Abstract
RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a JH-recombination signal sequence (RSS) within a recombination centre (RC) and then linearly scanning upstream chromatin, presented by loop extrusion mediated by cohesin, for convergent D-RSSs1,2. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning3. Scanning of RAG from the DJH-RC-RSS to upstream convergent VH-RSSs is impeded by D-proximal CTCF-binding elements (CBEs)2,3,4,5. Primary progenitor B cells undergo a mechanistically undefined contraction of the VH locus that is proposed to provide distal VHs access to the DJH-RC6,7,8,9. Here we report that an inversion of the entire 2.4-Mb VH locus in mouse primary progenitor B cells abrogates rearrangement of both VH-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both the utilization of cryptic VH-RSSs that are normally in opposite orientation and RAG scanning beyond the VH locus through several convergent CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary progenitor B cells promotes RAG scanning of the VH locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL)10, a cohesin-unloading factor, was low in primary progenitor B cells compared with v-Abl-transformed progenitor B cell lines that lacked contraction and RAG scanning of the VH locus. Correspondingly, depletion of WAPL in v-Abl-transformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.
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Data availability
HTGTS-V(D)J-seq, 3C-HTGTS, ChIP–seq and GRO-seq data reported in this study have been deposited in the GEO database under the accession number GSE151910. Source data are provided with this paper.
Code availability
HTGTS-V(D)J-seq, 3C-HTGTS, ChIP–seq and GRO-seq data were processed through the published pipelines as previously described14. Specifically, these pipelines are available at http://robinmeyers.github.io/transloc_pipeline/ (HTGTS pipeline), http://bowtie-bio.sourceforge.net/bowtie2/index.shtml (Bowtie2 v.2.2.8) and https://sourceforge.net/projects/samtools/files/samtools/1.8/ (SAMtools v.1.8).
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Acknowledgements
We thank Alt laboratory members for contributions to the study, particularly H.-L. Cheng for advice and help with the embryonic stem cell culture, M. Tian for the EF1 embryonic stem cell line, Y. Zhang for the RAG-expressing retrovirus plasmids, S. Jain for help with cell culture and data analysis, R. Judson and N. Manfredonia for help with the blastocyst injection, and J. Hu for data uploading; as well as E. Bekman at the University of Lisbon for advice on the construction of the degron system, particularly on the inducible expression of OsTIR1. This work was supported by NIH R01 AI020047 (to F.W.A.). H.-Q.D. is a fellow of the Cancer Research Institute (CRI) of New York. H.C. was an NRSA Fellow (T32 AI07386) and was supported by the Leukemia and Lymphoma Society. Z.B. was supported by a CRI fellowship. R.C. is partially funded by the NIH Regulome Project. C.-S.L. was previously supported by a CRI fellowship and is now funded by the Ministry of Science and Technology in Taiwan (MOST109-2636-B-007-004). F.W.A. is an investigator of the Howard Hughes Medical Institute.
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Authors and Affiliations
Contributions
H.-Q.D., C.-S.L. and F.W.A. designed the studies. R.C. and A.C. contributed to the design of some experiments. H.-Q.D. and H.H. performed most of the experiments with important contributions from J.L., C.-S.L. and X.Z. Z.B. and S.L. established the RAG1-deficient, Eμ-Bcl2-expressing C57BL/6 v-Abl pro-B line. H.S.Y. established the homozygous 3′Igh-deleted embryonic stem cells. A.Y.Y., N.K. and H.C. designed the bioinformatics pipelines for data analysis and applications. A.Y.Y. performed the statistical analyses of data. A.M.C.-W. and K.J. performed the embryonic stem cell analyses and blastocyst injections. H.-Q.D., H.H. and F.W.A. analysed and interpreted the data, designed the figures and wrote the manuscript with important input from A.Y.Y. for statistical analyses. R.C., J.L., A.Y.Y., Z.B., L.Z., Y.Z., X.Z. and A.C. provided insights and helped polish the manuscript. F.W.A. supervised the study.
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F.W.A. is a co-founder of Otoro Biopharmaceuticals. The remaining authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Generation and characterization of the IgH VH locus inversion mouse model.
a, Schematic diagram showing CRISPR-Cas9-mediated entire Igh VH locus inversion upstream VH81X in embryonic stem cells (ES cells) on the Igh allele in C57BL/6 genetic background. Cut1 and Cut2 showing the location of 2 sgRNAs. Details as shown in Fig. 1a. b, Confirmation of the upstream and downstream inversion junctions by Sanger sequencing. The sgRNA-targeting sequence is underlined, and the PAM sequence is labelled in red. sgRNAs and oligos used are listed in Supplementary Table 6. c, Schematic showing the generation of Igh VH locus inversion mouse model and further assays for phenotype and mechanism analyses. d, e, Representative flow cytometry analysis of IgM- bone marrow (BM) B cell populations in 4~6-week-old WT (d) and Igh VH locus inversion (e) mice. B220+IgM- B cells were gated and shown in the left plot (d, e). The black arrow lines indicate the gating strategies. B220+CD43+ pro-B and B220+CD43- pre-B cell populations are indicated in the right plot (d, e).
Extended Data Fig. 2 RAG utilization of cryptic RSSs across normal and inverted Igh loci in primary pro-B cells.
a, 6 examples (labelled in Fig. 2b, I-VI) of RAG off-target peaks in WT (repeat #1, middle) and Igh VH locus inversion (repeat #1, bottom) primary pro-B cells plotted at single-base-pair resolution. Top panels are WT 2.4 Mb Igh locus and upstream 4 Mb region track. For comparison, the sequence of RAG off-target peaks (III-VI) in the inverted region are shown in inverted orientation. b, c, RAG off-target junction profiles at Igh locus and upstream 4 Mb region in WT (repeat #2, #3, middle) and Igh VH inversion (repeat #2, #3, bottom) primary pro-B cells. For comparison, RAG off-target junction profiles in the inverted region are shown in normal orientation (c). The same regions of RAG off-target peaks (shown in a) are highlighted with pink lines (b, c). The Igh VH inverted region is highlighted in transparent green (WT, a, b) and transparent gold (Igh VH inversion, a, c), respectively. Del (+) indicates deletional junction. Inv (-) indicates inversional junction. d, Normalized RC-3C-HTGTS reads ± s.d. of WT (n = 3) and INV (n = 3) in entire upstream Igh locus and J558/3609 VH region are shown. See Supplementary Data 1 for individual repeats of RC interactions with whole Igh locus and entire upstream Igh locus. Indicated P values were calculated using unpaired two-tailed t-test. n, number of independent experiments.
Extended Data Fig. 3 The major RC interactions, transcription patterns and binding of key looping factors are maintained in the inverted Igh loci in cultured RAG1-deficient primary pro-B cells.
For comparison, all the data in the inverted region are shown in normal orientation. Related to Fig. 3b. Zoom-in profiles of 3C-HTGTS, GRO-seq, RAD21/CTCF ChIP-seq signals for ± 10 kb regions of 15 representative peaks/clusters in Fig. 3b from WT (blue) and Igh inversion (INV, Red) cultured RAG1-deficient primary pro-B cells along with relevant bona fide CBE motif sites are shown, data are presented as average signal counts ± s.e.m., PAIR elements (green bars) that located in each peak were also shown above. 3C-HTGTS: WT, n = 3; INV, n = 3. GRO-seq: WT, n = 4; INV, n = 4. RAD21 ChIP-seq: WT, n = 3; INV, n = 3. CTCF ChIP-seq: WT, n = 3; INV, n = 3. n, number of independent experiments. A similarly detailed view of these parameters across the entire VH locus is shown in Supplementary Data 2. Discussion, overall these patterns indicate that proximal 7183/Q52 highly rearranged VHs are not transcribed but frequently have CTCF-bound closely associated CBEs; whereas distal J558/3609 highly rearranged VHs are often transcribed and/or have CBEs within 5kb that are CTCF-bound, which in cases where both occur may cooperate to enhance rearrangement. In the J558 and Middle VH domains, some highly rearranged VHs are transcribed and/or have associated CTCF/cohesin-bound CBEs; but some highly rearranged VHs in these regions appear to have neither, suggesting other unknown mechanisms for their RAG-targeting during scanning. In some latter examples we cannot rule out very low level sense and/or anti-sense transcription as having a contributory role; since the threshold for transcriptional targeting of RAG scanning activity has not been determined. Overall, these patterns are somewhat similar to those discussed in recent report on study involving CTCF-depletion in v-Abl cells2.
Extended Data Fig. 4 Cohesin/CTCF binding patterns and germline VH transcription patterns are shown in normal and inverted Igh loci in cultured RAG1-deficient primary pro-B cells.
Related to Fig. 3b, Supplementary Data 2. a, b, For comparison, the ChIP-seq data in the inverted locus are shown in normal orientation. Average signal counts ± s.e.m. of RAD21 (a) / CTCF (b) ChIP-seq across the 4 VH domains as indicated in WT (upper, blue, n = 3) and Igh VH locus inversion (bottom, red, n = 3) cultured RAG1-deficient primary pro-B cells. n, number of independent experiments. c, Average signal counts ± s.e.m. of GRO-seq profiles across the 4 VH domains in WT (upper, n = 4) and Igh VH locus inversion (bottom, n = 4) cultured RAG1-deficient primary pro-B cells. n, number of independent experiments. For comparison, the GRO-seq results in the inverted locus is shown in inverted orientation (c, bottom). The WT and inverted VH locus/domains with PAIR elements are diagrammed at the top of each panel. Both the sense and antisense transcription are relative to the entire Igh VH locus upstream VH81X with or without inversion and indicated, respectively. For comparison, 15 representative major interaction peaks/clusters as shown in Fig. 3b were indicated with colour shades and numbers. The Igh VH inverted region is highlighted in transparent green (WT) and transparent gold (Igh VH inversion), respectively. See Supplementary Data 2 for more details.
Extended Data Fig. 5 The 3′CBEs are not required for VH utilization.
a, Diagram of the entire mouse Igh locus with or without ten 3′Igh CBEs, details as shown in Fig. 1a. b, Schematic of the strategy to generate the mouse ES cells with entire 3′CBE locus deletion. Ten 3′Igh CBEs are shown in grey peaks on the top panel. LoxP sites flanking the pGK-Neo selection marker are shown as black triangles. Red filled boxes ‘A’ and ‘B’ indicates the left and right homology arms, respectively, flanking all ten 3′Igh CBEs. c, Gel images show the confirmation results of two independent 3′CBE-deleted ES cell clones, 3′CBEsΔHOMO #1 and #2 (n = 2 biologically independent repeats with similar results). P1-P3 are the genotyping primers and listed in Supplementary Table 6. See Methods for more details. d, Average utilization frequencies ± s.d. of all VH segments in WT primary pro-B cells (n = 6). VH usage data were extracted from Ba et al.2 (GEO: GSE142781). n, number of independent experiments. e, f, Average utilization frequencies ± s.d. of all non-productive (NP) VH segments in splenic B cells from WT (e, n = 3) and 3′CBE deletion (f, n = 3) mice, respectively. n, number of independent experiments. All the data are from 129SV background mice (d–f). Prior studies show that non-productive allele rearrangement patterns of splenic B cells are highly correlated with those productive and non-productive patterns in primary pro-B cells5. See Supplementary Table 3 and Methods for all further details of this analysis. Discussion, termination of loop extrusion-mediated VH locus RAG-scanning within the VH locus in normal primary pro-B cells may be largely impeded by bona fide convergent VH-RSS-mediated rearrangements. In this regard, inversion of the VH locus would eliminate any potential contribution of VH-RSS-mediated contributions. Beyond this, such termination of RAG-scanning also may be contributed by cooperative activities of highly frequent distal VH locus scanning impediments including active transcription sites and CBEs. Due to the number of such impediments, and the possibility that their activity may be collective, assessing their functions in this context may be difficult via targeted mutational analyses. However, our findings of differential effects of the inversion on proximal versus distal VH locus CBEs interactions with convergent 3′CBEs in the VH inverted locus provides indirect support of such a potential role for distal CBEs (Fig. 3c, bottom).
Extended Data Fig. 6 Generation and characterization of RAG1-deficient WAPL-degron v-Abl cell lines.
a, Scatter plots of average transcriptome-wide GRO-seq counts in G1-arrested v-Abl cells (x axis, n = 3) and primary pro-B cells (y axis, n = 4). n, number of independent experiments. Representative known requisite genes implicated in the cohesin-complex function for V(D)J recombination and chromatin interactions are highlighted by red circles and blue arrows. Representative known genes implicated in the DNA repair and B cell development were also analysed to determine if there were any potential transcriptional defects in these essential genes for V(D)J recombination and none were found and highlighted. Analyses of scatter plots indicate that Wapl is expressed at significantly higher levels in G1-arrested v-Abl cells than in primary pro-B cells (Spearman’s correlation coefficient (rho) and P values determined by two-sided Spearman’s correlation test are presented). These transcription finding were confirmed by western blotting studies (Fig. 4a). b, Comparison of Wapl transcription levels by GRO-seq in primary pro-B cells and G1-arrested v-Abl cells from 4 and 3 independent repeats, respectively. Data are presented as average signal counts ± s.e.m. of GRO-seq. n, number of independent experiments. Although other genes upstream and downstream of the Wapl gene show altered transcription in primary pro-B cells compared to the v-Abl cells, their products thus far have not been implicated in loop extrusion or chromatin scanning. c, Schematic of the targeting strategy for introducing Tet-On OsTIR1 expression cassette at the mouse Rosa26 locus. d, Strategy to generate WAPL-degron v-Abl cell lines. Positions of homology arms (grey box), Cas9/sgRNAs and Southern blot probe are indicated. e, Southern blot confirmation of two correctly targeted clones (#5-3 and #5-9) with WAPL-mAID on both alleles (n = 2 biologically independent repeats with similar results). f, Diagram of the experimental strategy to specifically deplete mAID-tagged WAPL protein in G1-arrested v-Abl cells. g, Western blotting to detect WAPL and WAPL-mAID protein. The indicated clones (#5-3 and #5-9) were grown without or with WAPL depletion at indicated time points before immunoblotting (n = 2 biologically independent repeats with similar results). The specific western blotting bands of WT WAPL and WAPL-mAID were labelled. OsTIR1 was detected by anti-V5 antibody. Primary #5 clone was used for the WT WAPL control and β-actin was a loading control. h, Western blotting to detect WAPL protein levels in G1-arrested RAG1-deficient primary #5 v-Abl cells and cultured RAG1-deficient primary pro-B cells. Each sample was loaded with 3 indicated titrations. One of the two experiments is shown. i, Western blotting results to determine relative WAPL protein levels in cycling and G1 arrested WAPL-degron v-Abl cell clones (#5-3 and #5-9) with indicated conditions. For comparison, the intensity of the WAPL band in G1-arrested primary #5 v-Abl cells is set as 1.0. Average value is indicated at each bar. n, number of independent repeats. Indicated P values were calculated using unpaired two-tailed t-test. j, Representative flow-cytometry plots showing the percentage of Clover-positive WAPL-degron v-Abl cells that are without (Untreated) or with (IAA + Dox) WAPL depletion at indicated time points (n = 2 biologically independent repeats with similar results). Primary #5 was processed as a Clover-negative control. k, Cell viability assay for G1-arrested v-Abl cells with indicated conditions. Data are presented as average percentage ± s.d. of viable cells for each condition. n, number of independent experiments. l, Representative flow-cytometry plots of propidium iodide (PI) stained G1-arrested v-Abl cells with indicated conditions (n = 2 biologically independent repeats with similar results). Percentages in the top-right corner represent the percentage of cells at G1, S and G2/M stage. One of the two experiments is shown. Indicated P values were calculated using unpaired two-tailed t-test (i, k). Plasmids, sgRNAs and oligos used are listed in Supplementary Table 6.
Extended Data Fig. 7 Characterization of WAPL/ CTCF/ RAD21-binding in G1-arrested RAG1-deficient WAPL-degron v-Abl cells.
a, Average signal counts ± s.e.m. of WAPL ChIP-seq across the entire Igh locus are plotted as indicated for G1-arrested RAG1-deficient v-Abl cells without (Untreated, blue) or with (IAA + Dox, red) WAPL depletion and cultured RAG1-deficient primary pro-B cells (green). n, number of independent experiments. WAPL ChIP-seq showed that IAA + Dox treatment leads to a depletion of chromatin-bound WAPL at Igh locus, which largely resembles that of primary pro-B cells at Igh locus. b, Three independent repeats of WAPL ChIP-seq signal within ± 1.0 kb region across all peaks genome-wide called in G1-arrested RAG1-deficient v-Abl cells without (Untreated) or with (IAA + Dox) WAPL depletion. Top: Average enrichment. c, d, Average signal counts ± s.e.m. of CTCF (c) and RAD21 (d) ChIP-seq across the entire Igh locus are plotted as indicated for G1-arrested RAG1-deficient v-Abl cells without (Untreated, blue) or with (IAA+Dox, red) WAPL depletion, and cultured RAG1-deficient primary pro-B cells (green). n, number of independent experiments. RAD21 ChIP-seq showed that WAPL depletion in G1-arrested v-Abl cells influenced RAD21 (cohesin) redistribution across the Igh locus to give a pattern significantly similar to primary pro-B cells (Spearman’s correlation r = 0.84, P < 2e-16) (d). e, Spearman correlation analyses of VH usage with RAD21 and CTCF binding in WT primary pro-B cells and WAPL depleted v-Abl cells. Data for Spearman correlation analyses are from panels c, d, Figs. 1b, 4d and Extended Data Fig. 4a, b. Indicated P values were calculated by a two-sided Spearman’s correlation test and shown as: *: 0.01 ≤ P < 0.05, **: 0.001 ≤ P < 0.01, and ***: P < 0.001. Discussion, as noted in the text, we found significant correlations between VH usage and RAD21 or CTCF binding in 3 of the 4 VH families including the distal VH J558/3609 family in primary pro-B cells. However, in the IAA + Dox-treated v-Abl lines, we did not observe a significant correlation between VH usage and RAD21 or CTCF binding in the distal VH J558/3609 family. To explore the reason for this correlation discrepancy, we did an analysis of the correlation of primary pro-B cell VH usage patterns with cohesin and CTCF binding patterns in IAA + Dox-treated v-Abl cells, which indeed revealed significant correlations of these patterns in distal J558/3609 VH usage for both cohesin and CTCF (e). This finding indicates that the lack of correlation of distal J558/3609 VH usage with cohesin and CTCF binding patterns in WAPL-depleted v-Abl cells stems from their VH usage patterns. In this regard, primary pro-B cells generate their VH repertoires from a steady state population in which these cells presumably have RAG scanning across the locus fully engaged and coordinated with VH locus contraction. However, in WAPL-depleted v-Abl cells, RAG scanning and VH locus contraction may not be physiologically matched due to ectopic RAG expression and ectopically-induced WAPL depletion. The further impact of the nearly complete WAPL-depletion in IAA + Dox-treated v-Abl cells versus WAPL expression levels in primary pro-B cells (Fig. 4a) is reflected by greatly reduced levels of proximal versus distal VH rearrangements in v-Abl lines versus those primary pro-B cells (Fig. 4d, bottom, Extended Data Fig. 9c; Supplementary Table 2, 5). Moreover, complete WAPL depletion decreases overall V(D)J recombination activity per se in v-Abl lines as noted in the text, which might also impact these correlations.
Extended Data Fig. 8 Characterization of Igh gene transcription, D usage and Igκ rearrangements in G1-arrested WAPL-degron v-Abl cells.
a, Average signal counts ± s.e.m. of GRO-seq across the 4 VH domains (left) and RC region (right) are plotted as indicated in G1-arrested RAG1-deficient v-Abl cells without (Untreated, upper) or with (IAA + Dox, middle) WAPL depletion and cultured RAG1-deficient primary pro-B cells (bottom). n, number of independent experiments. For comparison, 15 representative major interaction peaks/clusters as Fig. 3b are indicated. PAIR elements are indicated as purple lines with grey background. b, Scatter plots of average transcriptome-wide GRO-seq counts in G1-arrested RAG1-deficient v-Abl cells without (Untreated, x axis) and with (IAA + Dox, y axis) WAPL depletion. n, number of independent experiments. Representative known requisite genes for V(D)J recombination and chromatin interaction are highlighted by red circles and blue arrows in each of the three scatter plots. Spearman’s correlation coefficient (rho) and P value determined by two-sided Spearman’s correlation test are presented. c, Diagram of the experimental strategy including retrovirus-mediated RAG complementation in WAPL-degron v-Abl cells for HTGTS-V(D)J-seq assay. d, Average utilization frequencies ± s.d. of all D segments from DJH plus VHDJH joins in RAG1-complemented, G1-arrested Untreated primary, Untreated WAPL-degron and IAA+Dox-treated WAPL-degron v-Abl lines. n, number of independent experiments. Indicated P values were calculated using unpaired two-tailed t-test. e, Average 3C-HTGTS signal counts ± s.e.m. baiting from RC for interactions with the RC domains that includes 3′CBE downstream and the most proximal VHs upstream in RAG1-deficient cultured primary pro-B cells (top), G1-arrested RAG1-deficient v-Abl cells without (middle) or with WAPL depletion (bottom). n, number of independent experiments. See Supplementary Data 1 for whole Igh locus interactions. Related very low-level peaks of RC interactions with IGCR1 in IAA + Dox treated cells suggest IGCR1 impediments were neutralized by WAPL depletion in v-Abl cells. f, Absolute individual Vκ usage in total reads. Each library was normalized to 20,000 total reads including Igκ junctions and germline reads. Data are presented as average absolute Vκ usage ± s.d. across the Igκ locus in WT primary pre-B cells and untreated WAPL-degron v-Abl cells (G1_Untreated). For the IAA + Dox-treated WAPL-degron v-Abl cells (G1_IAA + Dox), data are presented as average absolute Vκ usage. Spearman correlation of Vκ usage patterns between WT primary pre-B cells and untreated WAPL-degron v-Abl cells is statistically significant (r = 0.96, P < 0.001). P values are calculated by two-sided Spearman’s correlation test. n, number of independent experiments. g, Relative Vκ to Jκ rearrangement levels in RAG1-complemented cycling v-Abl cells and G1-arrested WAPL-degron v-Abl cells without (G1_Untreated) or with (G1_IAA + Dox) WAPL depletion. Relative Vκ to Jκ rearrangement levels = Igκ junctions / (Igκ junctions + germline reads) %.Data are presented as mean percentage values ± s.d. for cycling v-Abl cells and untreated WAPL-degron v-Abl cells (G1_Untreated). For IAA + Dox treated WAPL-degron v-Abl cells (G1_IAA + Dox), data are presented as mean percentage values. Average Igκ recombination levels are indicated at each bar. n, number of independent experiments. Vκ usage data of RAG1-complemented G1-arrested WAPL-degron v-Abl cells without or with WAPL depletion from panel f (middle and bottom) are used to generate the bar graphs (G1_Untreated and G1_IAA + Dox). n, number of independent experiments. h, Percentage of individual Vκ usage in total Vκ to Jκ rearrangements. The same library as panel f was normalized to 20,000 total Igκ junctions. Data are presented as average percentage ± s.d. in WT primary pre-B cells and untreated WAPL-degron v-Abl cells (G1_Untreated). For the IAA + Dox treated WAPL-degron v-Abl cells (G1_IAA + Dox), data are presented as average percentage. n, number of independent experiments. See Supplementary Table 2, 4 and Methods for all further details of the analyses.
Extended Data Fig. 9 The large VH inversion has similar effects on normal and cryptic RSS utilization within and beyond the VH locus in G1-arrested WAPL-degron v-Abl cells and primary pro-B cells.
a–d, Average utilization frequencies ± s.d. of all VH segments in RAG1-complemented, G1-arrested v-Abl cells with indicated conditions. Average percentage ± s.d. of VHDJH and DJH rearrangements are shown. Untreated primary, n = 3 (a); Untreated WAPL-degron (single Igh allele), n = 3 (b); IAA + Dox WAPL-degron (single Igh allele), n = 3 (c); IAA + Dox WAPL-degron Igh VH inversion (single Igh allele), n = 3 (d) were used for independent experiments for statistical analyses with error bars. n, number of independent experiments. All VH segments divided into four domains from most proximal to distal. See Supplementary Table 2, 5 and Methods for more details. e, Pooled HTGTS junction profiles at Igh locus and upstream 4 Mb region for deletional and inversional joining in RAG1-complemented, G1-arrested v-Abl cells without (Untreated, upper, pooled n = 3) or with WAPL depletion (IAA + Dox, middle, pooled n = 3), or with 2.4 Mb inversion with WAPL depletion (IAA + Dox, Igh VH inversion, bottom, pooled n = 3). For comparison, the VH usage (d) and RAG off-target (e, bottom) data in the VH-loci inverted v-Abl cells are shown in normal Igh orientation. A smaller scale is used to present recombination to cryptic recombination signal sequences in WAPL-degron v-Abl lines (e) compared with that in primary pro-B cells (Fig. 2b); the need for this likely reflects lower levels of overall V(D)J recombination following WAPL depletion. f, Average frequencies ± s.d. of plus strand (red, +) and minus strand (blue, -) joining events within indicated regions in RAG1-complemented, G1-arrested v-Abl cells: Untreated WAPL-degron, n = 3; IAA + Dox WAPL-degron, n = 3; IAA + Dox WAPL-degron Igh VH inversion, n = 3. n, number of independent experiments. Indicated P values were calculated using unpaired two-tailed t-test. See Methods for more details.
Extended Data Fig. 10 Working model for loop extrusion-mediated Igh locus contraction for RAG chromatin scanning.
a, In C57BL/6 mice, all 109 VH segments are located within one or another four VHs domains as indicated in figure from proximal to distal: 7183/Q52 (blue), Middle (pink), J558 (green) and J558/3609 (red). Beyond VH domains relevant elements and proteins including the DJHRC, 12 and 23 RSSs, cohesin, and RAG1 and RAG2 subunits and complexes are indicated in the box. b–j, Model for loop-extrusion-mediated physiological locus contraction for distal VH utilization based on RAG1-deficient background analyses and RAG on- and off-target analyses in primary pro-B cells with reduced WAPL expression. See text for overall description. In brief: Loop extrusion past the nascent (non-RAG-bound) DJH-RC may cover much greater distances of upstream chromatin (that would vary from cell) than would occur when RAG is bound to the RC, as directly suggested by results of a recent publication from our lab1 (b–f). If RAG bound to the DJH-RC subsequently in such cells, it could form active DJHRCs that could initiate loop-extrusion-mediated scanning at different extrusion points across the VH locus (g–j), which could avoid downstream rearrangements and potential impediments in those cells to provide more equal scanning access to all VHs across the locus6 for V(D)J recombination and contribution to diverse antibody repertoires. This model and findings that form its basis1 may also be relevant to why VH utilization patterns, which must be done in RAG sufficient cells, in some cases, do not correlate as well as might be anticipated with various peaks found for CTCF/cohesin-binding and 3C-HTGTS interaction studies done in RAG-deficient cells observed in this study and a parallel study2. In this regard, future experiments with catalytically dead RAG mutants in which RAG still binds to the RC, as opposed to RAG-deficient cells, may be informative. Finally, please see related discussion in Extended Data Fig. 7.
Supplementary information
Supplementary Figure 1
Uncropped images obtained by electrophoretic separation scans. This file contains uncropped images for DNA gel panel (Extended Data Fig.5c), southern blot (Extended Data Fig. 6e) and western blots (Fig. 4a; Extended Data Fig. 6g, h) used in the main Figures and Extended Data Figures, as well as statistical quantification analysis related relative Wapl protein expression levels (Fig. 4b; Extended Data Fig.6i). Western blots in each big black box were from the same samples and running in the same gel, incubating with indicated antibodies separately according to different protein molecular weights. β-actin inside each big black box was used as a loading control and was from the same samples, running on the same gel. The small black boxes inside the big black box were used to show the cropped bands or used for quantification integrated density by Image J (version 1.42q). The relative Wapl expression level was compared based on ratio of Wapl/β-actin and set the indicated sample as 1.0 to better observe their relative Wapl expression levels. The western blots used for quantification analysis in Fig. 4b and Extended Data Fig. 6i were including big black box scans from No.1 to No.6 labeled in the top right. See corresponding Source Data for exact relative Wapl/β-actin ratio and more details.
Supplementary Tables
This file contains Supplementary Tables 1-6.
Supplementary Data 1
3C-HTGTS interactions in cultured RAG1-deficient primary pro-B cells with normal and inverted VH-loci; Wapl depletion activates IgH VH locus contraction in G1-arrested Wapl-degron v-Abl cells. Related to Fig. 3b-c and 4c; Extended Data Fig.2d and 8e. For comparison, all related inversion data are shown in normal IgH orientation (a, d, f). a, Individual 3C-HTGTS chromatin interaction profiles of RC/iEμ bait across the whole IgH locus and entire upstream IgH region in cultured RAG1-deficient primary pro-B cells with normal (WT, blue, n=3) and inverted VH-loci (IgH VH inversion, red, n=3). b, Zoom-in profiles of individual 3C-HTGTS across the entire upstream IgH region. c, d, Zoom-in profiles of individual replicates of 3C-HTGTS across the whole IgH locus in normal and inverted VH-loci. n, number of biological repeats (a-d). e, f, Individual replicates of 3C-HTGTS interaction profiles of 3’CBE bait across the entire IgH locus in normal (e, blue, n=3) and inverted VH-loci (f, red, n=4) in cultured RAG1-deficient primary pro-B cells. n, number of biological repeats. The IgH VH inverted region is highlighted in transparent green (a, c, e, WT) and transparent gold (a, d, f, IgH VH inversion), respectively. g, Average 3C-HTGTS signal counts ± s.e.m. across the entire IgH locus in cultured RAG1-deficient primary pro-B cells (top), G1-arrested RAG1-deficient v-Abl cells without (middle) or with Wapl depletion (bottom). n, number of independent repeats. All VH segments are divided into four VHs domains as indicated by graphic at the top and dashed vertical lines (a, c-g).
Supplementary Data 2
VH usage, 3C-HTGTS, GRO-seq, ChIP-seq, CBE motifs sites and PAIR elements for all VHs in primary pro-B cells. Related to Fig. 1b, 3b; Extended Data Fig. 3, 4. Average VH usage ± s.d. of all VHs segments in cultured RAG1-deficient normal (WT, blue, n=3 biologically independent experiments) or inverted VH loci (IgH VH inversion, red, n=4 biologically independent experiments) primary pro-B cells and zoom-in profile of 3C-HTGTS, GRO-seq, Rad21/CTCF ChIP-seq signals for ± 10kb region of all VHs in cultured RAG1-deficient WT (blue, n=4 biologically independent experiments for GRO-seq and all other assays n=3) or IgH VH inversion (red, n=4 biologically independent experiments for GRO-seq and all other assays n=3) primary pro-B cells along with relevant bona fide CBE motif sites are presented, PAIR elements (blue bars) that located near indicated VH were also shown above. For easy comparison with WT loci, all inverted regions are shown in normal IgH orientation. Both the sense (S) and antisense (AS) transcription are relative to the entire IgH VH locus upstream VH81X with or without inversion and indicated, respectively. Utilization data of VH segments was normalized to 118,475 total recovered junctions. Indicated p value was calculated by unpaired two-tailed t-test.
Supplementary Data 3
VH usage, GRO-seq, 3C-HTGTS, ChIP-seq, CBE motifs sites and PAIR elements for all VHs in G1-arrested Wapl-degron v-Abl cells. Related to Fig. 4c, d; Extended Data Fig.7c, 7d, 8a, 8e. Average VH usage ± s.d. of all VHs in G1-arrested Wapl degron v-Abl lines without (Untreated, blue, n=3 independent experiments) or with (IAA&Dox, red, n=3 independent experiments) Wapl depletion and zoom-in profile of GRO-seq, 3C-HTGTS, Rad21/CTCF ChIP-seq signals for ± 10kb region of all VHs in G1-arrested, Untreated (blue, n=4 independent experiments for Rad21 ChIP-seq and all other assays, n=3) and IAA&Dox treated (red, n=4 independent experiments for Rad21 ChIP-seq and all other assays, n=3) Wapl degron v-Abl cells along with relevant bona fide CBE motif sites are presented, PAIR elements (blue bars) that located near indicated VH were also shown above. S, sense transcription; AS, antisense transcription. Utilization data of VH segments was normalized to 100,000 total recovered junctions. Indicated p value was calculated by unpaired two-tailed t-test.
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Dai, HQ., Hu, H., Lou, J. et al. Loop extrusion mediates physiological Igh locus contraction for RAG scanning. Nature 590, 338–343 (2021). https://doi.org/10.1038/s41586-020-03121-7
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DOI: https://doi.org/10.1038/s41586-020-03121-7
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