA antibody is generated in chicken” appears to be inaccurate and should be clarified or revised.\nMinor\n1. Figure1: Could the authors please explain their interpretation of how the whole CHs region inversion increases the RC and 3’RR interaction?\n2. Figure 1: The authors examine the importance of chromatin configuration including transcriptional orientation and CH region distance in orientation-specific CSR by inverting the entire CH region. However, the most downstream S 3 predominantly undergoes deletional joining with Sμ despite its opposite transcription orientation relative to Cμ. The authors could further discuss this observation and his the contest of their model.\n3. In Figure 5, why is there no CSR to other CHs in the two indicated lines?\n4. Figure 7: A more detailed figure legend is needed to improve readability and interpretation of the data. In particular, the left panel requires clarification — please explain what the different colors denote.", "reviewer_3": "In this study, Luo et al. examined the orientation-specific class switch recombination (CSR) in the altered chromatin context. Through a series of elegant genetic manipulations, the authors changed the transcription orientation of the acceptor CH unit (relative to the donor), shortened the distance between the recombination partners, or separated them into different loop domains. All these changes led to increased levels (to various degrees) of inversional recombination. This study is a nice extension of previous works on the orientation specificity of CSR and the loop extrusion model. All data were generated by cutting edge technologies and rigorous approaches. These findings contribute to our knowledge of CSR and chromatin structures and dynamics. It should be of great interest to a broad readership.\nOne perceived weakness is that the manuscript lacks a coherent model to explain on how distance and transcription orientation affect del:inv recombination. The authors should elaborate (or at least speculate) on the possible mechanisms (e.g., within the framework of loop extrusion). The authors probably should acknowledge that CSR is still predominantly deletional even when the acceptor CH orientation is inverted, and perhaps comment on whether this is expected or unexpected and why.\nSpecific points that the authors may want to address:\n1. In the γ3-αInv cells, Sa transcription is negligible, it is difficult to appreciate what the ratio of del:inv (Fig. 1d, e) really means. It’s perhaps more appropriate to examine the Sγ3 region where transcription and 3D interactions are clearly abundant. These data can then be compared to those depicted in Fig. 2.\n2. Pro-seq and 3C-HTGTS data are not included for any of the cell lines used in Fig. 2. Those data are important for evaluation of the effects caused by changes in transcription and 3D interactions.\nMinor points:\n3. It is interesting to see two peaks (CSR-HTGTS-seq) at the fused Sγ3-Sα region in Iγ3-FS-Cα cells (Fig. 4a). Is there a plausible explanation for the dip at the Sγ3-Sα junction? Is it because of non-S DNA sequences at that position? It is important to determine the splicing pattern of the Sγ3-Sα fusion region which may shed light to the molecular basis of the two peaks.\n4. The Pro-seq data of Fig. 4a and 4b are quite interesting. In Fig. 4a, the Pro-seq signals are mostly mapped to Sα, and few to Sγ3, yet switch junctions are mapped comparably to both switch regions. Any explanation (speculation)?\n5. Fig. 1f, mislabeling of Sµ vs. Cµ."}, "author_response": "Response: Thanks for the reviewer’s careful review and comment.\nWe also thank this reviewer for pointing out the key question that we want to solve in the field and the experiments that we have done to answer the question.\n[...]\n(Full author response text included in the provided document)"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have satisfactorily addressed the issues raised. This revised manuscript is a much improved version from the original submission.", "reviewer_2": "As in my first review, I found this manuscript to be important for the field and related fields and mentioned why. The methodology is sound and probably exceeds standards for the field . I mentioned some comments, mostly minors for improving the presentation . In reading the response to my review and reading the changes to the text in response to my review, I am fully satisfied that the authors addressed my comments in the reivsion and see no need for further revision in response to my review. This is a terrific paper.", "reviewer_3": "The authors addressed most of my questions and the manuscript is suitable for publication. Yet, I don't quite understand how the RNAseq data in Fig. R4b address the question on RNA splicing. However, this is of minor concern."}, "author_response": "Response: Thanks for the reviewer’s positive feedback and we are pleased to know that the reviewer find our revised manuscript to be a much-improved version.\nResponse: Thanks for the reviewer’s positive assessment of our work and for emphasizing the importance of this work.\nResponse: Thank for the reviewer’s positive feedback and we are pleased that the reviewer stated “the manuscript is suitable for publication”.\nFor the new RNA-seq experiments, our main goal in performing this experiment was to address the reviewer's original question regarding the potential transcriptional activity and splicing pattern of the fused Sγ3–Sα region to shed light to the molecular basis of the two peaks (CSR-HTGTS-seq).\nWe observed robust RNA-seq reads mapping to the Cα exons, but very few or no reads spanning the Sγ3–Sα fusion region. This result suggests that: the locus is transcriptionally active. However, the fused Sγ3–Sα region does not appear to be efficiently incorporated into a stable, spliced mRNA product. This finding indirectly supports our interpretation that the two CSR peaks detected by CSR-HTGTS-seq (Fig. 4a) reflect recombination hotspots within the genomic DNA of the core S regions, rather than being a product of RNA splicing or transcript mapping.\nSo, the dip at the Sγ3-Sα junction reflects the lack of core S region sequences with high AID targeting motifs at this specific genomic location."}]

Decision letter