CTCF couples long-range loop extrusion and diffusion to mediate a diverse Igκ repertoire

Domain: neuroscience
Published: 2025-12-11
Editorial outcome: minor_revision

Abstract
Dynamic genome folding is important for V(D)J recombination at the immunoglobulin kappa (Igκ) locus, which recombines Jκ and Vκ gene segments across a 3.2 Mb region in both deletional and inversional orientations. Chromatin loop extrusion and diffusion are considered two key mechanisms underlying Igκ locus folding, but how they coordinate remains unclear. Here we show that CTCF is a key regulator coupling loop extrusion and diffusion during Igκ V-J rearrangement, promoting recombination in both orientations across long genomic distances. Mechanistically, the CTCF N-terminus promotes long-range loop extrusion that facilitates distal Vκ usage by stabilizing cohesin against WAPL release, and also forms loop barriers enabling chromatin diffusion for inversional Vκ joining. In CTCF N-terminal-deficient B cells, defects in inversional Vκ joining are not restored by WAPL depletion but are instead largely rescued by a dCas9-blockade targeted to the Vκ-Jκ intergenic region, mimicking the CTCF barrier. Our findings thus highlight how CTCF coordinates distinct genome-folding mechanisms through its dual roles in cohesin stabilization and extrusion barrier formation to ensure the generation of a diverse Igκ repertoire.

Introduction
The dynamic spatial organization of mammalian genomes play critical roles in differentiation and development, serving as the structural foundation for coordinated genetic and epigenetic activities1–15. A prominent mechanism involved in folding the genome into functional units is chromatin loop extrusion, which employs the cohesin complex to linearly drive progressive loop formation and synapsis of functional elements during this process16–26. The architectural protein CTCF binds to the asymmetric CTCF sites27,28 and interacts with cohesin through its N-terminus, functioning as a directional barrier for cohesin extrusion to generate loop boundaries29–34. Structural studies revealed that a Tyr-Asp-Phe (YDF) motif in the CTCF N-terminus is critical for this polar interaction29,34. The other key mechanism of genome folding is diffusional capture that occurs via stochastic interactions35, which lacks orientation and is not considered to be efficient for long-range interactions36,37.

A concrete example of how dynamic genome folding instructs cellular function is V(D)J recombination, a programmed somatic recombination process that occurs in developing lymphocytes and generates diverse antigen receptor repertoires essential for adaptive immunity14. This process is initiated in the G1 phase of cell cycle by the RAG endonuclease, which cleaves a pair of recombination signal sequences (RSSs) flanking the V, D, and J coding segments38. The cleavage occurs in a stepwise manner: RAG first binds to one RSS, then finds and captures a complementary partner RSS for subsequent cleavage38. Given that antigen receptor loci can span several megabases (Mb), mechanisms are needed to overcome the bias of genomic distance and promote efficient synapsis of widely separated RSSs. In this regard, studies revealed that loop extrusion promotes RAG scanning along the chromatin within loop domains to locate partner RSS in convergent orientation for deletional joining7,39,40. This mechanism plays a fundamental role in mediating V(D)J recombination at the mouse immunoglobulin heavy (Igh) chain locus, initially facilitating the physiological deletional joining of D to J segments in pre-pro-B cells40. This is followed by deletional V-to-DJ recombination in pro-B cells, where developmental depletion of the cohesin releaser WAPL21,22,41–43 promotes extended loop extrusion, enabling the bypass of a number of CTCF barriers at the Igh locus and allowing for the use of distal V segments across the entire 2.4 Mb V region44,45.

Immunoglobulin light (Igκ, Igλ) chain loci undergo recombination following assembly of the Igh locus to generate B cell receptors. Unlike the Igh locus, which contains only forward V segments for deletional joining, the Igκ locus includes both forward and reverse Vκ segments that recombine efficiently in deletional and inversional orientations, respectively46. The presence of robust Vκ-RSSs has been demonstrated to be important to promote inversional Vκ joining, however, this phenomenon cannot be solely explained by linear loop extrusion and requires a diffusion-based mechanism47. How the two processes coordinate to promote a diverse Igκ repertoire is largely unknown.

The 103 functional Vκ segments are distributed across the 3.15 Mb 5’ Igκ region, with reverse Vκ segments located in the middle and forward Vκ segments at both the very proximal and distal ends46 (Fig. 1a). Broad Igκ recombination potential is mainly regulated by the intronic (iEκ) and 3’ (3’Eκ) enhancers located adjacent to the four functional Jκs, which together form a recombination center to recruit partner Vκ-RSSs for recombination48–51. In addition to the recombination center enhancers, the Vκ region contains two recently identified enhancers, E88 and E34, which modulate nearby Vκ gene segment usage52,53. Importantly, Vκs are insulated from the recombination center by the Cer and Sis elements interacting with the Vκ region and recombination center, respectively47. Each element contains two CTCF sites, with those in Cer oriented toward Vκs and those in Sis oriented toward the recombination center, together supporting a balanced Vκ repertoire54,55 in a manner dependent on the orientation of these CTCF sites56. Additionally, multiple CTCF sites are spread throughout the Vκ region46,57,58. Conditional depletion of CTCF in pre-B cells skews Vκ repertoire toward the use of proximal Vκ segments59. These studies implicated CTCF as an important regulator of Igκ recombination, however, the underlying mechanisms of its action remain elusive.

The molecular basis of how distinct chromatin-folding processes cooperate to enable efficient Igκ recombination in both deletional and inversional orientations remains unknown. In this study, we identify a central role for CTCF in coordinating long-range chromatin loop extrusion and short-range diffusion to balance recombination outcomes across the Igκ locus, through its dual functions in cohesin stabilization and loop barrier formation. These findings reveal that CTCF regulates immune diversity through tightly coupled yet distinct mechanisms and highlight its versatility in genome regulation, which may extend to other fundamental processes such as transcription and DNA repair.

Results
Multifaceted regulation of Igκ repertoire by CTCF N-terminus
To investigate the molecular basis of CTCF’s role in Igκ recombination, we disrupted the CTCF N-terminal YDF motif critical for the polar interaction between CTCF and cohesin via the CTCF(Y226A/F228A) mutation29 (thereafter referred to as CTCF-Nm). The homozygous CTCF-Nm was generated via CRISPR/Cas9 targeting60 combined with a single-stranded DNA oligonucleotide (ssODN) template61 in a RAG1 deficient Eμ-Bcl2+ v-Abl pro-B cell line48 (Supplementary Fig. 1a, b). This cell line lacks endogenous RAG1 and carries a transgenic catalytically-dead RAG1 (D708A)48, which allows inducible V(D)J recombination62 in G1-arrested cells upon wild-type (WT) RAG1 complementation. v-Abl pro-B cell lines have been demonstrated to be a reliable system to study Igκ rearrangement mechanisms47. We employed high-throughput genome-wide translocation sequencing-adapted V(D)J-sequencing (HTGTS-V(D)J-Seq)7 with a Jκ1 bait to compare Vκ-to-Jκ joining profiles in WT and CTCF-Nm cells, which were RAG1-complemented and G1-arrested for 4 days.

WT cells underwent robust Vκ-to-Jκ1 recombination utilizing both forward and reverse Vκs across the 3.15-Mb Vκ region, generating deletional and inversional VκJκ joins, respectively (Fig. 1b, left panel). However, in the CTCF-Nm mutants, there was a considerable change in the pattern of Vκ utilization, with significantly decreased middle/distal Vκ usage accompanied by a substantial increase in the frequency of proximal Vκ usage (Fig. 1b, left panel). This suggests that the CTCF N-terminus plays an important role in supporting long-range Vκ joining, though it remains unclear at this point whether this effect occurs indirectly by suppressing proximal Vκ joining or directly by promoting distal Vκ joining. This pattern is similar to that reported for CTCF depletion59, indicating CTCF N-terminus is essential for CTCF’s function in Igκ recombination. We also noted a reduced level (3.7-fold) of absolute Vκ junctions in the CTCF-Nm mutants compared to WT cells, which might further indicate a negative impact on Igκ recombination center48 activity or RAG activity by the CTCF-Nm mutation (Supplementary Fig. 1c).

Unexpectedly, while the mutation caused a consistent decrease in the usage of middle/distal Vκ segments regardless of Vκ orientation, it had a differential impact on proximal Vκs. We observed a 3.6-fold overall increase in the percentage of deletional, but not inversional, Vκ joins (Fig. 1b, right panel). Inspection of the utilization frequency of individual proximal Vκs showed a range of 2 to 15-fold increase in deletional Vκ usage (Fig. 1c), while inversional Vκ usage was either unchanged or reduced up to 5-fold (Fig. 1d). These observations strongly suggest a suppression of inversional Vκ joining in the CTCF-Nm mutants.

Vκ-RSS orientation prescribes Vκ recombination activity in CTCF N-terminal mutants
We next investigated whether it is indeed the orientation, rather than the intrinsic sequence feature of Vκs, that contributes to the differential recombination potential in the CTCF-Nm mutant. To test this, we inverted a proximal Vκ region to see if the inversion, which reverses the Vκ orientation while maintaining the same sequence, would alter the joining frequency of the individual Vκ segments. To build the line, we first generated a CTCF-Nm Igκ+/- line harboring only a single Igκ allele by CRISPR/Cas9-mediated deletion of the entire Igκ locus on the other allele to facilitate the generation of mutant lines (Supplementary Fig. 2a). The CTCF-Nm Igκ+/- line demonstrated very similar Vκ joining pattern across the Igκ locus with the parental line containing both alleles (Supplementary Fig. 2b). Next, on the intact Igκ allele, we inverted a 377-kb proximal Vκ region spanning from 6.9-kb upstream of Vκ8-34 to 7.3-kb downstream of Vκ6-15, which includes 4 forward Vκs and 15 reverse Vκs, to generate the CTCF-Nm Igκ377kb-inv line (Fig. 2a, Supplementary Fig. 2c). Remarkably, upon this inversion, all 4 forward Vκs, now in reverse orientation for inversional Vκ-to-Jκ recombination, demonstrated greatly reduced joining activity with a 6 to > 28-fold decrease in the frequency of utilization (Fig. 2b, c). In contrast, most of the reverse Vκs (11 out of 15), now in forward orientation for deletional Vκ-to-Jκ recombination, showed significantly increased joining activity with a 2 to 39-fold gain in the frequency of utilization (Fig. 2b, d). These results strongly support the orientation of Vκ as a major determinant of proximal Vκ recombination potential in the context of CTCF N-terminal deficiency.

A potential limitation of the 377-kb inversion is that it spans a large chromosomal distance, which alters the relative linear proximity of the Vκ segments it contains to the Jκ segments. To minimize the impact of positional change, we also performed a localized mutation of the Vκ6-17 segment, in which only the 28-bp Vκ-RSS and its adjacent 10-bp coding region were inverted (Supplementary Fig. 3a, b). We found that reversing the orientation of this forward Vκ led to a substantial 9-fold decrease in its utilization frequency, from 5.58 to 0.64% (Fig. 2e, f, Supplementary Fig. 3c). This trend closely mirrors the one observed in the 377-kb inversion.

Collectively, the HTGTS-V(D)J-Seq analysis indicated two major roles of the CTCF-N terminus in Igκ recombination: (1) supporting long-range Vκ usage, and (2) promoting the utilization of inversional Vκs. We then sought out to explore the mechanistic basis.

CTCF N-terminal mutation compromises loop regulation at Igκ locus
We hypothesized that the decreased utilization of middle and distal Vκs resulted from impaired long-range Igκ chromosomal interactions. To test this, we compared Igκ interaction profiles between WT and CTCF-Nm cells. We performed HTGTS-adapted chromosome-conformation-capture sequencing (3C-HTGTS)63 with the Cer locale as bait in RAG deficient cells arrested in G1 for 4 days. The 3C-HTGTS analysis revealed that in WT cells, Cer interacted with genomic sequences across the Vκ region, with stronger interactions observed in the more proximal Vκ regions (Fig. 3a, Supplementary Fig. 4a). Most of the interaction peaks overlapped with CTCF or the E2A transcription factor64,65 binding sites (BS) (Fig. 3a, Supplementary Fig. 4b). However, the CTCF-Nm mutation exhibited a significant reduction in long-range interactions with middle and distal Vκ regions, with this reduction affecting all peaks in these regions (Fig. 3a–c, Supplementary Fig. 4a, c). In the proximal Vκ region, mid-range interactions up to 670-kb were observed. However, while peaks at E2A BS were either maintained or increased, peaks at the CTCF BS were largely abolished in this region (Fig. 3a, c, Supplementary Fig. 4c). These findings suggest a dual role for the CTCF N-terminus in facilitating Igκ interactions: (1) mediating loop boundary formation for CTCF-dependent loops, and (2) promoting long-range interactions beyond this boundary function.

We wondered if the defective long-range Igκ loop formation in the CTCF-Nm cells resulted from compromised loop extrusion activity. Consistent with this hypothesis, Chromatin Immunoprecipitation Sequencing (ChIP-Seq) analysis of SMC3, a key component of the cohesin ring complex, showed a substantial reduction of cohesin accumulation on chromatin both genome-wide and at the Igκ locus in the CTCF-Nm cells compared to WT cells (Fig. 3d, e, Supplementary Fig. 5a), supporting that CTCF N-terminal deficiency may promote increased removal of cohesin from chromatin.

CTCF has been implicated in cohesin processivity by protecting cohesin from WAPL release29,66. To further investigate, we performed label-free quantitative mass spectrometry (qMS) via nanoflow reversed-phase liquid chromatography (RPLC) on SMC3 immunoprecipitants from chromatin fraction of G1 arrested cells, normalizing to the chromatin-bound SMC3 for quantitative analysis. The IP-qMS results showed that the CTCF-Nm mutation led to a 3-fold reduction in the interaction between CTCF and SMC3, and notably, a 1.9-fold increase in the interaction between cohesin and WAPL (Fig. 3f). As a control, interactions between SMC3 and other core cohesin components, including SMC1a and RAD21, were shown to be unaffected (Fig. 3f). Chromatin loops are sensitive to changes in WAPL activity. It has been reported that a 2.2-fold increase in WAPL protein levels greatly alters genomic organization in pre-B cells compared to pro-B cells67. Therefore, the 1.9-fold increase in cohesin-WAPL interaction in CTCF-Nm cells is significant and likely accounts for decreased loop extrusion activity and the decreased long-range Igκ interactions observed in these cells.

We also noted that while the mutation only mildly reduced CTCF binding genome-wide and at the Igκ Cer-Sis elements, binding at Vκ locus was significantly decreased at many loci, suggesting many Vκ CTCF sites have relatively weak affinity that is dependent on a functional N-terminus of CTCF (Supplementary Fig. 5b). However, differences in CTCF binding among Vκ sites did not translate into differences in Vκ usage. For example, Vκs in a 400-kb distal region containing CTCF sites comparatively less affected by the mutation exhibited similar decreases in usage as other distal Vκs (Supplementary Fig. 5c). This suggests that although differential binding likely reflects intrinsic CTCF site strength, the binding retained in CTCF-Nm mutants may be non-functional due to the N-terminal mutation. In the proximal Vκ region, several inversional Vκs, such as Vκ6-32, Vκ8-30, Vκ8-21, Vκ8-16, and Vκ6-14, showed little change in usage upon CTCF-Nm mutation. To determine whether this was due to retained nearby CTCF binding, we examined the surrounding regions and found that these Vκs lack adjacent CTCF binding (Supplementary Fig. 5d). Instead, they were located near E2A sites, suggesting that retained or increased E2A-mediated interactions, rather than CTCF, may help preserve their usage in the mutant background.

Finally, comparison of CTCF sites that retained versus lost binding in the CTCF-Nm mutants, both genome-wide and within the Igκ locus, did not reveal obvious differences in DNA sequence composition, implying that factors beyond the core CTCF motif and its immediate flanking sequences may contribute to N-terminal-dependent binding sensitivity (Supplementary Fig. 5e).

We further performed Precision Run-On sequencing (PRO-Seq) in WT and CTCF-Nm cells to assess whether the CTCF-Nm mutation affects expression of factors involved in loop extrusion26 or diffusion68–72. This analysis revealed no significant changes in key genes for these processes (Supplementary Fig. 6a). Moreover, germline transcription across the Vκ region was largely preserved in CTCF-Nm mutants (Supplementary Fig. 6b), indicating that altered chromatin accessibility across the Vκ region does not explain the observed Igκ recombination defects in long-range Vκ usage and inversional joining.

On the other hand, transcription across the recombination center, including the Jκ segments, iEκ, and 3’Eκ, was substantially reduced (Supplementary Fig. 6b), correlating with lower overall Igκ recombination and supporting a role for CTCF N-terminus in regulating recombination center activity (Supplementary Fig. 1c). Expression of V(D)J recombination factors was minimally affected, aside from enrichment of RAG1(D708A) and RAG2 that were transgenically expressed in these cells (Supplementary Fig. 6c), confirmed by RAG1 western blotting (Supplementary Fig. 6d). While retroviral complementation with abundant WT RAG1 may partly mitigate this effect for V(D)J recombination assays, potential WT heterodimerization with RAG1(D708A) may reduce fully active WT-WT dimers, contributing to lower absolute Vκ utilization alongside with reduced recombination center activity in CTCF-Nm cells.

Collectively, these findings support a direct role for the CTCF N-terminus in shaping the pattern of the Igκ repertoire by promoting loop barrier formation and protecting cohesin from WAPL release.

Restoring long-range extrusion rescues distal deletional but not inversional joins
Given the increased cohesin removal by WAPL in CTCF-Nm mutants, we next asked whether depleting WAPL to enhance loop extrusion activity could rescue the defects in Igκ recombination. To test this, we first generated a line allowing for rapid degradation of WAPL using auxin-inducible degron (AID) technology. To minimize leaky degradation, we employed the AID version 2 (AID2) system73. In this system, we targeted OsTIR1(F74G) to the Rosa26 locus on both alleles to ensure robust OsTIR1 expression (Fig. 4a, Supplementary Fig. 7a). WAPL was then tagged with AID2 and fused to tdTomato for inducible degradation with 5-Ph-IAA and tracking of WAPL level via flow cytometry analysis. We refer to this line as Wapl-AID2. Near complete WAPL degradation was achieved after 4h of 1uM 5-Ph-IAA treatment and maintained during G1 arrest when 5-Ph-IAA was continuously present (Fig. 4b, Supplementary Fig. 7b, c). Finally, we introduced the CTCF(Y226A/F228A) mutation into the Wapl-AID2 line to generate the Wapl-AID2 CTCF-Nm line. Comparative analyses were then conducted between untreated and WAPL-depleted cells using this line.

3C-HTGTS analysis using the Cer bait showed that as expected, untreated cells exhibited an Igκ interaction profile similar to that of CTCF-Nm cells, with the majority of interactions occurring in the proximal Vκ region (Fig. 4c, d, Supplementary Fig. 7d). This was characterized by prominent peaks at E2A sites and lack of strong interaction peaks at CTCF sites (Supplementary Fig. 7e). On the other hand, WAPL-depleted CTCF-Nm cells showed significantly enhanced long-range interactions, extending into the middle and distal Vκ regions, as well as beyond the Igκ locus into neighboring domains, consistent with increased loop extrusion activity (Fig. 4c, d, Supplementary Fig. 7d). These changes were accompanied by reduced shorter-range interactions in the proximal Vκ region and, notably, the near-complete removal of interaction peaks at E2A sites seen in the untreated cells (Fig. 4c, d, Supplementary Fig. 7d, e). This suggests that E2A sites may act as weak barriers to loop extrusion, which can be readily bypassed when extended extrusion activity is enabled.

Next, we performed HTGTS-V(D)J-Seq analysis on RAG complemented cells to assess Igκ recombination. The untreated Wapl-AID2 CTCF-Nm cells exhibited a Vκ repertoire similar to that of the CTCF-Nm line, which is biased toward use of proximal deletional Vκ segments (Fig. 4e). Notably, WAPL depletion significantly increased the utilization frequency of distal deletional Vκ segments at the cost of proximal ones, a change closely correlated with alterations in genomic interactions within these regions (Fig. 4e). Comparable fold enrichment patterns of weak and strong distal Vκ-RSSs indicates that these changes result from increased loop-extrusion-mediated RAG scanning activity, rather than from selective targeting of certain strong Vκ-RSSs (Supplementary Fig. 7f). Unexpectedly, despite enhanced interactions within the middle/distal inversional Vκ region, there was no improvement in the utilization of middle/distal inversional Vκ segments (Fig. 4e). These results provide direct evidence that loop extrusion-mediated long-range interactions are adequate to mediate deletional, but not inversional, distal Vκ joining. Therefore, enabling diffusion that promotes orientation-independent interactions is necessary for inversional Vκ joining. We next investigated how this process is achieved.

CTCF loop barrier function is required for inversional Vκ joining
Previous reports showed that WAPL depletion alone in pro- and pre-B cells has a mild impact on Igκ repertoire45,67. Our HTGTS-V(D)J-seq analysis revealed that treatment of Wapl-AID2 cells with 5-Ph-IAA resulted in increased distal Vκ deletional joining and decreased proximal Vκ joining, consistent with enhanced loop extrusion activity after WAPL depletion (Fig. 5a). On the other hand, most inversional Vκ joining was retained, in stark contrast to WAPL-depleted CTCF-Nm cells, which showed minimal inversional joining (Fig. 5a, b).

Unlike WAPL-depleted CTCF-Nm cells, cells with only WAPL depletion retain functional CTCF. We hypothesized that the loop barrier function of CTCF is critical to promote diffusion driving inversional Vκ joining. 3C-HTGTS with Cer bait showed that 5-Ph-IAA treated Wapl-AID2 cells exhibited extended chromatin interactions, similar to WAPL-depleted CTCF-Nm cells (Fig. 5c, Supplementary Fig. 8a). However, unlike the WAPL-depleted CTCF-Nm cells that lacked detectable peaks in the Vκ region, 5-Ph-IAA-treated Wapl-AID2 cells displayed multiple strong Cer-interaction peaks. Moreover, all of the interaction peaks detected within the Vκ region in 5-Ph-IAA-treated Wapl-AID2 cells overlapped with CTCF sites (Fig. 5d, Supplementary Fig. 8a, b). These results confirmed that WAPL-depleted cells preserved strong CTCF barriers, which is required for inversional Vκ joining, though it remained unclear if the CTCF barriers at the Igκ locus, specifically the Vκ-Jκ intergenic sites versus Vκ region CTCF sites, are of equal importance.

Targeted dCas9-SunTag blockade rescues inversional Vκ joining
The CTCF barriers likely enable diffusion by preventing direct extrusion between Jκs and Vκs, a process that would otherwise inhibit inversional RAG recombination. This hypothesis predicts that extrusion barriers at the Vκ-Jκ intergenic region are of critical importance.

To directly assess it, we tested whether targeted roadblocks mimicking CTCF barriers could rescue inversional Vκ joining in CTCF-Nm cells. Previous studies have shown that DNA-bound nuclease-dead Cas9 (dCas9) proteins can simulate extrusion barriers both in vivo and in vitro34,40. Additionally, it has been reported that similar to CTCF, a dCas9-blockade acts as a polar barrier to cohesin, halting its translocation when cohesin encounters the proto-spacer adjacent motif (PAM)-proximal side34. We generated the dCas9-blockade system in the untreated Wapl-AID2 CTCF-Nm cells. One copy of the Igκ allele was further deleted in the cells to facilitate targeting. To generate an efficient blockade, we leveraged multiple dCas9 BS with the bulky dCas9-SunTag system74. We inserted a synthetic sequence with 38 consecutive dCas9 BS into the Vκ-Jκ intergenic region, close to the Sis regulatory element. Of these, 20 dCas9 BS were oriented toward the Vκ region, and 18 were oriented toward the Jκ region, mimicking the CTCF site orientations in the Cer and Sis elements, respectively (Fig. 6a). This line is referenced as BS-only line. Next, we introduced a doxycycline-inducible, modified dCas9-SunTag system into the BS-only cell line using PiggyBac vectors (Fig. 6a). This modified system retained all components of dCas9-SunTag except for the VP64 transcription activator to avoid affecting the local epigenetic environment. This line is termed the dCas9-SunTag blockade line.

We confirmed stable expression of dCas9 protein and gRNA in the dCas9-SunTag blockade line (Fig. 6b, c), and ChIP-qPCR analysis showed strong dCas9 binding at the targeted locus (Fig. 6d). Furthermore, 3C-HTGTS analysis with iEκ bait demonstrated the formation of a targeted dCas9-SunTag-mediated loop barrier, which was evidenced by significantly decreased bait interaction with Cer and Sis elements located upstream of the dCas9 BS, while interaction with regions adjacent to the BS within the newly formed loop was enhanced (Fig. 6e). Therefore, direct loop extrusion from Jκ to Vκ was largely blocked in the dCas9-blockade line, enabling diffusional access between Jκs and Vκs. We did not detect increased interactions at the BS itself, likely because the dCas9-SunTag complex sterically hindered cleavage by NlaIII, the restriction enzyme used in 3C-HTGTS. This interpretation is consistent with previous reports showing that dCas9 can block restriction enzyme digestion75 (Supplementary Fig. 9a).

Both the BS-only and dCas9-SunTag blockade lines were complemented with RAG1 and subjected to G1 arrest for HTGTS-V(D)J-Seq analysis. The BS-only line exhibited a Vκ joining pattern similar to that of CTCF-Nm cells, which was primarily characterized by proximal deletional Vκ joining (Fig. 6f). Remarkably, the dCas9-SunTag blockade targeted to the Vκ-Jκ intergenic region largely rescued the Vκ repertoire deficiency. In the dCas9-SunTag blockade line, we observed a reduction in proximal deletional Vκ usage and a significant increase in middle/distal inversional Vκ joining as well as an increase in distal deletional Vκ usage, ultimately resulting in a Vκ joining pattern more akin to that of WT cells (Fig. 6f, Supplementary Fig. 9b). Collectively, these findings provide strong evidence that extrusion barriers, particularly at the Vκ-Jκ intergenic region, are crucial for inversional Vκ joining via diffusion.

Methods
Cell lines
The parental RAG1(D708A), Eμ-Bcl2+ v-Abl pro-B cell line48 was a gift from Dr. David Schatz. Parental and derivative v-Abl pro-B lines were maintained in RPMI 1640 medium (10-040-CV; Corning) supplemented with 10% FBS (Bio-techne, S11150H), 50 uM 2-Mercaptoethanol (Sigma-Aldrich, M3148), 100 U/ml Penicillin-Streptomycin (Gibco, 15140122) and 2 mM L-Glutamine (Gibco, 25030081) at 37 oC with 5% CO2.

Generation of mutant v-Abl pro-B cell lines
CTCF(Y226A/F228A) mutation. We employed CRISPR/Cas9 targeting combined with a short single-stranded DNA oligonucleotide (ssODN) template61 to generate the desired mutation. The ssODN (AAAACCAAAAAGAGCAAACTTCGTTACACAGAAGAGGGCAAAGACGTGGATGTGTCTGTGGCCGATGCTGAGGAAGAACAGCAAGAAGGACTGCTGTCTGAGGTTAATGCAGAGAAAGTAGTTGGTAAT) contained the target Y226A/F228A mutations and a silent mutation that disrupts the PAM motif of the gRNA targeting site. Cas9 protein (Integrated DNA Technologies, 1081059) and target-specific crRNA:tracrRNA duplex were assembled as ribonucleoprotein and introduced into cells along with the ssODN via nucleofection using Lonza’s 4D-Nucleofector system (SF solution, DN100 program). Cells were subcloned by dilution one day after nucleofection. Candidate clones were screened for homozygous mutation by PCR and validated by Sanger sequencing. Detailed oligo and primer information in this study are listed in Supplementary Table 1. All oligos and primers in this study were synthesized by Integrated DNA Technologies, unless otherwise specified.

Igκ allele modifications. The single Igκ allele mutation was generated via CRISPR/Cas9-mediated deletion of one entire 3.2-Mb Igκ allele, leaving the other allele intact. Candidate clones were screened by PCR and validated by Southern blotting. It served as the parental line for Igκ inversional mutations. The 377-kb proximal Igκ-inversion was made by inverting the region spanning from 6.9-kb upstream of Vκ8-34 to 7.3-kb downstream of Vκ6-15. Clones were validated by PCR and Southern blotting. The Vκ6-15 inversion was made by Cas9 aided targeting, along with an ssODN that contains the inverted Vκ6-15-RSS sequence and 10-bp flanking coding region. Clones were validated via PCR and Sanger sequencing.

WAPL-degron lines. Donor plasmid (pOsTIR1(F74G)-Rosa26) for OsTIR1-V5 insertion was generated based on pEN11318 (Addgene, 86233) using HiFi DNA Assembly kit (New England Biolabs, E2621S). The modifications included generating the OsTIR1(F74G) mutation and cloning homology arms for Rosa26 locus integration. Upon Cas9 aided integration, cells were selected by 400 ug/ml G418 (Gibco, 11811-031) for 5 days and subcloned. Candidate clones were screened for homozygous mutation by PCR and validated by western blotting. The clones were then treated to remove the FRT-flanked NeoR-cassette by introducing pCAG-Flpo81 (Addgene, 60662) carrying flippase to the cells to generate the Rosa26-OsTIR1(F74G) line. To further tag WAPL with mAID-tdTomato, pMK29382 (Addgene, 72831) was modified to include homology arms flanking the WAPL stop codon and had mCherry replaced by tdTomato gene amplified from ptdTomato-N183 (Addgene, 54642) to generate pWAPL-mAID-tdTomato. Upon Cas9 aided targeting, tdTomato positive cells were subcloned via fluorescence-activated cell sorting and further validated by PCR, western blotting and flow cytometry analyzer.

dCas9-SunTag blockade lines. To generate the dCas9-SunTag blockade system in the untreated Wapl-AID2 CTCF-Nm cells, we first deleted one Igκ allele to facilitate analysis. To insert consecutive dCas9 BS to the Vκ-Jκ intergenic region, a donor plasmid (pgRNA1) was generated using the HiFi DNA Assembly kit. It harbors a 1710 bp synthetic sequence (synthesized by GenScript.) containing 38 repeats of a non-murine gRNA target sequence (GGTATGTCGGGAACCTCTGA) flanked by desired homology arms in the pBluescript KS(-) backbone. The synthetic sequence was verified by JASPAR vertebrate database84 for lack of CTCF and E2A sites and inserted 376bp downstream of Sis regulatory element via Cas9-aided integration to generate the BS-only line. The doxycycline inducible dCas9-SunTag and gRNA template were then introduced to the cells via PiggyBac vectors pPB-TRE3Gp-dCas9-SunTag-Puro and pPB-gRNA1-TetOn-3G-Activator-Neo, respectively. The pPB-TRE3Gp-dCas9-SunTag-Puro vector was modified from pPB-Ins-TRE3Gp-dCas9-SunTag-VP64-EF1Ap-Puro85 (Addgene, 183409) by deleting VP64. The pPB-gRNA1-TetOn-3G-Activator-Neo was modified from pPB-Ins-U6p-sgRNAentry-EF1Ap-TetOn3G-IRES-Neo85 (Addgene, 183411) by cloning the gRNA template into the latter. Cells were selected by 400 ug/ml G418 (Gibco, 11811-031) and 2 ug/ml puromycin (Gibco, A1113803) for a week and then subcloned by limited dilution. Subclones were treated with 2 ug/ml doxycycline (Millipore Sigma, D9891) for 2 days and those with clear GFP induction were selected as positive clones for analysis. This line was then referenced as the dCas9-SunTag blockade line. Positive clones were further validated for dCas9 expression via western blot, gRNA expression via RT-qPCR, dCas9 binding via ChIP-qPCR and loop barrier formation via 3C-HTGTS. To induce dCas9-SunTag expression in G1 cells for analysis, 2 ug/ml doxycycline was added simultaneously with 3 uM STI-571 and again on Day 2 of G1 arrest.

Chromatin fractionation and Co-IP
Isolation of chromatin-bound proteins and co-IP was performed based on a published protocol with slight modifications86. 40 million cells were lysed with cell lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 M NaF, 1 M β-glycerophosphate, 1 mM dithiothreitol (DTT), 1X protease inhibitor cocktail (ThermoFisher Scientific, A32965)) on ice for 10 min, centrifuged to remove supernatant (cytoplasmic fraction) and resuspended in nuclei lysis buffer (10 mM Tris-HCl, pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 M NaF, 1 M β-glycerophosphate, 1X protease inhibitor cocktail) on ice for 15 min. Further centrifugation was performed to remove supernatant (soluble nucleic fraction) and obtain the pellet of chromatin fraction. The chromatin fraction was solubilized via resuspending the pellet in Benzonase buffer (50 mM Tris-HCl, pH 7.5, 20 mM NaCl, 1 mM MgCl2, 0.2% NP-40, 1 M NaF, 1 M β-glycerophosphate, 1X protease inhibitor cocktail, 50 U/ml Benzonase (Millipore Sigma, E1014)) and rotating at room temperature (RT) for 1 h. The supernatant was collected to measure the protein concentration, and an equal amount of total protein was used for the subsequent co-IP across all samples. During co-IP, the NaCl concentration of the chromatin fraction was adjusted to 100 mM. Samples were then pre-cleared with 20 ul Protein A Dynabeads (Invitrogen, 10001D) for 2 h at 4 oC and subjected to overnight incubation with 5 ug of SMC3 antibody (Fortis Life Sciences, A300-060A) or 5 ug of IgG isotype control (Invitrogen, 02-6102). The IP samples were captured by incubation with 40 ul Protein A beads for at least 2 h, washed five times with wash buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 0.2% NP-40, 1 M NaF, 1 M β-glycerophosphate, 1X protease inhibitor cocktail), five times with PBS and then proceeded to label-free qMS.

Label-Free qMS
Sample preparation. Sodium dodecyl sulfate (SDS) in 100 mM ammonium bicarbonate (ABC, pH = 8) was added to the protein-bound beads to achieve a final concentration of 2% (w/v). The sample was mixed thoroughly by pipetting, then sonicated, vortexed, and incubated at 90°C for 10 minutes to release and denature the bound proteins. After centrifugation at 14,000 X g for 10 minutes at 14°C, the supernatant was collected, and 1 µL of 500 mM DTT was added for reduction, followed by incubation at 95°C for 15 minutes. Then, 2 µL of 500 mM iodoacetamide (IAA) was added for alkylation and incubated in the dark at room temperature for 20 minutes. The reaction was quenched with 1 µL of 500 mM DTT. SDS was removed, and proteins were digested using the SP3 method87. Briefly, 4 µL of a 1:1 mixture of carboxylate-modified hydrophilic (Sera-Mag, 45152105050250) and hydrophobic (Sera-Mag, 65152105050250) paramagnetic beads were added to the sample, followed by the addition of acetonitrile (ACN, ~75% v/v). After incubation and bead separation, beads were washed with ACN, resuspended in 100 mM ABC, and digested with 50 ng trypsin overnight at 37°C. Digestion was quenched with formic acid, and the peptide solutions were magnetically separated for Nanoflow RPLC-MS/MS analysis88.

Nanoflow RPLC-MS/MS. Peptide separation prior to MS/MS analysis was performed using an EASY nanoLC-1200 (Thermo Fisher Scientific) with a 75 μm i.d. × 20 cm C18 column. Buffer A contained 0.1% (v/v) FA, and buffer B consisted of 80% (v/v) ACN and 0.1% (v/v) FA. 1 µL of peptide sample was loaded with buffer A at a maximum pressure of 800 bar. Peptides were separated at 200 nL/min using a gradient: 2-8% (v/v) B in 5 min, 8-40% (v/v) B in 60 min, 40-80% (v/v) B in 5 min, and 80% (v/v) B for 10 min. MS/MS analysis was performed on a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) with a full MS scan range of 300-1500 m/z, a resolution of 60,000, and AGC of 3E6. MS/MS resolution was 60,000 with AGC of 1E5 and a maximum injection time of 150 ms. The loop count was set to 15 (top 15), with a quadrupole isolation window of 2 m/z and normalized fragmentation energy of 28. The ion intensity threshold was 6.7E4, and dynamic exclusion was set to 15 s.

Protein identification was performed using MaxQuant 1.5.5.189 with the Mus musculus UniProt proteome database (UP000000589). Label-free quantification and match between runs were enabled with default parameters. Database search settings included Trypsin/P as the digestion enzyme, with oxidation of methionine and acetylation of lysine and protein N-termini as dynamic modifications, and carbamidomethylation of cysteine as a fixed modification. The maximum number of missed cleavages was set to 2. The first search peptide mass tolerance was set to 20 ppm, and the main search precursor mass tolerance to 4.5 ppm. A false discovery rate (FDR) of 1% was applied to proteins, peptides, and peptide-spectrum matches using the target-decoy approach. Samples include WT and CTCF-Nm SMC3 immunoprecipitants from three independent replicates, together with their IgG isotype controls. To determine the relative abundance of SMC3 immunoprecipitants, we used the protein intensity data from the database search and normalized them to the SMC3 level across the samples. As the CTCF-Nm cells contain the Y226A/F228A mutation, peptides with corresponding changes were included in the search and included for the analysis of CTCF intensity.

ChIP-Seq library preparation and data analysis
IP and Input DNA for ChIP-Seq were prepared from G1 arrested, RAG1 deficient cells. 20 million cells were crosslinked in 10 ml prewarmed culture medium with 1% formaldehyde at RT for 10 min, then lysed on ice for 10 min in 1 ml cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40) followed by 150 ul nuclei lysis buffer (50 mM Tris-Cl pH 8.1, 10 mM EDTA, 1% SDS) at RT for 10 min. 850 ul dilution buffer (16.7 mM Tris-HCl pH 8.1, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl) was added to chromatin, followed by sonication at 4°C by Bioruptor (Diagenode, B01060001) (20 cycles of 30 sec on and 30 sec off). Lysates were precleared with 20 ul Dynabeads Protein A at 4°C for 2 h. 1/30 of lysate was reserved as input, the remainder incubated overnight at 4°C with 2.5 µg SMC3 (Fortis Life Sciences, A300-060A) or 2.5 µg CTCF (Millipore Sigma, 07-729) antibody, followed by capture with 40 ul Dynabeads Protein A for 2-4 h. Immunoprecipitates were washed sequentially in 1 ml each of cold dilution buffer, low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.1), high salt wash buffer (0.1% SDS, 1% Trion X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl pH 8.1), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1% NaDOC, 1 mM EDTA, 50 mM Tris-HCl pH 8.1) and 1X TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA), and eluted in elution buffer (100 mM NaHCO3, 1% SDS) at 65°C for 25 min. Both IP and input DNA had the concentration of EDTA and NaCl adjusted to 0.1 mM and 0.2 M, respectively, and were de-crosslinked at 65°C overnight and purified using MinElute PCR Purification Kit (Qiagen, 28004). ChIP-Seq libraries were prepared with NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, E7645S) and sequenced via Illumina paired-end 150 bp sequencing. Samples were aligned to mm9 using Bowtie 2, processed using SAMtools90 v1.8, deduplicated using Picard (https://broadinstitute.github.io/picard/) and normalized using bamCoverage91 with the --CPM flag. The E2A ChIP seq data was reanalyzed from public dataset (GSM546523)65. CTCF and E2A peak sites in the Igκ locus were called by MACS292 v2.11 narrow peak calling function using WT CTCF ChIP-Seq data in this study and the published E2A data, respectively. For genome-wide peak analysis, reference peak annotation was generated using MACS2 peak calling function on WT samples and retaining only peaks shared between replicates. Heat plots were generated using deepTools291 and excluded outliers that are larger than Q3 + 50x IQR (Q3: 75% percentile; IQR: Interquartile Range) of each column. To investigate potential differences in CTCF binding element sequences between WT and CTCF-Nm mutants, high-confidence peaks (MACS2 p-value < 0.05, further filtered by q-value < 0.01, and overlapping between replicates) were divided into two groups: (1) peaks shared between WT and mutant, and (2) peaks unique to WT. CTCF motifs within each peak were identified using a 19-bp core motif (MA0139.1) from the JASPAR vertebrate database. When multiple motif instances were present in a single peak, only the motif closest to the peak summit was retained for downstream analysis. Motif logos, including the core 19-bp motif plus 16 bp upstream and 18 bp downstream sequences, were generated using WebLogo93 and compared between the two peak groups at both the genome-wide level and within the Igκ locus.

WAPL depletion
Cell lines carrying the WAPL-degron system were treated with 1 uM 5-Ph-IAA (BioAcademia, 30-003). WAPL degradation was evaluated via flow cytometry analyzer comparing parental line, untreated and treated cells after 4 h. Cells were then G1 arrested by 3 uM STI-571 (Sigma-Aldrich, SML1027) with continuous presence of 1 uM 5-Ph-IAA for 4 days. WAPL degradation was checked again by flow cytometry analyzer with G1 arrested parental line and untreated cells as controls. Cells were then collected and subjected to various analysis as described below.

HTGTS-V(D)J-seq library preparation and data analysis
To prepare for HTGTS-V(D)J-Seq, RAG1 was reconstituted in RAG1 deficient Eμ-Bcl2 v-Abl cells via retroviral infection of the pMSCV-RAG1-IRES-Bsr plasmid (a gift from Dr. David Schatz), followed by 4 days of 50 ug/ml blasticidin (Gibco, A1113903) selection to enrich for cells with virus integration. Cells were then arrested by 3 uM STI-571 for 4 days, extracted for genomic DNA and prepared for HTGTS V(D)J-seq libraries7,94. 2–4 ug of DNA were sonicated using the Diagenode Bioruptor (two cycles of 20s on and 60s off) and linearly amplified by Phusion polymerase (Thermofisher Scientific, F530L) with a Jκ1 biotinylated primer (1 uM). Single-stranded products were purified with Dynabeads MyOne Streptavidin C1 beads (Invitrogen, 65002) and ligated to a bridge adaptor (2.5 uM) via on-bead ligation using T4 DNA ligase (New England Biolabs, M0202L) at 16°C overnight. Adaptor-ligated products were amplified by nested PCR with indexed Jκ1 and adaptor primers, followed by tag PCR with P5-I5 and P7-I7 primers. PCR Products (400–700 bp) were gel-purified and sequenced on an Illumina MiSeq with 300-bp paired-end reads, unless otherwise specified. Sequenced libraries were processed via a published pipeline94 (http://robinmeyers.github.io/transloc_pipeline/) with the following filters applied: f.unaligned=1, f.baitonly=1, f.uncut=G10, f.misprimed=L10, f.freqcut=1, f.largegap=G30, f.mapqual=1, f.breaksite=1, f.sequential=1 and f.repeatseq=1. Duplicate reads were then removed. Sequencing reads were aligned to the mm9 genome for strains containing WT Igκ allele. For strains with Igκ modifications, which include the 377-kb proximal Igκ-inversion, Vκ6-15 inversion and dCas9 BS insertion, reads were aligned to modified mm9 genomes incorporating the relative targeted mutations. To analyze the Vκ utilization pattern, relative frequency of individual Vκ usage was presented as a percentage, which was calculated by dividing the read count of each Vκ segment by the total Vκ usage in the library.

3C-HTGTS library preparation and data analysis
3C-HTGTS63 was performed on G1 arrested, RAG1 deficient cells. Briefly, 10 million cells were crosslinked in 12 ml culture medium with 2% formaldehyde (RT, 10 min), quenched with 0.125 M glycine, and lysed on ice for 10 min in cell lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, 1X Protease inhibitor cocktail). Pelleted nuclei were treated sequentially with 0.3% SDS and 1.8% Triton X-100 (37°C, 1 h each), digested overnight with NlaIII (700 U, New England Biolabs, R0125L), heat-inactivated at 65°C for 20 min, and ligated under dilute conditions with T4 DNA ligase (100 U, New England Biolabs, M0202L) at 16°C overnight. Ligated chromatin was de-crosslinked with Proteinase K (Millipore Sigma, 3115852001) at 65°C overnight, treated with RNase A (Invitrogen, 12091021) at 37°C for 1 h, purified by phenol-chloroform extraction, and resuspended in 200 µl 1X TE. 4 ug DNA per sample was processed for Illumina library preparation using the HTGTS-V(D)J-Seq method with Cer or iEκ bait and sequenced via Illumina paired-end 300 bp sequencing. Libraries were processed as described for HTGTS-V(D)J-seq. Sequencing reads were aligned to the mm9 genome, or modified genome for strains with the dCas9 BS insertion. In addition, we filtered reads aligned to telomeric and satellite repeat regions to avoid potential PCR artifacts. We also removed bait region reads (chr6:706586310-70660396 for Cer bait in the mm9 genome, and chr6:70677069-70678882 for iEκ bait in the modified genome with dCas9 BS insertion) to minimize the impact of self-ligation on quantitative analysis. The libraries were then size-normalized to total junctions of the smallest library in the set of libraries for comparison. 3C-HTGTS peaks were called using published code47 (https://github.com/Yyx2626/HTGTS_related) and only recurrent peaks called in at least two independent replicates were kept for analysis.

PRO-Seq library preparation and data analysis
PRO-Seq libraries were generated from G1 arrested, RAG1 deficient cells following a published protocol95 with slight modifications. 5 million nuclei were collected by sucrose gradient centrifugation. Nuclear run-on was performed at 37°C for 5 min by mixing nuclei with 2X run-on master mix (10 mM Tris-HCl pH 8.0, 5 mM MgCl₂, 1 mM DTT, 300 mM KCl, 50 uM Biotin-11-CTP (Revvity, NEL542001EA), 0.5 uM CTP, 250 uM each ATP/CTP/UTP, 10 U RNase inhibitor, 1% sarkosyl), followed by Trizol RNA extraction (Invitrogen, 10296010). RNA was hydrolyzed with 0.2 N NaOH on ice for 12 min, neutralized with 0.55 M Tris-HCl pH 6.8, and buffer-exchanged with Bio-Rad P30 columns (Bio-Rad, 7326250). Biotin-labeled RNA was enriched using Dynabeads MyOne Streptavidin C1 beads, treated with RNA 5’ Pyrophosphohydrolase (New England Biolabs, M0356S) and T4 Polynucleotide Kinase (New England Biolabs, M0201S), and sequentially ligated to 5’ and 3’ RNA adaptors by T4 RNA ligase (New England Biolabs, M0204S) with Streptavidin bead enrichments after each ligation. Reverse transcription was performed using Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific, EP0751) and followed by PCR with indexed Illumina adaptors (8 cycles). 200-500 bp products were selected by SPRIselect beads (Beckman Coulter, B23318) to remove adaptor dimers. Libraries were then amplified to full scale with cycle number determined by test PCR, SPRIselect beads-purified, and sequenced via Illumina paired-end 150-bp sequencing. Reads were aligned to the mm9 genome, and duplicate reads were removed using samtools markdup. Libraries were strand-separated based on alignment orientation and normalized to reads per million. Gene expression was quantified using the HTSeq tool96 by counting reads aligned to gene exons, and results were presented as the ratio of mutant to WT within the same experiment.

Antibodies
Detailed information of all antibodies used in this study is provided in Supplementary Table 2.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 9 via unpaired, two-tailed Student’s t test, except for Supplementary Fig. 7f, in which P value was calculated by the Kolmogorov-Smirnov test. P values are shown as exact values, and when P ≥ 0.05, it is also labeled as ns (non-significant).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper tackles a fundamental mechanistic question – what are the forces that organize the immense length of the immunoglobulin loci such that the VDJ recombination machinery can operate effectively.\nThe study focuses on chromosome organization of Jκ and Vκ gene segments (over a 3.2 Mb region)\nThey note” Loop extrusion and diffusion are considered two key mechanisms underlying genome folding. While both processes are thought to facilitate Igκ recombination, their cooperative mechanisms remain unclear. Our study demonstrates CTCF as a key regulator coupling loop extrusion and diffusion to enhance Vκ recombination “\nMAIN findings\n* CTCF's N-terminus promotes long-range loop extrusion important for distal Vκ usage by stabilizing cohesin against Wapl release.\n* CTCF's N-terminus promotes loop barrier formation crucial for the joining of inversional V  segments by blocking direct extrusion between Jκ s and Vκ s, thereby enabling diffusion.\n* In CTCF N-terminal mutants, defects in inversional Vκ joining were not corrected by Wapl depletion but largely rescued by a targeted dCas9-blockade mimicking the CTCF barrier.\nThis is a technically impressive dissection of the role of CTCF and Wapl in the regulation of deletion vs inversional Vκ joining and the conformation of the kappa locus.\nFor completeness, they should reference https://pmc.ncbi.nlm.nih.gov/articles/PMC10917333/ and mention the role of the E34 enhancer\n(review by Rachel Gerstein, UMASS Chan Medical School)", "reviewer_2": "This manuscript represents an outstanding follow-up to prior work by the senior author addressing the mechanisms by which Vk gene segments are brought into proximity of Jk gene segments for Igk recombination. The prior work had demonstrated that RAG cannot scan from Jk to Vk RSSs; rather, loop extrusion within the Vk array followed diffusion-based RSS synapsis is key to Vk-Jk recombination. Here the authors use a v-Abl pro-B system to analyze mechanisms by which the CTCF N-terminus regulates these processes. Mutation of the CTCF N-terminus caused increased usage of proximal V segments in deletional orientation while suppressing the use of distant V segments of either orientation. The former was inferred to result from reduced Cer-Sis barrier formation, and the latter from reduced long-range interactions with Cer. Defective looping was associated with substantially reduced chromatin-associated SMC3, and reduced SMC3-CTCF but increased SMC3-Wapl interactions, leading to the conclusion that increased cohesin release by Wapl in the mutant impairs long-range loop extrusion. Consistent with this, in CTCF Nm, Wapl degradation led to increased long-range interactions/loop extrusion across the Vk segments, and increased rearrangement of distant V gene segments. But in contrast to Wapl depletion with WT CTCF, rearrangement increased only for those Vk segments that rearrange by deletion, implicating impaired Cer-Sis barrier function. Finally, the authors used an elegant dCas9 blockade system to prove that it is indeed the loss of Cer-Sis barrier function in CTCF Nm that drives the locus towards deletional rearrangement, and that blockade of loop extrusion from Jks to Vks is critical for diffusion to be the dominant mechanism for Vk-Jk recombination. It is concluded that the CTCF N-terminus facilitates Igk repertoire formation by enforcing Cer-Sis barrier function and by facilitating cohesin-mediated loop extrusion across the Vk segments. This work is technically sound and represents a substantial advance for the field. The manuscript is very well written.\n1. The authors acknowledge that CTCF-Nm results in mild reduction of CTCF-binding genome-wide, while the occupancy at Igk locus is significantly reduced. In that case, how do they conclude the resulting phenotype is not simply due to reduced CTCF binding, rather than its impaired interaction with cohesin? It will be informative to show actual CTCF binding peaks, instead of “called” CTCF sites in the highlighted regions in the figures. Also, binding at some IgK CTCF sites seems to be less affected by the mutation than others. For the Vks with no difference in recombination frequencies (e.g. Vks 8-30, 8-21, 8-16), it will be interesting to know the status of nearby CTCF binding.\n2. It may be interesting to compare CBE sequences of sites that lose or retain CTCF binding in CTCF Nm, both within Igk and genome-wide.\n3. Can the authors confirm that there is minimal effect of CTCF-Nm on CTCF binding at Cer and Sis?\n4. In Li et al., Nature, 2020, CTCF YDF mutation caused differential gene expression of >2000 genes in HAP1 cells. Have the authors studied the changes in gene expression in their system? Are there changes in the expression of any genes known to affect loop extrusion and diffusion?\nMinor:\nline 231 – CTCF deficient should be CTCF Nm\nline 251 – Can the authors comment on the fact that interactions with the BS region do not appear to increase with dCas9-blockade?\nLines 261,2 – Mention of increased cohesin processivity, without further explanation or discussion, is a bit obtuse, and is perhaps not helpful.\nLines 318-321 – Since the authors note that secondary rearrangements occur after the Cer-Sis barrier is removed, it may be helpful to note that although these rearrangements should occur primarily by deletion, they can involve V segments that were initially in the wrong orientation since their orientation can be flipped by inversional primary rearrangement.", "reviewer_3": "The question of how numerous V gene segments, scattered over megabase-scale distances, successfully participate in V(D)J recombination to generate a diverse antigen receptor repertoire has been a central issue in the field for decades. While RAG chromatin scanning was definitively shown by this group to drive most D-to-J_H recombination, the mechanism for Vκ-to-Jκ joining is more complex. In the Igκ locus, RAG utilizes Vκ-RSSs from both deletional- and inversional-oriented gene clusters, inconsistent with a purely unidirectional linear scanning model.\nA recent study (Zhang et al., PMID: 38811728) proposed that Igκ recombination is uniquely \"diffusion-friendly,\" relying on strong RSSs and limited extrusion distances to support both deletional and inversional joining. In contrast, IgH recombination is more dependent on long-range loop extrusion and weaker RSSs, thereby biasing strongly toward deletional outcomes.\nThis manuscript makes a significant mechanistic contribution by using an established CTCF N-terminal (CTCF-Nm) mutation to dissect how the architectural protein CTCF governs Igκ recombination. The authors convincingly demonstrate that the CTCF-Nm mutation reduces both distal Vκ usage and long-range interactions with the Cer/Sis elements, while also decreasing inversional recombination events.\nThrough use of Wapl-degron and dCas9-SunTag tethering, the authors provide strong evidence that CTCF has dual roles:\n1. Cohesin stabilization: CTCF’s N-terminus resists Wapl-mediated unloading, facilitating long-range loop extrusion to engage distal Vκs. This aligns with Zhang et al.’s observations on local scanning constraints.\n2. Extrusion barrier formation: CTCF halts cohesin progression at specific boundaries, creating a chromosome architecture and topological window that permits diffusion-mediated inversional pairing.\nBy establishing these two distinct yet complementary roles, the authors extend the \"extrusion + diffusion\" model of Igκ recombination. This work represents a major contribution to our understanding of how genome architecture, recombination signal strength, and chromatin dynamics are integrated to regulate immune diversity.\nThe only major comment is that the paper needs to be revised to make the hypotheses, findings, and conclusions clearer and easy to conceptualize.\nMinor Points for Clarification or Revision:\n1. Line 27: \"Defects in inversional V joining were not corrected by Wapl.\"\n→ Consider revising to: “...were not fully restored by Wapl depletion” for better accuracy.\n2. Line 29–30: “CTCF regulates immune diversity via diverse mechanisms…”\n→ It would strengthen the statement to explicitly mention the two mechanistic roles uncovered in this study: (1) stabilizing loop extrusion via cohesin retention and (2) acting as a chromatin barrier that enables diffusion-based inversional joining.\n3. Line 99: The authors observe a 3.8 fold reduction in recombination with CTCF-Nm. They should confirm that RAG1 expression levels are comparable between the WT and CTCF-Nm cells (assuming it is feasible to perform RAG1 westerns). Normally this would not be a concern but the experiments are being performed in a cell line that also expresses substantial levels of RAG1 D708A. RAG1 forms very tight, stable dimers and WT and D708A RAG1 will dimerize, soaking up a significant fraction of the WT RAG1 into heterodimers with only one functional active site. As a result, even small differences in WT RAG1 expression could result in large differences in levels of fully active WT-WT homodimers. The authors should acknowledge this issue somewhere in the manuscript.\n4. Line 135-137: the way the conclusion is stated is incomplete and a bit misleading. First, the clearest change in the recombination pattern with CTCF-Nm is the large increase usage of proximal forward Vk segments, so one obvious function of CTCF is suppression of high level usage of these segments—and yet the authors do not mention this function. Second, the claim of “mediating” long range recombination goes beyond the data at this point in the manuscript. The decrease in long range recombination could be entirely due to the increase is proximal recombination, in which case CTCF is not positively supporting long range recombination (as implied by “mediating”), but “supports” or “enables” long range recombination by suppressing short-range.\n5. Line 171: “lack” is too strong. “decreased” would be appropriate.\n6. Line 216: It is not clear what is “remarkable” about this finding. Data from Fig. 1 had already shown that inversional recombination was reduced with CTCF-Nm and retained with WT CTCF. What is remarkable about the fact that inversional recombination is unchanged by loss of WAPL with WT CTCF? Please explain more clearly or rewrite.\n7. Line 222: please cite the relevant figure panels.\n8. Line 259: Figure 6f does not allow the reader to compare the recombination pattern in the blockage line to that of WT cells. A figure providing a direct comparison would be helpful.\n9. Clearer presentation requested: when referring to the dCas9 blockade (for example, line 28 in the abstract) it is vital that the authors make clear where the blockade is located. When I read the abstract, I inferred that the blockade was located in the Vk region, only to discover when I got to Figure 6 that the blockade was in a completely different place, between Jk and Vk. The last paragraph of the discussion is very important as it is the first place where the authors distinguish between CTCF sites and functions in Vk versus in the Jk-Vk interval (and throughout the paper, I thought that it was the CTCF sites in Vk that were the focus of the analysis, only to discover that they were not the focus at all). The authors are strongly encouraged to make it clear what they have done with the blockade and why, from the very beginning of the manuscript, and to make clear what they are focusing on and why throughout the manuscript. And see point 14 below: a well-illustrated model figure will help greatly in allowing the authors to specify the role of CTCF at Cer/Sis versus in the Vk region.\n10. Figure labeling (e.g., Fig. 1b): The use of “forward Vκs” and “reverse Vκs” is explained in the text but is not always easy to work with in the figures. For example, in Fig. 1b, it would be helpful if the labels “forward” and “reverse” had a second label in parentheses saying “deletional” and “inversional” to align more clearly with the bar graph labels.\n11. Mechanistic clarification requested: The CTCF-Nm mutation weakens the Cer–Sis boundary, allowing loop extrusion and RAG scanning into Vκs. And strong RSSs in the Vκ region are capable of mediating inversional joining via diffusion. Chromatin loop extrusion will bring the reverse Vk segments close to the Jk RC and yet they don’t recombine efficiently, and this observation is central to the manuscript. The authors should carefully and clearly explain how their findings and model explain this observation. Again, a good graphical model will be a big help.\n12. Rearrangement of weak RSSs: Do weak distal Vκ RSSs or cryptic RSSs (cRSS) show increased recombination when loop extrusion remains intact but barrier function is compromised (e.g., in Wapl-AID2 CTCF-Nm cells)? Any observed cRSS recombination upstream of the Igκ locus would support the model. Information regarding cRSS recombination might be available in the HTGTS data already collected.\n13. Chromatin accessibility and epigenetics: Were chromatin activation marks such as H3K27ac or H3K4me3 profiled across the Vκ region in CTCF-Nm mutant cells? This could help differentiate direct architectural changes from secondary effects on enhancer activity and chromatin changes associated with transcription. This would be a nice addition but is not required.\n14. Model illustration requested: A graphical model illustrating deletional vs. inversional recombination routes, highlighting the roles of CTCF, cohesin, Wapl, and dCas9, would be very helpful for readers to visualize the \"extrusion + diffusion\" framework. In addition, it would help orient readers to have a schematic in Figure 1 showing the conceptual framework that guided the design of the experiments: that is, the RC looping to SIS and the V regions looping to CER.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point responses to reviewer comments\nREVIEWER COMMENTS\nReviewer #1 (B cell development, VDJ) (Remarks to the Author):\nThis paper tackles a fundamental mechanistic question – what are the forces that organize\nthe immense length of the immunoglobulin loci such that the VDJ recombination\nmachinery can operate effectively.\nThe study focuses on chromosome organization of Jκ and Vκ gene segments (over a 3.2 Mb\nregion)\nThey note” Loop extrusion and diffusion are considered two key mechanisms underlying\ngenome folding. While both processes are thought to facilitate Igκ recombination, their\ncooperative mechanisms remain unclear. Our study demonstrates CTCF as a key regulator\ncoupling loop extrusion and diffusion to enhance Vκ recombination “\nMAIN findings\n* CTCF's N-terminus promotes long-range loop extrusion important for distal Vκ usage by\nstabilizing cohesin against Wapl release.\n* CTCF's N-terminus promotes loop barrier formation crucial for the joining of inversional\nV segments by blocking direct extrusion between Jκ s and Vκ s, thereby enabling\ndiffusion.\n* In CTCF N-terminal mutants, defects in inversional Vκ joining were not corrected by Wapl\ndepletion but largely rescued by a targeted dCas9-blockade mimicking the CTCF barrier.\nThis is a technically impressive dissection of the role of CTCF and Wapl in the regulation of\ndeletion vs inversional Vκ joining and the conformation of the kappa locus.\nResponse: We thank the reviewer for this very positive assessment and for recognizing\nthe ‘impressive’ strength of our experimental design and technical approach in\nsupporting our model.\nFor completeness, they should\nreference https://pmc.ncbi.nlm.nih.gov/articles/PMC10917333/ and mention the role of\nthe E34 enhancer\n(review by Rachel Gerstein, UMASS Chan Medical School)\nResponse: This is a great suggestion, we have now added this reference of E34 and\ndescribed its relevant function in the Introduction.\nReviewer #2 (VDJ recombination, epigenetic regulation, DNA repair) (Remarks to the\nAuthor):\naddressing the mechanisms by which Vk gene segments are brought into proximity of Jk\ngene segments for Igk recombination. The prior work had demonstrated that RAG cannot\nscan from Jk to Vk RSSs; rather, loop extrusion within the Vk array followed diffusion-based\nRSS synapsis is key to Vk-Jk recombination. Here the authors use a v-Abl pro-B system to\nanalyze mechanisms by which the CTCF N-terminus regulates these processes. Mutation\nof the CTCF N-terminus caused increased usage of proximal V segments in deletional\norientation while suppressing the use of distant V segments of either orientation. The\nformer was inferred to result from reduced Cer-Sis barrier formation, and the latter from\nreduced long-range interactions with Cer. Defective looping was associated with\nsubstantially reduced chromatin-associated SMC3, and reduced SMC3-CTCF but\nincreased SMC3-Wapl interactions, leading to the conclusion that increased cohesin\nrelease by Wapl in the mutant impairs long-range loop extrusion. Consistent with this, in\nCTCF Nm, Wapl degradation led to increased long-range interactions/loop extrusion\nacross the Vk segments, and increased rearrangement of distant V gene segments. But in\ncontrast to Wapl depletion with WT CTCF, rearrangement increased only for those Vk\nsegments that rearrange by deletion, implicating impaired Cer-Sis barrier function. Finally,\nthe authors used an elegant dCas9 blockade system to prove that it is indeed the loss of\nCer-Sis barrier function in CTCF Nm that drives the locus towards deletional\nrearrangement, and that blockade of loop extrusion from Jks to Vks is critical for diffusion\nto be the dominant mechanism for Vk-Jk recombination. It is concluded that the CTCF N-\nterminus facilitates Igk repertoire formation by enforcing Cer-Sis barrier function and by\nfacilitating cohesin-mediated loop extrusion across the Vk segments. This work is\ntechnically sound and represents a substantial advance for the field. The manuscript is\nvery well written.\nResponse: We thank the reviewer for this strongly positive review which clearly points\nout the high quality of our experiments and the high impact of our studies for the field.\nWe appreciate that the review found our work to be ‘outstanding’, ‘technically sound’\nand ‘represents a substantial advance for the field’.\nWe also thank the reviewer for the compliments on our writing.\n1. The authors acknowledge that CTCF-Nm results in mild reduction of CTCF-binding\ngenome-wide, while the occupancy at Igk locus is significantly reduced. In that case, how\ndo they conclude the resulting phenotype is not simply due to reduced CTCF binding,\nrather than its impaired interaction with cohesin? It will be informative to show actual\nCTCF binding peaks, instead of “called” CTCF sites in the highlighted regions in the figures.\nAlso, binding at some IgK CTCF sites seems to be less affected by the mutation than\nothers. For the Vks with no difference in recombination frequencies (e.g. Vks 8-30, 8-21, 8-\n16), it will be interesting to know the status of nearby CTCF binding.\nResponse: This is an excellent question. Several lines of evidence strongly suggest\nthat the CTCF-Nm phenotype results from defective N-terminal-dependent stabilization\nof cohesin, rather than reduced CTCF binding per se: 1. Distal Vκs within regions of\nrelatively preserved CTCF binding still showed significantly reduced usage, indicating\nthat retained CTCF was non-functional (Supplementary Fig. 5c). 2. WAPL depletion,\n2\nwhich enhances cohesin processivity without affecting CTCF binding, rescued distal\ndeletional joining in the CTCF-Nm mutants (Fig. 4e). 3. Despite only mild reductions in\nbinding at the Cer-Sis region, long-range interactions with distal Vks, including those\nmediated by E2A, were substantially diminished (Supplementary Fig. 5b; Fig. 3a).\n4. Targeted dCas9 blockade near Cer-Sis, which is unlikely to affect CTCF binding,\nstrongly rescued recombination across the locus (Fig. 6f). We have now incorporated\nthese points into the Discussion section (highlighted in yellow).\nWe also appreciate the reviewer’s suggestion to overlay Vκ segments with interaction\nprofiles and actual CTCF ChIP-Seq signals, particularly for CTCF sites less affected by\nthe mutation and Vκs whose usage was less impacted. To fully address this, we have\nincluded a zoomed-in view of a 400-kb distal Vκ region containing CTCF sites relatively\ninsensitive to CTCF-Nm, presented after the introduction of the CTCF ChIP-Seq data;\nVk segments in this region exhibited similar decreases in usage as other distal Vκs\n(Supplementary Fig. 5c). This indicates that, although differential binding likely reflects\nintrinsic CTCF site strength, retained binding in CTCF-Nm mutants may be non-\nfunctional due to the N-terminal mutation.\nAdditionally, we included zoomed-in views of a proximal Vk region containing multiple\ninversional Vks showing little change in usage, and found that these Vks lack adjacent\nCTCF binding sites (Supplementary Fig. 5d). This indicates that retained CTCF binding\nis not responsible for their preserved usage.\n2. It may be interesting to compare CBE sequences of sites that lose or retain CTCF binding\nin CTCF Nm, both within Igk and genome-wide.\nResponse: This is a good point. We have now included a comparative analysis of\nCTCF sites that retain or lose binding in CTCF-Nm mutant cells, both genome-wide and\nwithin the Ig locus (Supplementary Fig. 5e). The analysis encompassed the 19-bp core\nmotif plus 16 bp upstream and 18 bp downstream sequences, but revealed no obvious\ndifferences in DNA sequence composition. This may suggest that factors beyond the\ncore motif and its immediate flanking sequences contribute to the N-terminal-dependent\nbinding sensitivity, warranting further investigation in future studies.\n3. Can the authors confirm that there is minimal effect of CTCF-Nm on CTCF binding at Cer\nand Sis?\nResponse: We agree with the reviewer that this is an important point to highlight. We\nhave now added zoomed-in views of CTCF binding at the Cer-Sis region in\nSupplementary Fig. 5b, which demonstrated that unlike many V CTCF sites, CTCF\nbinding at Cer and Sis is only mildly impacted in CTCF-Nm cells. This observation is\nnow cited in the revised Results section.\n4. In Li et al., Nature, 2020, CTCF YDF mutation caused differential gene expression\nof >2000 genes in HAP1 cells. Have the authors studied the changes in gene expression in\ntheir system? Are there changes in the expression of any genes known to affect loop\nextrusion and diffusion?\nResponse: This is a great question. We have now performed and added PRO-Seq\ndata for WT and CTCF-Nm mutants from three independent biological replicates.\nsignificantly impact the expression of key factors known or hypothesized to be involved\nin loop extrusion or diffusion (Supplementary Fig. 6a).\nMinor:\nline 231 – CTCF deficient should be CTCF Nm\nResponse: Thanks for pointing this out, we have now corrected it.\nline 251 – Can the authors comment on the fact that interactions with the BS region do not\nappear to increase with dCas9-blockade?\nResponse: This is a great question. We believe the lack of interaction peaks at the BS\nregion is due to the dCas9-SunTag complex physically obstructing NlaIII digestion\nduring 3C-HTGTS library preparation, thereby preventing detection of these fragments.\nThe BS region contains four NlaIII recognition sites (CATG), all within 5 bp of BS,\nmaking them particularly susceptible to steric hindrance by dCas9 binding. This\nexplanation is consistent with previous reports that dCas9 occupancy can block\nrestriction digestion (Saifaldeen et al., 2021, PMID: 33876957). We have illustrated the\npositions of the NlaIII sites relative to BSs in Supplementary Fig. 9a and now include\nthis interpretation in the Results section.\nLines 261,2 – Mention of increased cohesin processivity, without further explanation or\ndiscussion, is a bit obtuse, and is perhaps not helpful.\nResponse: Thanks for this pointing this out, we agree with the reviewer and have now\ndeleted this sentence.\nLines 318-321 – Since the authors note that secondary rearrangements occur after the Cer-\nSis barrier is removed, it may be helpful to note that although these rearrangements should\noccur primarily by deletion, they can involve V segments that were initially in the wrong\norientation since their orientation can be flipped by inversional primary rearrangement.\nResponse: We have now edited the Discussion to include this point.\nReviewer #3 (VDJ recombination, DNA repair, SHM) (Remarks to the Author):\nThe question of how numerous V gene segments, scattered over megabase-scale\ndistances, successfully participate in V(D)J recombination to generate a diverse antigen\nreceptor repertoire has been a central issue in the field for decades. While RAG chromatin\nscanning was definitively shown by this group to drive most D-to-J_H recombination, the\nmechanism for Vκ-to-Jκ joining is more complex. In the Igκ locus, RAG utilizes Vκ-RSSs\nfrom both deletional- and inversional-oriented gene clusters, inconsistent with a purely\nunidirectional linear scanning model.\nA recent study (Zhang et al., PMID: 38811728) proposed that Igκ recombination is uniquely\n\"diffusion-friendly,\" relying on strong RSSs and limited extrusion distances to support both\ndeletional and inversional joining. In contrast, IgH recombination is more dependent on\nlong-range loop extrusion and weaker RSSs, thereby biasing strongly toward deletional\noutcomes.\nThis manuscript makes a significant mechanistic contribution by using an established\ngoverns Igκ recombination. The authors convincingly demonstrate that the CTCF-Nm\nmutation reduces both distal Vκ usage and long-range interactions with the Cer/Sis\nelements, while also decreasing inversional recombination events.\nThrough use of Wapl-degron and dCas9-SunTag tethering, the authors provide strong\nevidence that CTCF has dual roles:\n1. Cohesin stabilization: CTCF’s N-terminus resists Wapl-mediated unloading, facilitating\nlong-range loop extrusion to engage distal Vκs. This aligns with Zhang et al.’s observations\non local scanning constraints.\n2. Extrusion barrier formation: CTCF halts cohesin progression at specific boundaries,\ncreating a chromosome architecture and topological window that permits diffusion-\nmediated inversional pairing.\nBy establishing these two distinct yet complementary roles, the authors extend the\n\"extrusion + diffusion\" model of Igκ recombination. This work represents a major\ncontribution to our understanding of how genome architecture, recombination signal\nstrength, and chromatin dynamics are integrated to regulate immune diversity.\nResponse: We thank the reviewer for this highly positive review, highlighting that our\nevidence strongly supports our proposed model and represents ‘a significant\nmechanistic advance’ in the field.\nThe only major comment is that the paper needs to be revised to make the hypotheses,\nfindings, and conclusions clearer and easy to conceptualize.\nResponse: Thanks for this valuable feedback. In response, we have revised the\nmanuscript to improve the clarity and presentation of the hypotheses, findings, and\nconclusions as suggested. Importantly, we have added two model figures, one main\n(Fig. 7) and one supplementary (Supplementary Fig. 10) to help explaining our findings.\nMinor Points for Clarification or Revision:\n1. Line 27: \"Defects in inversional V joining were not corrected by Wapl.\"\n→ Consider revising to: “...were not fully restored by Wapl depletion” for better accuracy.\nResponse: Thanks for the suggestion. We have revised the wording, and now the\nsentence reads as “Defects in inversional V joining were not restored by WAPL”. We\nopted not to use “fully,” as it could suggest a partial rescue, whereas our data indicate\nthat Wapl depletion did not restore the inversional joining defects to any extent.\n2. Line 29–30: “CTCF regulates immune diversity via diverse mechanisms…”\n→ It would strengthen the statement to explicitly mention the two mechanistic roles\nuncovered in this study: (1) stabilizing loop extrusion via cohesin retention and (2) acting as\na chromatin barrier that enables diffusion-based inversional joining.\nResponse: Thanks for the suggestion. We have revised this sentence to “These\nfindings highlight how CTCF coordinates distinct genome-folding mechanisms through\nits dual roles in cohesin stabilization and extrusion barrier formation to ensure efficient\nVκ recombination, providing insights into antibody diversification and genome regulation\nmore broadly.” We opted not to restate the full details of the specific roles of CTCF in\nthis final sentence because they are already described earlier in the abstract: “We show\n5\nthat CTCF's N-terminus promotes long-range loop extrusion important for distal Vκ\nusage by stabilizing cohesin against Wapl release,” and “Furthermore, CTCF's N-\nterminus promotes loop barrier formation crucial for the joining of inversional Vκ\nsegments.”\n3. Line 99: The authors observe a 3.8 fold reduction in recombination with CTCF-Nm. They\nshould confirm that RAG1 expression levels are comparable between the WT and CTCF-\nNm cells (assuming it is feasible to perform RAG1 westerns). Normally this would not be a\nconcern but the experiments are being performed in a cell line that also expresses\nsubstantial levels of RAG1 D708A. RAG1 forms very tight, stable dimers and WT and D708A\nRAG1 will dimerize, soaking up a significant fraction of the WT RAG1 into heterodimers with\nonly one functional active site. As a result, even small differences in WT RAG1 expression\ncould result in large differences in levels of fully active WT-WT homodimers. The authors\nshould acknowledge this issue somewhere in the manuscript.\nResponse: Thanks for this insightful suggestion. We have now performed PRO-Seq\nanalysis and found that, while the CTCF-Nm mutation has little impact on other key\nfactors involved in V(D)J recombination, it increases the expression of RAG1 and RAG2\n(Supplementary Fig. 6c). Western blot analysis, as suggested, further confirmed\nenrichment of RAG1 protein (Supplementary Fig. 6d). One potential explanation is that\nRAG1 and RAG2 are expressed as transgenes in these cells and might be more\nsusceptible to the effects of the CTCF-Nm mutation. We have included these results\nand acknowledged this issue in the manuscript.\nOf note, PRO-Seq analysis also showed a significant impact of CTCF-Nm on\ntranscriptional activity at the recombination center (RC), but not at the Vκ region\n(Supplementary Fig. 6b), consistent with our prior hypothesis that CTCF-Nm negatively\nimpacts RC activity. Future studies will help to determine the relative contributions of\nthese mechanisms to the overall reduction in Igκ recombination.\n4. Line 135-137: the way the conclusion is stated is incomplete and a bit misleading. First,\nthe clearest change in the recombination pattern with CTCF-Nm is the large increase\nusage of proximal forward Vk segments, so one obvious function of CTCF is suppression of\nhigh level usage of these segments—and yet the authors do not mention this function.\nSecond, the claim of “mediating” long range recombination goes beyond the data at this\npoint in the manuscript. The decrease in long range recombination could be entirely due to\nthe increase is proximal recombination, in which case CTCF is not positively supporting\nlong range recombination (as implied by “mediating”), but “supports” or “enables” long\nrange recombination by suppressing short-range.\nResponse: Thanks for pointing this out. We have revised the text accordingly where\nwe first introduced the Igk recombination defect phenotype “However, in the CTCF-Nm\nmutants, there was a considerable change in the pattern of Vk utilization, with\nsignificantly decreased middle/distal Vk usage accompanied by a substantial increase in\nthe frequency of proximal Vk usage (Fig. 1b, left panel). This suggests the CTCF N-\nterminus plays an important role in supporting long-range Vk joining, though it remains\nunclear at this point whether this effect occurs indirectly by suppressing proximal Vκ\njoining or directly by promoting distal Vκ joining.”, Additionally, we replaced “mediating”\n6\nwith “supporting” as suggested in the reference sentence “Collectively, the HTGTS-\nV(D)J-Seq analysis indicated two major roles of the CTCF-N terminus in Igk\nrecombination: (1) supporting long-range Vk usage, and (2) promoting the utilization of\ninversional Vks. We then sought out to explore the mechanistic basis.”\n5. Line 171: “lack” is too strong. “decreased” would be appropriate.\nResponse: We have now replaced “lack” with “decreased.”\n6. Line 216: It is not clear what is “remarkable” about this finding. Data from Fig. 1 had\nalready shown that inversional recombination was reduced with CTCF-Nm and retained\nwith WT CTCF. What is remarkable about the fact that inversional recombination is\nunchanged by loss of WAPL with WT CTCF? Please explain more clearly or rewrite.\nResponse: We have now replaced “Remarkably, however” with “On the other hand”.\n7. Line 222: please cite the relevant figure panels.\nResponse: We have now added the relevant figure citation.\n8. Line 259: Figure 6f does not allow the reader to compare the recombination pattern in\nthe blockage line to that of WT cells. A figure providing a direct comparison would be\nhelpful.\nResponse: We have now added a new figure panel (Supplementary Fig. 9b) to provide\na direct comparison of Vκ utilization frequency between the dCas9-blockade and WT\nlines.\n9. Clearer presentation requested: when referring to the dCas9 blockade (for example, line\n28 in the abstract) it is vital that the authors make clear where the blockade is located.\nWhen I read the abstract, I inferred that the blockade was located in the Vk region,\nonly to discover when I got to Figure 6 that the blockade was in a completely different place,\nbetween Jk and Vk. The last paragraph of the discussion is very important as it is the first\nplace where the authors distinguish between CTCF sites and functions in Vk versus in the\nJk-Vk interval (and throughout the paper, I thought that it was the CTCF sites in Vk that\nwere the focus of the analysis, only to discover that they were not the focus at all). The\nauthors are strongly encouraged to make it clear what they have done with the blockade\nand why, from the very beginning of the manuscript, and to make clear what they are\nfocusing on and why throughout the manuscript. And see point 14 below: a well-illustrated\nmodel figure will help greatly in allowing the authors to specify the role of CTCF at Cer/Sis\nversus in the Vk region.\nResponse: We have made the following revisions to clarify the design and location of\nthe dCas9 blockade, as well as to distinguish the potential functional differences\nbetween CTCF sites at the Igκ Cer-Sis region and other Vκ CTCF sites:\n1. Abstract: We now explicitly indicate the location of the dCas9 blockade: “In CTCF N-\nterminal mutants, defects in inversional Vκ joining were not restored by WAPL depletion\nbut largely rescued by a dCas9-blockade targeted to the Vk-Jk intergenic region,\nmimicking the CTCF barrier.”\n7\n2. Introduction: further highlighted the role of Cer-Sis: Importantly, Vks are insulated\nfrom the recombination center by the Cer and Sis elements interacting with the Vk\nregion and recombination center, respectively47. Each element contains two CTCF sites,\nwith those in Cer oriented toward Vks and those in Sis oriented toward the\nrecombination center, together supporting a balanced Vκ repertoire54, 55 in a manner\ndependent on the orientation of these CTCF sites56.\n3. Results, CTCF ChIP-Seq profiles: We have now added a zoomed-in profile of Igk\nCer-Sis region, showing that unlike many Vk CTCF sites, CTCF binding at Cer-Sis is\nlargely retained: “We also noted that while the mutation only mildly reduced CTCF\nbinding genome-wide and at the Igk Cer-Sis elements, binding at Vk locus was\nsignificantly decreased at many loci, suggesting many Vk CTCF sites have relatively\nweak affinity that is dependent on a functional N-terminus of CTCF (Supplementary Fig.\n5b)”\n4. Results, dCas9 blockade experiments: We have added a sentence explaining the\nrationale for targeting the blockade to the Vκ–Jκ intergenic region: “The CTCF barriers\nlikely enable diffusion by preventing direct extrusion between Jκs and Vκs, a process\nthat would otherwise inhibit inversional RAG recombination. This hypothesis predicts\nthat extrusion barriers at the Vk-Jk intergenic region are of critical importance.”\n5. Results, ending sentence of the dCas9-blockade section: We have emphasized the\ngreater importance of the Vκ-Jκ intergenic barrier compared with other locus-wide\nbarriers: “Collectively, these findings provided strong evidence that extrusion barriers,\nparticularly at the Vk-Jk intergenic region, are crucial for inversional Vκ joining via\ndiffusion.”\n6. Discussion/Model figure: Importantly, we have added two model figures, one main\nand one supplementary, to more clearly illustrate our findings. We have added a\nschematic model in Fig. 7 to highlight the role of Cer-Sis (Fig. 7a) and the impact of the\ndCas9 blockade (Fig. 7c). In addition, we have included a model in Supplementary Fig.\n10 to illustrate the potential roles of Vκ CTCF sites during primary and secondary\nrearrangement, respectively.\n10. Figure labeling (e.g., Fig. 1b): The use of “forward Vκs” and “reverse Vκs” is explained in\nthe text but is not always easy to work with in the figures. For example, in Fig. 1b, it would\nbe helpful if the labels “forward” and “reverse” had a second label in parentheses saying\n“deletional” and “inversional” to align more clearly with the bar graph labels.\nResponse: Thanks for the suggestion. We have now added 'Del' (standing for\ndeletional) and 'Inv' (standing for inversional) in parentheses throughout the main and\nsupplementary figures.\n11. Mechanistic clarification requested: The CTCF-Nm mutation weakens the Cer–Sis\nboundary, allowing loop extrusion and RAG scanning into Vκs. And strong RSSs in the Vκ\nregion are capable of mediating inversional joining via diffusion. Chromatin loop extrusion\nwill bring the reverse Vk segments close to the Jk RC and yet they don’t recombine\nefficiently, and this observation is central to the manuscript. The authors should carefully\nand clearly explain how their findings and model explain this observation. Again, a good\ngraphical model will be a big help.\nTo aid clarity, we have now added a new model (Figure 7) summarizing our findings.\nWe believe this model, together with the following discussion, highlights the central\nobservation mentioned above, which showed that efficient inversional RSS joining\nrequires the barrier function of CTCF.\n“Our findings also highlight that long-range loop extrusion is necessary but not sufficient\nfor recombination of most inversional Vks, which also depend on CTCF’s role as an\nextrusion barrier. This contrasts with most deletional Vκ recombination that can occur\nefficiently without CTCF barriers. Accordingly, WAPL depletion in CTCF-Nm cells\nrestores distal deletional Vκ joining but does not rescue inversional Vκ joining (Fig. 4e,\nFig. 7b). Moreover, a striking difference was observed in inversional Vk joining\nfrequency between WAPL-depleted CTCF-Nm cells and WAPL-depleted only cells\nretaining CTCF barrier function (Fig. 5b).\nAlthough long suspected, our study provided the direct evidence that mechanistically,\nthe ability of CTCF barriers to block direct extrusion between Jks and Vks is essential\nfor inversional Vk joining as loop extrusion strongly favors deletional joins. In this\nregard, inversion of the proximal Vk region in CTCF-Nm cells compromising CTCF\nbarriers largely reversed the recombination strength of the contained Vks based on their\nRSS orientations (Fig. 2b-d). Moreover, introducing a CTCF-barrier mimicking dCas9\nblockade targeting the Vκ-to-Jκ intergenic region significantly enhances inversional Vκ\njoining across the Igk locus in the CTCF-Nm background (Fig. 6f).”\n12. Rearrangement of weak RSSs: Do weak distal Vκ RSSs or cryptic RSSs (cRSS) show\nincreased recombination when loop extrusion remains intact but barrier function is\ncompromised (e.g., in Wapl-AID2 CTCF-Nm cells)? Any observed cRSS recombination\nupstream of the Igκ locus would support the model. Information regarding cRSS\nrecombination might be available in the HTGTS data already collected.\nResponse: As suggested, we examined rearrangement of weak distal deletional Vκs\n(the 11 least-used prior to Wapl depletion) compared with strong distal deletional Vκs\n(the 11 most-used prior to Wapl depeletion) following Wapl depletion in CTCF-Nm cells.\nNo significant differences were observed, consistent with a model in which the changes\nreflect increased RAG scanning rather than selective targeting of certain strong Vk-\nRSSs. This analysis has been added to the Results (Supplementary Fig. 7e).\nWe also examined cRSS usage, but these events were detected only at very low\nfrequencies in Wapl-depleted CTCF-Nm cells and showed minimal recurrence across\nreplicates. Given this low signal, which is consistent with limited RC activity and possibly\nrapid bypass of potential cRSS sites during extended loop extrusion in the absence of\nbarriers in these cells, further detailed analysis was prevented.\n9\n13. Chromatin accessibility and epigenetics: Were chromatin activation marks such as\nH3K27ac or H3K4me3 profiled across the Vκ region in CTCF-Nm mutant cells? This could\nhelp differentiate direct architectural changes from secondary effects on enhancer activity\nand chromatin changes associated with transcription. This would be a nice addition but is\nnot required.\nResponse: Thanks for the suggestion. Although we do not currently have H3K27ac or\nH3K4me3 ChIP-Seq data, we have now performed PRO-Seq analysis (Supplementary\nFig. 6b) that directly measures transcription activity. The results show that germline\ntranscription across the Vκ region is largely maintained in CTCF-Nm mutant cells,\nindicating that altered transcriptional/chromatin accessibility across the Vk region does\nnot explain the observed Igκ recombination defects in long-range Vk usage and\ninversional joining. This new analysis has been incorporated into the Results section.\n14. Model illustration requested: A graphical model illustrating deletional vs. inversional\nrecombination routes, highlighting the roles of CTCF, cohesin, Wapl, and dCas9, would be\nvery helpful for readers to visualize the \"extrusion + diffusion\" framework. In addition, it\nwould help orient readers to have a schematic in Figure 1 showing the conceptual\nframework that guided the design of the experiments: that is, the RC looping to SIS and the\nV regions looping to CER.\nResponse: Thanks for this excellent suggestion. We have now added a model in the\nmain Figure 7 to illustrate the roles of Cer-Sis, cohesin, Wapl, and dCas9. In addition,\nSupplementary Figure 10 has been included to illustrate the potential roles of CTCF\nsites in Vk region.\nWe also appreciate the suggestion of highlighting the findings in a previous elegant\nstudy that the RC looping to SIS and the V regions looping to CER. We have\nincorporated the corresponding text in both Introduction to provide relevant background\nand Discussion for the clarity of the model.\nIntroduction: “Importantly, Vks are insulated from the recombination center by the Cer\nand Sis elements interacting with the Vk region and recombination center,\nrespectively47. Each element contains two CTCF sites, with those in Cer oriented toward\nVks and those in Sis oriented toward the recombination center, together supporting a\nbalanced Vκ repertoire54, 55 in a manner dependent on the orientation of these CTCF\nsites56”.\ninto close proximity, within a subdiffusive range, at which point diffusion takes over. This\naligns with a recent elegant study showing that the Vk region loops to the Cer element\nand the Jk recombination center loops to the Sis element47, a configuration likely\nenabled by the specific orientation organization of CTCF sites within these elements,\nwith their N-termini facing Vks and Jks, respectively (Fig. 7a). “\nReviewer #4 (EC) (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We thank the reviewer for the careful and insightful evaluation of our work.\nREVIEWERS' COMMENTS\nReviewer #2 (Remarks to the Author):\nThe authors have satisfactorily addressed all of my questions and comments and I am strongly\nin favor of publication.\nResponse: We thank the reviewer for this strongly positive assessment of our revision.\nI suggest one minor edit:\nLines 112-13: rewrite to \"which might further indicate a negative impact on Igk recomination\ncenter activity or RAG activity by the CTCF-Nm mutation\"\nResponse: Thanks. We have edited the text as suggested.\nReviewer #3 (Remarks to the Author):\nThe authors have done an excellent job addressing our comments and those of the other\nreviewers. The model figures are a big help in making the results understandable and the\nconclusions clear. The manuscript is much improved and we support publication.\nResponse: We thank the reviewer for this highly positive assessment of our revision.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for the careful evaluation of our work.\n1"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have satisfactorily addressed all of my questions and comments and I am strongly in favor of publication.\nI suggest one minor edit:\nLines 112-13: rewrite to \"which might further indicate a negative impact on Igk recomination center activity or RAG activity by the CTCF-Nm mutation\"", "reviewer_3": "The authors have done an excellent job addressing our comments and those of the other reviewers. The model figures are a big help in making the results understandable and the conclusions clear. The manuscript is much improved and we support publication.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point responses to reviewer comments\nREVIEWERS' COMMENTS\nReviewer #2 (Remarks to the Author):\nThe authors have satisfactorily addressed all of my questions and comments and I am strongly\nin favor of publication.\nResponse: We thank the reviewer for this strongly positive assessment of our revision.\nI suggest one minor edit:\nLines 112-13: rewrite to \"which might further indicate a negative impact on Igk recomination\ncenter activity or RAG activity by the CTCF-Nm mutation\"\nResponse: Thanks. We have edited the text as suggested.\nReviewer #3 (Remarks to the Author):\nThe authors have done an excellent job addressing our comments and those of the other\nreviewers. The model figures are a big help in making the results understandable and the\nconclusions clear. The manuscript is much improved and we support publication.\nResponse: We thank the reviewer for this highly positive assessment of our revision.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for the careful evaluation of our work.\n1"}]

Decision letter