Charlotte Bruzeau

Justine Pollet

Morgane Thomas

Zhaoqing Ba

David Roulois

Eric Pinaud
email: eric.pinaud@unilim.fr.

Sandrine Le Noir
email: sandrine.le-noir@unilim.fr

Core enhancers of the 3'RR optimize IgH nuclear position and loop conformation for oriented CSR

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

3'RR core enhancers are necessary and sufficient to preorganize resting B cell nuclei to facilitate a deletional CSR mechanism at activated stage. We demonstrated that the 3'RR core enhancers regulate IgH locus addressing in the nuclei, control IgH locus accessibility and orchestrate IgH loops formation.

Our findings pinpointed an additional regulation level of mechanisms underlying B cell diversification.

Introduction

Late stages of B cell development are characterized by immunoglobulin genes remodeling to produce highly specific antibodies. Somatic hypermutation (SHM) and class switch recombination (CSR) are the two mains Immunoglobulin Heavy Chain (IgH) locus modifications that both require transcription of target sequences and cytidine deamination by the activation-induced cytidine deaminase (AID) enzyme [START_REF] Muramatsu | Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme[END_REF]. It has been extensively documented in literature that these events are under the transcriptional control of the super-enhancer located at IgH locus 3'end: the IgH 3'Regulatory Region (3'RR) [START_REF] Pinaud | The IgH locus 3' regulatory region: pulling the strings from behind[END_REF]. In mice, this region is composed of four core enhancers, (hs3a, hs1.2, hs3b and hs4), which are surrounded by inverted repeated sequence (IRIS) centered on hs1.2 enhancer and delimited by hs3a and hs3b (3) (Figure 1A). IgH locus forms a topologically associated domain (TAD) as demonstrated by chromosome conformation capture (3C)-based studies [START_REF]Extremely Long-Range Chromatin Loops Link Topological Domains to Facilitate a Diverse Antibody Repertoire -PubMed[END_REF]. The 3' end of the locus is characterized by an insulator region composed of 10 CTCF (CCCTC-binding factor) binding site named 3' CTCF binding elements (3'CBE) which delimited the 3' IgH TAD border [START_REF] Zhang | Physiological role of the 3'IgH CBEs super-anchor in antibody class switching[END_REF]. Several mouse models carrying partial or total deletions of 3'RR region allow to decipher the exact role of both core enhancers and quasipalindromic structure on SHM, CSR, transcription and immunoglobulin production during B cell development [START_REF] Bruzeau | Panorama of stepwise involvement of the IgH 3' regulatory region in murine B cells[END_REF]. The 3'RR entire deletion leads to a 2-fold decrease of IgH germline transcription (GLT), and consequently, totally abolishes SHM and CSR events [START_REF] Vincent-Fabert | Genomic deletion of the whole IgH 3' regulatory region (hs3a, hs1,2, hs3b, and hs4) dramatically affects class switch recombination and Ig secretion to all isotypes[END_REF]. Interestingly 3'RR core enhancers (c3'RR mouse model) are mandatory for full activity of the 3'RR super-enhancer, and the surrounding sequences do not constitute useless junk DNA, but contribute to 3'RR activity: while CSR is supported by enhancers alone, SHM requires both 3'RR enhancers and its palindromic architecture [START_REF] Le Noir | Functional anatomy of the immunoglobulin heavy chain 3΄ super-enhancer needs not only core enhancer elements but also their unique DNA context[END_REF][START_REF] Saintamand | Deciphering the importance of the palindromic architecture of the immunoglobulin heavy-chain 3' regulatory region[END_REF].

Beyond transcriptional control of SHM and CSR, additional levels of regulation occurred during late B cell maturation. Most of Ig gene regulation studies have been so far performed at nucleosomal scale (epigenetic modifications and regulatory transcription of loci and gene segments). The increasing interest for the understanding of gene regulation at the whole nuclei scale raises the necessity to revisit previous models at both supranucleosomal (DNA loops and A/B compartments) and nuclear (chromosome territories and gene positioning) levels (9). To date, numerous studies showed that nuclear positioning of IgH loci is dynamic and regulated during early and late B cell development and IgH remodeling regulation, especially CSR recombination, also occurs at supranucleosomal level [START_REF] Bruzeau | Contribution of Immunoglobulin Enhancers to B Cell Nuclear Organization[END_REF].

CSR mechanism involves DNA double strand breaks (DSB) at Sµ donor and Sx acceptor regions [START_REF] Jung | Shutdown of class switch recombination by deletion of a switch region control element[END_REF].

Mammalian switch (S) regions, located upstream of each constant gene except C consist of 1 to 10 kb-long highly repetitive G-rich DNA sequences containing clusters of RGYW AID deamination hotspots [START_REF] Stavnezer | Immunoglobulin class switching[END_REF]. AID-induced DSB at S regions are followed by the deletion of DNA segment located between both DNA DSB and finally replacing Cµ constant gene by one of the six Cx constant gene (IgG3, IgG1, IgG2a, IgG2b, IgA, IgE). The recombination event between DNA ends is essentially ensured by the NHEJ complex to achieve productive deletional CSR [START_REF] Iwasato | Circular DNA is excised by immunoglobulin class switch recombination[END_REF]. Mature resting B cells ensure an IgH locus configuration "prepared for CSR" by bringing in close contact the 3'RR and Eµ-regions, thereby forming a 50-200Kb "big chromatin loop" that contains all S regions. Upon stimulation, this configuration acquires additional contact between the acceptor S region involved in CSR and the previously interacting IgH enhancers [START_REF] Wuerffel | S-S Synapsis during Class Switch Recombination Is Promoted by Distantly Located Transcriptional Elements and Activation-Induced Deaminase[END_REF][START_REF] Zhang | Fundamental roles of chromatin loop extrusion in antibody class switching[END_REF][START_REF] Zhang | The role of chromatin loop extrusion in antibody diversification[END_REF]. In this new layout, S donor and S acceptor regions are aligned to favor productive deletional CSR. DSB response factors inhibition impact the ratio of deletional vs inversional CSR pathway, highlighting the important role of DNA repair pathway for efficient deletional CSR [START_REF] Dong | Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching[END_REF]. The current model proposes that such loops are formed by loop extrusion, a mechanism consisting in loading cohesin complexes at transcribed enhancers or promoters [START_REF] Zuin | A Cohesin-Independent Role for NIPBL at Promoters Provides Insights in CdLS[END_REF][START_REF] Dorsett | Cohesin at active genes: a unifying theme for cohesin and gene expression from model organisms to humans[END_REF][START_REF] Kagey | Mediator and Cohesin Connect Gene Expression and Chromatin Architecture[END_REF] followed by chromatin extrusion until cohesin meets convergently oriented CTCF sites [START_REF] Zhang | Physiological role of the 3'IgH CBEs super-anchor in antibody class switching[END_REF]. In the specific case of IgH locus, cohesin is loaded at enhancers (either 3'RR or Eµ regions) to first initiate the "big chromatin loop" between 3'RR and Iµ/Sµ regions at the resting stage. Upon B cell stimulation, new internal loops are shaped to bring together transcribed S regions in order to form the CSR center (CSRC) [START_REF] Zhang | Fundamental roles of chromatin loop extrusion in antibody class switching[END_REF][START_REF] Zhang | The role of chromatin loop extrusion in antibody diversification[END_REF], a structure that favors deletional CSR recombination [START_REF] Dong | Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching[END_REF].

The role of 3'RR and its components, core enhancers and palindromic structure, remains to be determined in the context of CSR center formation. Here, by taking advantage of two mouse models devoid of either the entire 3'RR (3'RR ( 6)) or only its palindromic structure (c3'RR model ( 7)), we evaluated by multiple approaches the role of 3'RR core enhancers in B cell nuclear organization and IgH locus architecture. Interestingly, we showed that the four 3'RR core enhancers are necessary and sufficient to pre-organize resting B cell nucleus to allow deletional CSR recombination at activated stage.

Materials & Methods

Mice. 8-to 10-weeks old wt, 3'RR, c3'RR or hs5-7 (kindly gifted by Barbara Birshtein) mice were bred and maintained in SOPF conditions at 21-23°C with a 12-h light/dark cycle. Procedures were reviewed and approved by the Ministère de l'Education Nationale de l'Enseignement Supérieur et Recherche autorisation APAFIS#16151-2018071716292105v3.

Cell Culture. Splenocytes were collected, non-B cells were removed with mouse B cell kit isolation from stem cell (REF#19854) to collect resting B cells. In vitro activated B cells were obtained after three or four days of culture at 1.10 6 cells/mL in RPMI 1640 medium supplemented with 10% FCS, 2mM Glutamine (Eurobio), 1% Eagle's Non-essential Amino Acids (Eurobio), 50U/ml of penicillinstreptomycin (Gibco), 1mM sodium pyruvate (Eurobio), 129 µM 2-βmercaptoethanol (Sigma-Aldrich).

Cells were activated by adding 1µg/mL lipopolysaccharide (LPS EB ultrapure Invivogen). DNA 3D-FISH. DNA 3D FISH was performed as previously described [START_REF] Le Noir | The IgH locus 3' cis-regulatory super-enhancer co-opts AID for allelic transvection[END_REF]. Briefly, resting and in vitro stimulated B cells for three days were dropped onto poly-L-lysine slides and fixed with 4% paraformaldehyde for 10min at room temperature (RT). After PBS washes, cells were permeabilized with pepsin 0.02%/HCl 0.1M for 10min at 37°C. Then cells were washed with PBS and post-fixed with 1% paraformaldehyde for 5min at RT. DNA and probes were denatured in 70% formamide/2X SSC at 72°C and at 95°C for 5min respectively. DNA and probes were hybridized at 37°C overnight. The IgH and -satellite probes (which highlight heterochromatin) were respectively labelled with dCTP-biotin or dUTP-digoxigenin (Invitrogen REF#19518018 or #11573152910) and dUTP-Alexa Fluor 488 (Invitrogen REF#C11397). Slides were washed in 1X SSC at 72°C for 5min and then incubated with streptavidin-Alexa Fluor 594 (Molecular Probes (life) REF#S32356) (1/300 e in 4X SSC) during 1h at RT and mounted with vectashield containing DAPI (Vector Labs). Images were acquired with an epifluorescence microscope (NIKON). Optical sections separated by 0.2µm were captured and stack were deconvoluted and analyzed using Huygens and Volocity softwares, respectively. Separation of alleles was measured from the center of each signal. Volumetric pixel size was 0.065µm in xy and 0.2µm in z-direction. ATAC-Seq. 10,000 in vitro stimulated cells for three days were centrifuged at 500g at 4°C during 10min.

Flow cytometry and Cell

Then cells were incubated during 3min in lysis buffer (0,1% Tween-20, 0,1% Nonidet P40, 0,01% Digitonin, 10mM Tris-HCl pH7.4, 10mM NaCl, 3mM MgCl2) on ice and resuspended in 10µl of transposition mix (1X TD Buffer from Illumina, Transposase from Illumina, 0,01% digitonin, 0,1% Tween-20). Tagmentation was performed on a thermomixer at 1000rpm 37°C during 30min. Transposed DNA was cleaned-up with minElute Qiagen kit (#28006). Transposed DNA was amplified by PCR using primers Ad1 (5'-AATGATACGGCGACCACCGAGATCTACACTCGTTCGGCAGCGTCAGATGTG-3') and Ad2.X (5'-CAAGCAGAAGACGGCATACGAGATBARECODEGTCTCGTGGGCTCGGAGATGT-3'). Libraries were sequenced 2*100bp on NovaSeq 6000. ATAC-qPCR. DNA accessibility was quantified in resting B cells by qPCR using Bioline SYBR Hi-ROX kit (BIO-92020). Accessibility of Cµ region was quantified using Cµ-Forward (5'-CTTCCCAAATGTCTTCCCCC-3') and Cµ-Reverse (5'-TGCGAGGTGGCTAGGTACTTG-3') as well as C1 using C1-Forward (5'-GCCCAAACTAACTCCATGGTC-3') and C1-Reverse (5'-CAACGTTGCAGGTGACGGT-3'). Cµ and C1 accessibility were normalized to Lig1, using Lig1-Forward (5'-CTCTTCTCCCCGACTGTCAC3') and Lig1-Reverse (5'-GAGGCTGCTGGGAGTTGTAG-3'), gene identified as invariant between our sample as described in the "analysis of ATAC-Seq section".

3C-HTGTS. 3C-HTGTS was performed as previously described [START_REF] Zhang | Physiological role of the 3'IgH CBEs super-anchor in antibody class switching[END_REF]. Briefly, 10 million of resting or three days stimulated B cells were crosslinked with 2% formaldehyde 10% FCS PBS for 10min at RT under rotation. Crosslinking was stopped by adding glycine at 0.1M. Then, cells were lysed in 50mM Tris, 150mM NaCl, 5mM EDTA, 0.5%NP-40,1% TX-100 supplemented with protease inhibitor (ROCHE #11873580001). Nuclei were resuspended in 0,3% SDS for 1h at 37°C at 900rpm and then neutralized with Triton TX-100 for 1h. DNA restriction was performed using CviQ1 (Thermo Fisher ER0211) in B buffer (Thermofisher #BB5) overnight at 37 °C, before heat inactivation for 25 min at 65 °C. Overnight ligation was performed at 16°C at 300rpm. Next, DNA was treated by proteinase K and RNase and cleaned by phenol/chloroform. After 3C step, the LAM-HTGTS protocol was performed [START_REF] Hu | Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated highthroughput genome-wide translocation sequencing[END_REF]. Briefly, 3C

DNA was sonicated using the Bioruptor (Diagenode; two pulses at low intensity for 20s), and 10 μg was

LAM-HTGTS.

Genomic DNA (gDNA) was extracted from 10 million of 4 days stimulated B cells. LAM-HTGTS was performed as previously described ( 22) with a Sµ bait. Data analysis of MiSeq sequencing reads was performed as previously described [START_REF] Frock | Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases[END_REF]. All sequence alignments were done with the mouse mm10 genome. Analysis of junction structure has been performed with CSReport tool [START_REF] Boyer | CSReport: A New Computational Tool Designed for Automatic Analysis of Class Switch Recombination Junctions Sequenced by High-Throughput Sequencing[END_REF].

Analysis of ATAC-seq. Nextera sequences were trimmed by TrimGalore [START_REF] Krueger | Zenodo[END_REF] with only pairs of reads with their two reads of at least 55bp each were reported in the resulting fastqs and then processed by the nf-core/atacseq pipeline (26) using mm10 for reference genome. Differentially opened chromatin regions from comparisons of different conditions were extracted using DESeq2 ( 27) from the DESeqDataSet previously produce by the pipeline [START_REF] Patel | v2.0 -Iron Iguana Zenodo[END_REF].

The normalized counts extracted from the DESeqDataSet were used to calculate the coefficient of variation (CV) of each consensus peaks. Peaks with coverage greater than or equal to the mean were ranked by increasing CV to find a suitable region for ATAC-qPCR normalization controls among the samples compared. The third least variable consensus peak hitting Lig1 gene from positions 13278266

to 13279324 of chromosome 7 on mm10 were selected to design primers for ATAC-qPCR normalization controls. Chromatin A/B compartments were inferred from the consensus peaks of nf-core/atacseq analysis ( 26) at 3000kb resolution by an extended version (github trichelab/compartmap repository) of the original R package Compartmap [START_REF] Fortin | Reconstructing A/B compartments as revealed by Hi-C using long-range correlations in epigenetic data[END_REF].

Analysis of 3C-HTGTS.

Sequencing reads were aligned to the mm10 genome and processed as previously described [START_REF] Hu | Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated highthroughput genome-wide translocation sequencing[END_REF]. Each 3C-HTGTS library plotted for comparison was normalized by randomly selecting a number of junctions equal to the total number of junctions present in the smallest library in the comparison set.

Statistical analysis. Mann Whitney two-tailed tests were used for statistical analysis using GraphPad

Prism software (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Results

3'RR core enhancers maintain IgH loci in an active compartment in mature resting B cells.

To study the role of 3'RR core enhancers vs 3'RR palindromic structure, we compared two mouse models carrying total or partial deletion of the 3'RR to wt mice. We took advantage of c3'RR model [START_REF] Le Noir | Functional anatomy of the immunoglobulin heavy chain 3΄ super-enhancer needs not only core enhancer elements but also their unique DNA context[END_REF] carrying only the four core enhancers and 3'RR model ( 6) carrying total deletion of the 3'RR region (Figure 1A). We compare, in these models, transitional splenic B cells (T) collected by flow cytometry (Supplementary Figure S1A), mature resting B cells (R) and in vitro-activated B cells for 3 days with LPS (S). To avoid any impact of cell subtypes representation in bulk culture at day 3, we verified the % of CD138 positive cell after LPS stimulation in the three models. As expected, the percentage of newly generated plasmablasts in vitro in the presence of LPS was comparable (Supplementary Figure S1B).

By using ATAC-Seq (Assay for Transposase-Accessible Chromatin) technic, we determined, with a high throughput approach, chromatin accessibility across the genome in resting and in vitro activated B cells from wt, c3'RR and 3'RR mice. First of all, a principal component analysis was performed to verify reproducibility between samples (Supplementary Figure S2). Then, we first analyzed all differentially opened (DO) chromatin regions. As expected a large number of DO regions was identified upon B cell stimulation (Supplementary Table S1A), corresponding to the induction of B cell activation program. In contrast, when we compared mutants to wt in resting and in in vitro stimulated B cells, only few differentially open chromatin regions were identified (less than 80 regions) (Supplementary Tables S1B andS2). Moreover, when we confronted the % of A and B compartments, no drastic change was observed between mutants nor in resting B cell neither in in vitro stimulated B cells (Figure 1B). Then, we focused exclusively on the IgH locus, we observed a decrease of its accessibility in resting B cells from 3'RR mutants in comparison to wt (Figure 1C, Top). To confirm this trend, we quantified ATACseq normalized read count in three distinct regions across the IgH locus (R1: around Eµ/Sµ, R2: constant regions and R3: 3'CBE; whose coordinates are available in Supplementary Table S3) and we observed a global decrease of IgH accessibility throughout the 3' part of the locus (Figure 1C

3'RR core enhancers allow IgH loop conformation in mature resting and stimulated B cells

As a supplemental level of regulation, chromatin loop formation appears as a master regulator of gene remodeling, such as CSR, in B cells. But, the regulation of loop formation itself is not fully described.

Our mouse models allowed to assess the respective involvement of components of the 3'RR in IgH locus looping dynamics. To achieve this point, we performed, in resting and LPS-stimulated B cells from wt and mutants, high resolution 3C-HTGTS [START_REF] Zhang | Fundamental roles of chromatin loop extrusion in antibody class switching[END_REF] with two complementary viewpoints: one located within the Iµ/Sµ region (named Iµ/Sµ bait) (Figure 3A) [START_REF] Dong | Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching[END_REF] and another one located between hs5 and hs6 enhancers within the 3' IgH TAD border region conserved between the three models (named 3'CBE bait) (Supplementary Figure S4A) for quantifying chromatin loop from bait to prey (Supplementary Table S4A for prey coordinates).

First, 3C-HTGTS performed in wt resting B cells with Iµ/Sµ bait showed long-range contacts between

Iµ/Sµ and the 3'RR region (Figure 3B bedgraphs and right bargraph, Supplementary Figure S3B and Supplementary Table S4B for individual mice); these data reflect the first level of IgH locus folding ("big loop") and are consistent with both pioneer studies based on chromosome capture [START_REF] Wuerffel | S-S Synapsis during Class Switch Recombination Is Promoted by Distantly Located Transcriptional Elements and Activation-Induced Deaminase[END_REF] and the more recent studies built on high throughput techniques [START_REF] Zhang | Fundamental roles of chromatin loop extrusion in antibody class switching[END_REF]. In contrast and as expected, 3'RR resting B cells were not enable to perform any chromatin loop between Iµ/Sµ and the 3'RR region as a consequence of the total deletion of the 3'RR region (Figure 3B bedgraphs and middle bargraph and Supplementary Figure S3B). In this model, the decrease of long-range (3'RR) interactions from Iµ was not compensated by any new interaction inside the locus nor outside the locus at the eS region, a phenomenon previously described in the mouse model devoid of the 3'CBE region (5) (Figures 3B bedgraphs and left bargraph and C). Strikingly, in resting c3'RR B cells, devoid of the palindromic structure, chromatin loops between Iµ/Sµ and 3'RR regions still occurred notably thanks to the hs1.2 enhancer (Figure 3B bedgraphs and right bargraph and Supplementary Figure S3B). This finding was confirmed, albeit to lesser extent, by using the 3'CBE bait located downstream from 3' super-enhancer (Supplementary figure S4A). Similar chromatin loops were observed in wt and c3'RR model (Supplementary figure S4B), harboring both high interaction frequencies between the Iµ/Sµ and the 3'RR regions. In contrast, in 3'RR, 3C-HTGTS did not detect any chromatin loop between Iµ/Sµ and the 3' super-enhancer (Supplementary figure S4B). Altogether, these results highlight, for the first time, a major role of 3' regulatory enhancers in IgH locus folding ("big loop", involving Iµ/Sµ and the 3' superenhancer) of resting B cells. While the 3'RR core enhancers are necessary and sufficient for the IgH "big loop" formation, the 3'RR DNA packaging (supporting the quasi-palindromic organization) is not essential for such structure.

Secondly, we analysed in in vitro stimulated wt B cells with LPS for 3 days the occurrence of chromatin loop by using the Iµ/Sµ viewpoint and globally we found the same pattern as in resting B cells but with a more pronounced difference between genotypes. In wt, DNA loop detected by 3C-HTGTS revealed the "big loop" structure and the occurrence of new interactions between Iµ/Sµ and the targeted S3 and S2b regions that normally undergoes germline transcription in such conditions [START_REF] Zhang | Fundamental roles of chromatin loop extrusion in antibody class switching[END_REF]. In accordance with the literature, this data reflects the physiological IgH "internal loops" formed with targeted switch regions in a CSRC (Figure 3D, Supplementary Figure S3C and Supplementary Table S4C for individual mice) [START_REF] Zhang | Fundamental roles of chromatin loop extrusion in antibody class switching[END_REF]. In 3'RR model, the difference is even greater at this stage than in resting B cells, the IgH big loop is still not detectable, and a drastic decrease of contact frequencies between Iµ/Sµ and S3 or S2b regions is observed (Figure 3D bedgraphs and bargraphs and supplementary figure S3C). Just as in resting B cells, the reduction in long-range interactions upon 3'RR deletion (Figure 3D) is not compensated by any random interactions within IgH locus (Figures 3D bedgraphs and S4C). And, as expected, there is still a defect of interactions between Iµ/Sµ and targeted switch regions in stimulated 3'RR B cells (Supplementary Figure S4C).

These data support several hypotheses regarding the establishment of IgH loops during B cell activation.

Firstly, it seems quite clear that the 3'RR core enhancers alone, without the packaging of surrounding DNA, are sufficient for the formation of IgH chromatin loops, the "big loop" and the internal loops, in resting and in activated B cells. Second, the big loop formation at resting stage is a prerequisite for the formation of the internal loops and the CSRC formation in activated B cell. The internal loop defect identified by 3C-HTGTS, reflect the failure to form the CSRC which is in accordance with the strong CSR defect observed in the 3'RR model [START_REF] Saintamand | Elucidation of IgH 3′ region regulatory role during class switch recombination via germline deletion[END_REF] To fully conclude on the role of 3'RR core enhancers as IgH chromatin organizers, we performed 3C-HTGTS on a mice model carrying the partial deletion of 3'CBE region (deletion from hs5 to hs7) [START_REF] Volpi | Germline deletion of Igh 3' regulatory region elements hs 5, 6, 7 (hs5-7) affects B cell-specific regulation, rearrangement, and insulation of the Igh locus[END_REF] with a bait conserved upon deletion of the entire 3'RR localized downstream hs4 (bait 3'RR) (Supplementary Figures S5 A andB). In this model, we observe the formation of the IgH big loop in resting B cells as well the internal loops in stimulated B cells. All together these results showed that the 3'CBE region is not involved in IgH chromatin loops formation (Supplementary Figures S5 C andD).

3'RR core enhancers are required for proper deletional CSR mechanism

Productive CSR events are the result of deletional recombination events that occur by joining the correctly juxtaposed Sµ donor and Sx acceptor regions in such a way as to achieve deletion of intermediate DNA sequences. In some cases, the recombination event breaks and religates the large intermediate DNA segment between the two S regions in an inverted manner, resulting in an inversion of the IgH C gene, hence called inversional CSR, thereby prohibiting functional expression of heavy chain (Figure 4A). To further investigate the involvement of 3'RR core enhancers in CSR center formation, we questioned the CSR mechanism involved (ie: CSR deletional vs CSR inversional) in our mutants. After in vitro activation with LPS for four days, we first compared the occurrence of CSR junctions from Sµ to S3 and S2b by LAM-HTGTS using a bait localized within Sµ (Figure 4B) as previously described [START_REF] Dorsett | Cohesin at active genes: a unifying theme for cohesin and gene expression from model organisms to humans[END_REF][START_REF] Frock | Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases[END_REF] and the resulting sequences have been analyzed through the dedicated pipeline. As previously described, the frequency of CSR events in the c3'RR model is almost comparable to the wt (7) (Figure 4C). Despite the drastic decrease of CSR characterizing the 3'RR-deficient model ( 6), few CSR junctions could thus be detected in LPS-stimulated cells (Figure 4C). To identify the CSR mechanism involved in our mutants we analyzed the orientation of the S acceptor region (i.e +1 or -1).

As expected, in the wt context, almost all CSR junctions were typical to the CSR deletional mechanism (Figure 4D). In accordance with internal IgH loop formation in the c3'RR model, all junctions were in favor of deletional CSR (Figure 4D). At the opposite, all the junctions identified in the context of the 3'RR deletion were characteristic of the inversional CSR mechanism. We then analyzed structural features of CSR junctions and found that the large majority of junctions are blunt, in all three models, a typical hallmark of the NHEJ repair pathway (Figure 4E). Increase of inversional CSR were reported in case of specific DNA repair actors inhibition/deletion [START_REF] Panchakshari | DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions[END_REF], however our findings suggest inversional CSR find in our 3'RR mutants is related to the impairment of loops and not to defect of DNA repair actor recruitment. Indeed, the junctions were almost all blunt suggesting a normal DNA repair pathway (Figure 4F). All together this result suggested that the chromatin loops at the IgH locus allow productive CSR recombination by preventing inversional CSR.

Conclusion

Our study clearly demonstrates that 3'RR core enhancers are necessary and sufficient to preorganize As previously described, in wt resting B cells, a small proportion of cells harbors an IgH locus localized in pericentromeric heterochromatin [START_REF] Skok | Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes[END_REF][START_REF] Holwerda | Allelic exclusion of the immunoglobulin heavy chain locus is independent of its nuclear localization in mature B cells[END_REF]. However, in in vitro stimulated B cells, their relative positioning is still debate. According to Skok's lab, the unproductive IgH allele colocalized with PCH and replicated later [START_REF] Skok | Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes[END_REF], implying that the non-productive IgH allele is excluded only by its nuclear location.

In contrast, we and the De Latt group showed, in in vitro activated B cells, that both IgH loci remain in euchromatin [START_REF] Holwerda | Allelic exclusion of the immunoglobulin heavy chain locus is independent of its nuclear localization in mature B cells[END_REF]. This finding is in accordance with IgH biallelic expression observed in mature B cells [START_REF] Daly | Transcription of productive and nonproductive VDJ-recombined after IgH allelic exclusion[END_REF][START_REF] Tinguely | Cross talk between immunoglobulin heavy-chain transcription and RNA surveillance during B cell development[END_REF]. In this study, we found that IgH locus from 3'RR B cells is less accessible, which is consistent with previous ChIP-qPCR experiments that demonstrated a decrease of active chromatin marks (using anti-H3K9Ac antibody) in both constant and Sµ regions in 3'RR activated B cells [START_REF] Saintamand | Elucidation of IgH 3′ region regulatory role during class switch recombination via germline deletion[END_REF]. This observation perfectly correlates with the BCR low phenotype found in this model in mature B cell subpopulations and enforces the role of 3'RR in late stages of B cell differentiation [START_REF] Garot | Sequential activation and distinct functions for distal and proximal modules within the IgH 3' regulatory region[END_REF]. Moreover, IgH locus accessibility is in agreement with IgH constant region transcription; in c3'RR model the level of constant regions transcription is similar to the wt context whereas in 3'RR mice the transcription is largely decreased 13 but not abolished [START_REF] Le Noir | Functional anatomy of the immunoglobulin heavy chain 3΄ super-enhancer needs not only core enhancer elements but also their unique DNA context[END_REF]. The reduced accessibility of the IgH locus in the 3'RR model may be explained by fixation of chromatin-regulating factors in the 3'RR. In particular, the 3'RR super-enhancer recruits Brg1, a protein forming part of the SWI/SNF chromatin remodeling complex, known to preferentially bind the enhancers with the aim to open the chromatin and activate the transcription. This factor was initially described to bind the 3'RR in immature B cells [START_REF] Holley | The BRG1 chromatin remodeler regulates widespread changes in gene expression and cell proliferation during B cell activation[END_REF][START_REF] Osipovich | Cutting edge: SWI/SNF mediates antisense Igh transcription and locus-wide accessibility in B cell precursors[END_REF]. More recently, Brg1 was found to bind the 3'RR in mature B cells especially during the GC formation [START_REF] Schmiedel | Brg1 Supports B Cell Proliferation and Germinal Center Formation Through Enhancer Activation[END_REF]. ZMYND8, a protein described as a "chromatin reader", is also known to bind the 3'RR. The conditional deletion of ZMYND8 in B cell results in a phenotype similar to the 3'RR deletion (reduced CSR and SHM) suggesting the involvement of this factor in regulation of IgH locus at supranucleosomal scale [START_REF] Delgado-Benito | The Chromatin Reader ZMYND8 Regulates Igh Enhancers to Promote Immunoglobulin Class Switch Recombination[END_REF].

On another scale, our data show that 3'RR core enhancers are required for the establishment of chromatin loops at the IgH locus during CSR. However the Pavri Group recently published that the IgH big loop was mediated only by the 3'CBE region and not by the 3'RR region [START_REF] Costea | A de novo transcription-dependent TAD boundary underpins critical multiway interactions during antibody class switch recombination[END_REF]. This study was carried out only under LPS+IL4 stimulation conditions, inducing a switch to IgG1, an isotype known to be slightly differently regulated from other isotypes, since the switch to IgG1 persists, to a lesser extent, in the 3'RR model. Moreover, in the hs567 mice (partial 3'CBE deletion), it was shown that the IgH loops occurred as in a wt context in line with a normal CSR recombination [START_REF] Volpi | Germline deletion of Igh 3' regulatory region elements hs 5, 6, 7 (hs5-7) affects B cell-specific regulation, rearrangement, and insulation of the Igh locus[END_REF]. In addition, in a model of 3'CBE total deletion, a decrease of CSR is observed to all isotypes, except IgG1, suggesting a particular link between the 3'CBE region and the CSR toward IgG1.

'3RR and c3'RR models showed a drastic decrease in SHM [START_REF] Le Noir | The IgH locus 3' cis-regulatory super-enhancer co-opts AID for allelic transvection[END_REF] suggesting that the mechanisms governing the nucleus organization and three-dimensional topology of IgH locus are different between GC and in in vitro-activated B cells. So "big loop", putting the EµMARs and 3'RR regions in contact, seems to not be involved in regulating the SHM process. This suggests a role of the palindromic structure in the formation of another loop, bringing the 3'RR and EµMARs regions into contact with the rearranged pVH.

Described in 2019 by F. Alt's team, chromatin loop formation at IgH locus is mediated by the loop extrusion mechanism [START_REF] Zhang | Fundamental roles of chromatin loop extrusion in antibody class switching[END_REF]. It involves loading of the cohesin complex onto DNA by the NIPBL protein, extrusion of DNA into the ring of cohesin complex until encountering two converging CTCF binding sites, followed by dissociation of the cohesin complex from DNA by the WAPL protein. In the specific case of IgH locus, cohesin is loaded at enhancers but in the absence of the entire 3'RR, no loop was found throughout the IgH locus. It is therefore tempting to speculate that the cohesin complex loading onto the IgH locus requires the 3'RR core enhancers. Relative DNA accessibility by qPCR in resting B cells. Normalization was done to Lig1 which is not differentially opened between models. q-PCR probes are indicated with black lines. D. As in B for in vitro LPS activated B cell from wt (n=2 independent mice), 3'RR (n=1 mouse) and c3'RR (n=2 independent mice).

Legend to figures

  Sorting. To sort transitional B cells, B splenocytes were labeled with anti-B220-APC (RA3-6B2 clone, BD 553092) and anti-CD93-BV421 (AA4.1 clone, BD 561990) conjugated antibodies. Transitional (B220+/CD93+) B cells were sorted on ARIA III FACS (BD Biosciences).Plasmablasts were labelled with anti-B220-BV421 (RA3-6B2 clone, BD 562922) and anti-CD138-APC (281-2 clone, BD 558626).

  used for the LAM-HTGTS step. A 3′CBE (5'biotin-CACTGTCCAGACAGCAAACC-3'), Iµ/Sµ (5'biotin-GCAGACCTGGGAATGTATGGT-3') or 3'RR (5'biotin-GGACTGCTCTGTGCAACAAC-3') bait was used for primer elongation. These single-stranded DNA fragments were incubated with streptavidin beads (Dynabeads C1 streptavidin beads; Invitrogen) overnight at RT and washed with BW buffer (1M NaCl, 5mM Tris-HCl pH7.4, 0,5mM EDTA pH 8.0). A universal I7 adaptor (5'-GCGACTATAGGGCACGCGTGGNNNNNN-3'NH2 and 5'-P CCACGCGTGCCCTATAGTCGC-3'NH2) was ligated before the nested PCR performed with 3′CBE-bait nested primer (5I7 and I5 Illumina primer, PCR products were cleaned using PCR clean-up kit (Macherey-Nagel REF#740609) and validated after migration on BioAnalyser (Agilent). 3C-HTGTS libraries were sequenced 300bp paired-end MiSeq V3 with 20% PhiX.

  , Middle). In parallel, we aimed to confirm this result by quantifying DNA accessibility by qPCR. For this purpose, we first identified an open region with low variability between samples to normalize qPCR. As expected, we noticed a slight decrease of IgH accessibility in both constant regions tested in 3'RR resting B cells (Figure1C, Bottom). Surprisingly, when we compared c3'RR deficient and wt mice, we showed that the c3'RR resting B cells harbour the same profile as the wt, demonstrating that the 3'RR core enhancers are sufficient to maintain IgH loci in an open chromatin conformation (Figure1C). In LPS-stimulated B cells, IgH locus is also globally less accessible in 3'RR model (Figure1D). A slight decrease of IgH accessibility was shown in the c3'RR model across the locus. Subsequently, we assessed addressing of IgH loci to pericentromeric heterochromatin (PCH) within B cells nuclei by a DNA 3D-FISH (Fluorescent In Situ Hybridization) approach using an IgH fluorescent probe (RP23-109B20) encompassing a region lying from the DH cluster to C2a segment (Figure2A) and a probe (major -satellite) specific to PCH as the "inactive zone". In a wild-type context, in all three cell types tested, approximately 20% of cells have at least one IgH allele in the PCH, with a minor proportion of cells having both IgH alleles in PCH (Figure2B, left). In 3'RR mice, around 15% of IgH alleles are localized in PCH at T stage as in wt mice. In contrast, in resting B cells, the proportion of B cells harboring at least one IgH allele colocalized to PCH reaches 35% and includes a higher proportion of cells for which both alleles localized to PCH (Figure2B, middle). This configuration is maintained in in vitro stimulated B cells. Surprisingly, when only 3'RR core enhancers, not the surrounding DNA packaging, are reintroduced (c3'RR model), almost the same profile as in wt was observed (Figure2B, right). Overall, DNA 3D-FISH and ATAC-seq experiments showed a global decrease of IgH loci accessibility in resting B cells and in in vitro stimulated B cells from 3'RR. In contrast, c3'RR B cells are in an open chromatin configuration almost as in the wt context. All together these results show that in wt resting B cells, IgH loci were widely in an open chromatin compartment and that PCH addressing is governed only by 3'RR core enhancers, and not by palindromic structure. These findings point out a crucial role of the 3'RR core enhancers in regulating the nuclear organization of mature resting B cells.3'RR core enhancers are sufficient to regulate IgH loci position dynamics in nuclei of mature B cells Using DNA 3D-FISH, we also investigated the position of IgH loci within nuclei of transitional, mature resting and in vitro stimulated B cells from the three mouse models. We measured the IgH inter allelic distance as well as the distance between IgH loci and nuclear center at each stage of differentiation cited above. In the wt context, we observed that during the transition from transitional to mature resting B cell stage, IgH loci move closer together (p<0.0001), and then, move away from each other during in vitro activation (p=0.0122) (Figure 2C, Top). In wt mice, the same dynamic is observed when we measured the distances between IgH loci and the nuclear center: during transition from transitional to resting stage, IgH loci move closer to the center of nucleus (p<0.0001), while they move away from it upon in vitro stimulation (p<0.0001) (Figure 2C, Bottom). In contrast, B cells deficient for the entire 3'RR (3'RR model) loss the dynamics observed in wt context. In fact, the IgH interallelic distance remains constant (Figure 2D, Top). Similarly, distance between IgH loci and nucleus center does not differ through transitional to resting and stimulated stages (Figure 2D, Bottom). Interestingly, the reintroduction of 3'RR core enhancers (c3'RR model) alone rescued normal dynamics: IgH loci get closer to each other during transitional to resting stage evolution (p<0.0001) and move away from each other upon in vitro stimulation (p=0.0004) (Figure 2E, Top). Comparably, in c3'RR resting B cells, IgH loci are closer to the nucleus center than in transitional B cells (p<0.0001) and then, IgH loci move away from nucleus center during activation (p<0.0001) (Figure 2E Bottom). These results pinpoint a dynamic relocalization of IgH loci during late B cell maturation orchestrated by the c3'RR enhancers alone and highlight the crucial role carried by the 3'RR core enhancers in the nuclear positioning of IgH loci. They allow both the dynamic relative positioning of IgH loci to each other, and to the center of nucleus, especially at resting B cell stage. So, it is tempting to speculate that the 3'RR core enhancers pre-organize resting B cell nucleus in order to properly position IgH loci for remodeling events occurring upon activation.

  3E). Interestingly, by using Iµ/Sµ bait in LPS-stimulated B cells from c3'RR model, the IgH big loop is still detected, but slightly decreased because of the lack of palindromic structure. As in resting B cells, interactions from Iµ/Sµ to 3'RR are mainly enabled by hs1.2 enhancer (Figure 3D bedgraphs and right bargraph). On the other hand, contact frequencies between Iµ/Sµ and S3 or S2b regions occurred in c3'RR model at the same level than in wt mice (Figure 3D bedgraphs and bargraphs). In LPS stimulated B cells, 3C-HTGTS performed with the 2 nd viewpoint (3'CBE bait) confirmed all these findings. Indeed, high levels of interaction between the bait and Iµ/Sµ region were detected in wt and c3'RR mice as well as internal loops involving the S3 or S2b regions (Supplementary figure

  the resting B cell nucleus. Firstly, we show that are mandatory for maintaining IgH loci in a transcriptionally active compartment. Secondly, we demonstrate that core enhancers keep IgH loci close to each other and close to the nucleus center in an active compartment at resting B cell stage. Finally, we prove that they are essential for forming the IgH big loop between Iµ/Sµ and the 3'RR regions prior the internal loops involving the S regions at activated B cells stage. Altogether our results highlight that 3'RR core enhancers, beyond their regulatory function on IgH transcription (32), regulate IgH chromatin loops formation to prepare IgH locus to CSR. We propose a simplistic model in which 3'RR core enhancers shape, by themselves, resting B cells at nuclear and supranucleosomal levels (See graphical abstract).

Figure 1 . 3 '

 13 Figure 1. 3'RR core enhancers maintained IgH loci in an active compartment in mature resting B cells. A. Murine IgH locus scheme (not to scale). IgH constant genes are represented by squares, arrows represent the palindromic structure. Deletion of 3'RR (6, 30) and c'3RR (7) mice used in this study are schemed. B. A and B compartment proportions in resting and stimulated B cells from wt and mutants C. IgH locus accessibility by ATAC-Seq in splenic resting B cells from wt (n=2 independent mice), 3'RR (n=2 independent mice) and c'3RR mice (n=2 independent mice). Bedgraphs are normalized to reads per million and visualized with IGV. IgH loci was split into three regions and the normalized reads coverage were counted in each region as indicated in each bargraph.

Figure 2 . 3 '

 23 Figure 2. 3'RR core enhancers are sufficient to maintained IgH loci close together and close to the center in mature resting B cells A. Localization of RP23-109B20 FISH probe is represented in red. B. Top: Bars graphs represent the % of IgH loci into pericentromeric heterochromatin (PCH, stained by -satellite major probe) in transitional (T) (wt = 116 nuclei analyzed from 3 independent mice) (3'RR = 75 nuclei analyzed from 3 independent mice) (c3'RR = 108 nuclei analyzed from 2 independent mice), in resting (R) (wt = 232 nuclei analyzed from 3 independent mice) (3'RR = 136 nuclei analyzed from independent mice) (c3'RR = 199 nuclei analyzed from 3 independent mice) and in activated (S) (wt = 239 nuclei analyzed from 4 independent mice) (3'RR = 249 nuclei analyzed from 4 independent mice) (c3'RR = 740 nuclei analyzed from 3 independent mice) B cells by DNA-3D-FISH. Light and dark green respectively corresponds to the % of cells harboring one or two IgH allele in PCH. Bottom: Representative nuclei of resting B cells from each model (IgH loci in red / PCH in green, scale bars: 5µm). C. Violin plots represent raw IgH-IgH interallelic distances in µm from transitional to stimulated B cell stages in each model. D. As in (C) for IgHnucleus center distances.

Figure 3 . 3 '

 33 Figure 3. 3'RR core enhancers are mandatory for IgH big loop and internal loops formation at resting and stimulated B cell stages. A. Representation of murine IgH locus, the yellow arrow represents the 3C-HTGTS bait. B. Top: Representative bedgraphs from splenic resting B cell sample for wt (n=5 independent mice), 3'RR (n=3 independent mice) and c3'RR (n=2 independent mice) models, visualized with IGV. Bedgraphs were normalized to smallest sample. Bottom: Bargraphs of normalized reads count. C. Quantification of normalized reads count in the entire IgH locus in splenic resting B cells from wt and mutant mice. D. Same as in (B) in wt (n=4 independent mice), 3'RR (n=3 independent mice) and c3'RR (n=3 independent mice) in vitro LPS-stimulated B cells. E. Same as in (C). Scale is adapted to see interactions of interest.

Figure 4 .

 4 Figure 4. Deletional CSR in stimulated B cells requires 3'RR core enhancers. A. Schematic representation of deletional and inversional CSR mechanisms B. Localization of Sµ bait used is represented by the yellow arrow in the IgH locus. C. Percentage of switch recombination to S3 determined by LAM-HTGTS in wt (n=5 independent mice), 3'RR (n=3 independent mice) and c3'RR (n=3 independent mice) from in vitro stimulated B cells. D. Proportion of deletional and inversional CSR determined by LAM-HTGTS, in in vitro stimulated B cells from wt and mutants' mice. E. Structure of CSR junctions analyzed with CSReport.

  Figure 1

Acknowledgments

We thank BISCEm and the animal core facility team for help with mouse work on both practical and regulatory aspects. We are grateful to Emeline Lhuillier and the Genotoul Plateau GeT-Santé facility () for technical assistance with ATAC-Seq sequencing. We thank Vonick Sibut for bio-informatic analysis advices on ATAC-Seq. We thank Adam Yongxin Ye and Xuefei Zhang for precious advices on 3C-HTGTS experiments (technical part and bio-informatic analysis), Mehdi Alizadeh and Emilie Guérin for advices on sequencing step. We are grateful to Dr Brice Laffleur for fruitfull scientific discussion and critical reading of the manuscript. We also thank Pedro P Rocha for scientific and technical discussions.

We thank Christelle Oblet for her invaluable help in creating the graphical abstracts. CB was by PhD fellowships from the French Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation. MT was supported by PhD fellowships from the French Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation and the Fonds Européen de Développement Régional (FEDER). JP was supported by Institut CARNOT CALYM. This work was supported Agence Nationale de la recherche (ANR-21-CE15-0001-0), La Ligue Contre le Cancer (comités 87, 23 to EP and SLN), the Fondation ARC pour la recherche sur le cancer (PhD continuation fellowship to CB and MT).

Data Availability. Raw data from ATAC-seq, 3C-HTGTS and LAM-HTGTS have been deposited in the European Nucleotide Archive database under access number PRJEB52320.

Author contribution

CB, MT and SLN performed experiments. EP and SLN conceived and supervised the study. JP performed the bio-informatic analysis. D.R and Z.B were very helpful respectively for ATAC-seq and 3C-HTGTS experiments. CB, EP and SLN wrote the manuscript.

Competing financial interest:

The authors declare no competing financial interests.

Graphical abstract.

Position of IgH loci through B cell development (from transitional to stimulated stages) is represented by the red spots. In wt and c3'RR context, IgH loci get closer from each other and from nucleus center during evolution from transitional to mature resting stage and they relocates more at nuclear periphery, away one from each other, upon in vitro stimulation. In 3'RR model, this dynamic is lost and, moreover, IgH loci are more localized to pericentromeric heterochromatin (represented by green area) since the mature resting B cell stage and remain in after in vitro stimulation. S1: A Bedgraphs, visualized with IGV, of replicates of splenic resting B cells from wt (n=2 independent mice), 3'RR (n=2 independent mice) and c3'RR (n=2 independent mice). C. Same as in B for in vitro stimulated B cells from wt (n=3 independent mice), 3'RR (n=2 independent mice) and c'3RR (n=2 independent mice) mice. Scale is adapted to see interactions of interest. S5: A

Supplementary Figure

Supplementary Figure