Contribution of Immunoglobulin Enhancers to B Cell Nuclear Organization

B cells undergo genetic rearrangements at immunoglobulin gene (Ig) loci during B cell maturation. First V(D)J recombination occurs during early B cell stages followed by class switch recombination (CSR) and somatic hypermutation (SHM) which occur during mature B cell stages. Given that RAG1/2 induces DNA double strand breaks (DSBs) during V(D)J recombination and AID (Activation-Induced Deaminase) leads to DNA modifications (mutations during SHM or DNA DSBs during CSR), it is mandatory that IgH rearrangements be tightly regulated to avoid any mutations or translocations within oncogenes. Ig loci contain various cis-regulatory elements that are involved in germline transcription, chromatin modifications or RAG/AID recruitment. Ig cis-regulatory elements are increasingly recognized as being involved in nuclear positioning, heterochromatin addressing and chromosome loop regulation. In this review, we examined multiple data showing the critical interest of studying Ig gene regulation at the whole nucleus scale. In this context, we highlighted the essential function of Ig gene regulatory elements that now have to be considered as nuclear organizers in B lymphocytes.


INTRODUCTION
To produce highly specific antibodies, B cells undergo genetic modifications of their immunoglobulin (Ig) genes. Among these events, V(D)J recombination takes place in the bone marrow during the early steps of B cell development and occurs in an antigen-independent manner. Mature B cells migrate towards secondary lymphoid organs and continue their differentiation once stimulated by antigens. This process integrates secondary beneficial DNA remodeling events including class switch recombination (CSR) and somatic hypermutation (SHM) but can also induce B cell death through locus suicide recombination (LSR), a detrimental rearrangement that abrogates surface B cell receptor expression (1). These events, all mediated by the activation-induced deaminase (AID) enzyme, characterize the late antigen-dependent phase of developing cells ( Figure 1).
Nuclear organization and chromosome distribution in B-lineage cells have to be considered as important parameters for the control of all these events. Chromosomes are not randomly scattered in the nucleus; their positions change in a dynamic fashion especially during the B cell lifespan. Indeed, distinct organizational levels co-exist in the genome on nucleosomal (genes and loci), supranucleosomal (chromatin domains and compartments) and nuclear (chromosome territories) scales (2)(3)(4). In addition to histone mark patterns that reflect the nucleosomal context, the emergence of new molecular biology approaches led to the discovery of Topologically Associated Domains (TADs) and the dynamic loop extrusion model (5). Such methods, based on chromosome capture conformation (6) (3C, 4C and Hi-C), make it possible to evaluate additional levels of gene regulation at the supranucleosomal scale. This particular level of nuclear topology includes TAD structures, A and B chromatin compartments, DNA loops and interchromatin space. Gene transcription takes place in the A-euchromatin compartment whereas B-heterochromatin prevents it. In a simplified scheme, A and B compartments are respectively positioned at the center of nuclei and at the nuclear periphery (4). More recently, the Volk's group showed that heterochromatin and euchromatin, respectively defined as B and A compartments, are localized at the nucleus periphery, leaving the nucleus center devoid of nucleosomes (7). As a structural unit of genome organization, a TAD is a large chromosomal region in which the contact frequencies between genes or regulatory regions are higher than elsewhere in the genome. TADs themselves are subdivided into multiple sub-TAD structures (chromatin loops) that undergo dynamic cell-type specific connections. The mouse genome contains around 2000 TADs, each with an approximate mean size of 1 megabase (Mb) (8). Indeed, some dynamic processes drive chromatin regions into a free space termed the interchromatin compartment in order to permit gene segment interactions. Such contacts between gene portions occur either in active (A) or inactive (B) chromatin compartments and it is widely recognized that interactions take place within the same TAD. Among chromatin compartments, long-range homologous contacts (A-A or B-B) are largely favored over heterologous contacts (A-B) (9). Moreover, additional TAD interactions exist since chromosome portions are not only able to establish close contacts in cis, but also in trans with other chromosomes (10). Some of these trans interactions have been documented in the case of olfactory receptor (11) and Th2 cytokines genes (12). At TAD extremities, TAD borders are enriched in CTCF (CCCTC-binding Factor) insulator protein, mediator complexes (MED1, MED12) as well as active histone marks (H3K4me3 and H3K36me3) (8). TAD borders display specific "insulating" features, preventing loci located on each side of this border to establish contacts (8).
By considering genome nuclear topology, these emerging models are particularly relevant for the tightly-regulated Ig gene loci. Most Ig gene regulation studies have so far been performed at the nucleosomal scale (epigenetic modifications and regulatory transcription of loci and gene segments). The increasing interest in understanding gene regulation at the whole nucleus scale prompted B cell scientists to revisit previous models at both supranucleosomal (DNA loops and TADs) and nuclear (chromosome territories and nuclear position) levels (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). To provide a clearer picture of how B cell development is tightly regulated by the nuclear location of Ig genes, including chromosome looping and loci positioning in the mouse, we will begin with an overview of Ig genes and their enhancers and then focus on the role of their main enhancers on 3D-nuclear organization. The relationship between Ig and respective enhancers will be discussed in this review.

OVERVIEW OF IMMUNOGLOBULIN GENES AND THEIR REGULATORY ELEMENTS
In mice, Ig genes are encoded by three loci located on three distinct chromosomes. The immunoglobulin heavy chain (IgH) locus lies on chromosome 12. Immunoglobulin light (IgL) chain loci contain either kappa immunoglobulin light chain (Igk) or lambda immunoglobulin light chain (Igl) genes and are respectively located on chromosomes 6 and 16. In mice, at least 95% of B cells express Igk light chains (13,14).

Immunoglobulin Heavy (IgH) Chain Loci
The IgH locus spans approximately 3Mb, in its germline configuration, and contains various cis-regulatory regions ( Figure 2A). From 5' to 3', the 5'hs123ab elements, are situated upstream from the first V H segment (15). The intergenic V H D H region, located between the most distal D H segment, DFL16, and the most proximal V H segment, V H7183a.2.3 , contains six DNase I sensitive sites (hs1 to 6) among which hs4 and hs5 are CTCF binding sites (17,18). This set is also called Intergenic Control Region 1 (IGCR1) (16,17). The promoter/enhancer pDQ52, is located just upstream from DQ52 (18). The Eµ-MARs region, spanning about 1kb, is composed of a 220-base pair (bp) core enhancer element (cEµ) flanked by two matrix attachment regions (MARs) located between the last J H exon and the Sµ region. Between the Cg1 and Cg2b constant genes, two transcriptional enhancers hRE1 and hRE2 (19) form the g1E regulatory element (20). At the 3'extremity, the 3'Regulatory Region (3'RR), spanning more than 30 kb, is composed of four enhancers, called hs3a, hs1.2, hs3b and hs4 (21). The central hs1.2 enhancer is flanked by inverted repeated intervening sequences (IRIS) that form a 25kb-long quasipalindrome (22). Downstream from this palindrome, hs4 is the most distal element harboring an enhancer activity within the 3'RR (23). Although highly divergent in different species, IRIS sequences always stand as inverted copies on both sides of hs1.2, conserving a singular symmetry within the 3'RR (24). Downstream from the 3'RR, four hs elements (hs5, hs6, hs7 and hs8) lie in a region containing ten CTCF Binding Elements (3'CBEs) (25,26). This region acts as an insulator and delimits the 3' IgH TAD border (25,26).

Immunoglobulin Light Chain Loci: Igk and Igl
The 3.2 Mb Igk locus contains regulatory elements within the Vk segments ( Figure 2B) which are partially homologous to those within the IgH: kRE1 (19) and E88 (38). Six DNase I hypersensitive sites (hs), hs1 to 6, are located in the intervening Vk-Jk region (39,40). Hs1-2 forms Cer (Contracting element for recombination) and hs3-6 comprises the Sis (Silencer in the intervening sequence) elements. A MAR-intronic Ek enhancer (MiEk) (41)(42)(43), located downstream from the last Jk segment, is composed of a 5'flanking matrix attachment region (MARk) and an intronic enhancer. The 3'Ek enhancer region is situated downstream from the unique Ck gene (44). A third regulatory region, called Ed (due to its downstream location within the Igk locus), is located distal to 3'Ek (45).
In comparison to IgH and Igk loci, the Igl locus is smaller (200kb) and uniquely organized. It comprises four families which contain a pair of J l and C l segments with only three V l segments (46) ( Figure 2C). The Igl locus contains two main enhancers called E l2-4, located between Cl4 and Vl1, and E l3-1 located downstream from Cl1 (47). Three supplementary elements featuring enhancer activity have been described: lRE3, lRE2 and lRE1 (19). lRE3 lies between E l2-4 and Vl1 while lRE1 and lRE2 are located close to the E l3-1 enhancer. Both Igl enhancers, E l2-4 and E l3-1 are involved in transcription and VJ rearrangement regulation (47). lRE elements, especially lRE1 and lRE3, have been shown to potentiate the enhancer activity of E l3-1 and E l2-4 , in pro-B and plasma cells respectively (19).

Immunoglobulin Joining Chain Loci: IgJ
Located on chromosome 5 in mice, the IgJ loci encodes the joining peptide (J) chain which promotes active IgA (48) or IgM (49) secretion by ensuring efficient assembly of these Ig subtypes in plasma cells.

IgH Sub-Nuclear Positioning and Chromatin Loops
Ig loci positioning within the nucleus is dynamic throughout early B cell development and it has been clearly shown that nuclear organization provides a critical level of regulation during V(D)J recombination, particularly for loci contraction/ decontraction, nuclear positioning (center vs periphery) and heterochromatin addressing ( Figure 3).

IgH Sub-Nuclear Positioning
Dynamic repositioning of the IgH locus throughout early stages is involved in IgH accessibility in order to correctly orchestrate V (D)J recombination ( Figure 3A). In CLP and pre-pro-B cells, IgH loci are preferentially located at the nuclear periphery within a repressive compartment (54). While only few changes occur at immature stages (55), the pro-B cell stage undergoes, in contrast, global genome reorganization since around 20% of genes switch to A-B or B-A compartments (56). Indeed, both IgH alleles are relocated to the active A compartment to allow D H -J H recombination on both alleles. At the large pre-B cell stage, once VDJ H recombination completed, pre-BCR signaling induces repositioning of one IgH allele into a repressive centromeric domain. Although pericentromeric recruitment has been associated with allelic exclusion (57), this nuclear location does not preclude transcriptional expression of the non-productive allele (58). Monoallelic IgH addressing to pericentromeric heterochromatin (PCH) remains until the immature B cell stage (57).

IgH Locus Contraction and Chromatin Loops
Beyond nuclear positioning, IgH allele contraction/decontraction, as well as TAD and loop reorganization represent an additional level of regulation, considered as critical for early B cell development (59). At the pre-pro B cell stage, both IgH alleles are decontracted (60) (60). This contraction is completed by looping within the V H region to facilitate VDJ recombination (57,61). This builds a rosette-like structure and allows equal usage between proximal, middle and distal V H genes, essential for the generation of a diversified immunoglobulin repertoire (62). After productive IgH recombination and in response to pre-BCR signaling, locus contraction is reversed in small pre-B cells (60) or in large pre-B cells (57) (17). These loops permit RAG scanning between two recognition signal sequences (RSS) and therefore focal RAG targeting activity during D H to J H joining. This loop extrusion, is thought to ensure better sequence recognition by the RAG complex, avoiding offtargeting (63). While IgH locus contraction and looping are important to permit equal usage of all V H genes, nuclear positioning of IgH loci seems more determinant than locus contraction in the orchestration of ordered V(D)J rearrangements (60).

Igk Nuclear Positioning and Chromosome Loops
In contrast to the IgH locus, the exact kinetics of Igk loci contraction and relocation within the nucleus remain unclear but it is admitted that dynamic changes also occur at Igk loci. In CLP and pre-pro B cells, both Igk alleles are located in PCH. At the pro-B cell stage, Igk loci relocate to a more central and active area within the nucleus (54,64,65). From large pre-B cells and until the immature stage, one Igk allele becomes more closely associated with active chromatin and the other Igk allele stays in the PCH (64) ( Figure 3A). This chronology is still debated. Rother et al. described relocation of both Igk loci only at pre-B cell stages (60). In small pre-B cells, contraction occurs at the Igk locus, in preparation for Vk-Jk recombination and this contracted status persists until the immature B cell stage (39, 57) ( Figure 3B). While some controversy still remains concerning contraction of Igk locus in all early developmental stages, it is clear that redistribution of intra-loci interactions occurs at the small pre-B cell stage (60,66). This redistribution, mediated by pre-BCR signaling, results in Igk looping through ordered coordination between MiEk, 3'Ek and Sis regulatory elements spread throughout the locus (67).

IgH V-D H Intergenic Region and IGCR1
Beyond the observation that IGCR1 interacts with the 3'CBEs insulator region in pro-B cells (68,69); recent studies suggest an insulation function for IGCR1 itself. In an IGCR1 deficient mouse model, interaction between the whole V-D H intergenic region and 3'CBEs was abolished while Eµ/3'RR interaction was not (69). In IGCR1 deficient pro-B cells, Eµ/IGCR1 interaction was decreased as expected but Eµ/V H 81 and Eµ/3'RR interactions still occurred (70). Moreover, the recombination center, in which DFL1.6/DQ52/Eµ/hs4 interact in a wt context, was disturbed in IGCR1 deficient pro-B cells. The absence of IGCR1 allows an alternative bigger loop to occur between V H 81X/DQ52/Eµ/hs4. By including proximal V H segments in the former loop, this new chromosome conformation alters V(D)J repertoire by decreasing distal V H usage (70). According to the actual model, IGCR1 seems to work as an insulator to delimit Em action/function. These findings are contradicted by Busslinger's group who carried out 4C-Seq experiments in IGCR1 deficient pro-B cells where interaction patterns remain unchanged compared to Rag -/pro-B cells (71). This discrepancy between studies could be explained by the restriction enzymes used in 4C experiments. When Busslinger's group used sequentially HindIII (6pb cutter) and Sau3AI enzymes, Sen's group used two 4pb cutter enzymes (MseI and NlaIII) that generate smaller fragments and probably offer higher resolution ( Table 1).

IgH DQ52 Enhancer and Eµ Region
Many studies have contributed to the identification and unraveling of the role of Ig gene enhancers on 3D loci conformation during early B cell development proving that such regulatory elements are clearly involved in subnuclear positioning and chromosome looping (intra-TAD modification). To decipher the role of pDQ52 and the Eµ enhancer, Guo and colleagues generated two mouse models (17): P -E -(both pDQ52/ DQ52 and Eµ enhancers are deleted) and P -E + (only promotor pDQ52 and DQ52 segments are deleted). Using 3D-FISH, authors demonstrated that the Em enhancer is required for IgH locus contraction in pro-B cells. Indeed, in P -E -, but not in the P -E + model, the large loops between V-D H intergenic regions (DFL16.1, IGCR1), Eµ and the 3'RR are drastically reduced underlining the important role of Eµ in loop formation. Moreover, ChIP-Seq experiments coupled with 3C experiments demonstrated that such large domain interactions are shaped by CTCF and cohesin proteins (68). In this way, CTCF-mediated IgH looping facilitates the generation of a diversified repertoire by juxtaposing distal V H to DJ H regions (71). It has also been shown that, in pro-B cells, cEµ deletion leads to IgH relocation in close proximity to the nuclear periphery (17). Taken together, these studies show that the Eµ region has a pivotal role during early V(D)J recombination by regulating both long range interactions and IgH sub-nuclear positioning. While Eµ seems to facilitate IgH loops in developing B cells, the presence of this enhancer is not strictly necessary since another study showed, by 4C-Seq, that several long-range interactions remain detectable in the absence of Eµ (71) ( Table 1).

Igk E88
The E88 element located in the intervening V-Jk region also participates in the generation of a diversified Ig repertoire (38). Endowed with enhancer activity starting at pre-B cell stages, E88 regulates long-range Igk chromatin interactions and participates in sub-TAD determination of the Igk locus. E88 deletion in pro-B cells disrupts interactions between Vk/Jk genes and three other known enhancers, especially MiEk, but also to some extent 3'Ek and 3'Ed (38). It is then reasonable to consider E88 as a major hub of Igk locus interactions critical for regulation of Igk repertoire diversity.

Igk Cer and Sis Elements
While of critical interest to generate a diversified antibody repertoire of the Igk chain (39,40); Cer and Sis regulatory elements located within the Igk locus are also involved in Ig loci PCH addressing. It is now established that IgH and Igk loci are monoallelically repositioned into PCH in pre-B cells (72). In Sis -/pre-B cells, both IgH and Igk alleles do not relocate to PCH. While Igk locus contraction and looping still occur normally in the absence of this element, Sis-deficient B cells harbor a biased Igk repertoire with increased proximal Vk gene usage accompanied by decreased distal Vk utilization. Taken together, these results show that the Sis element is required for Igk (cis-acting) and IgH (trans-acting) monoallelic positioning within the nucleus and somehow promotes a diversified antibody repertoire (39). Cer deletion leads to a strong increase in proximal Vk usage with decreased distal Vk usage. Cer -/pre-B cells show normal epigenetic marks throughout the Igk locus but the compaction level is clearly decreased suggesting that Cer regulates Igk locus contraction (40). As a whole, Sis seems to be more involved in nuclear Ig loci positioning and Cer in Igk locus conformation. The idea that nuclear positioning, rather than loci contraction, promotes Ig gene recombination (60) is thereby questioned by the reciprocal functions of Sis and Cer elements in the Igk locus.

Igk MiEk Element
Igk locus contraction, mediated by MiEk, seems to occur at the pro-B cell stage and remains until the pre-B cell stage. This observation indicates that locus contraction is lineage, but not stage specific (60). In pro-B cells, MiEk deletion both provokes positioning of both Igk alleles to PCH and reduces physical distance between IgH and Igk loci evidence of Ig loci crosstalk at this stage. In pre-B cells, MiEk deletion enforces both Igk and IgH allele positioning to PCH (76). The current model proposes pDQ52 Eµ-MAR *Loop formation between DFL16.1, IGCR1, Eµ and 3'RR. (17,69). *Maintain IgH loci at nuclear periphery in pro B cells (17,69).

3'Ed
In the absence of study depicting the role of Ig enhancers the boxes are empty. Bold correspond to the name of the enhancers. that MiEk is more implicated in Ig loci sub-nuclear organization than in locus looping.

3' Ek Region
Involvement of 3'Ek in early rearrangement of the Igk locus is clearly established. This enhancer region is necessary for Igk germline transcription activation, which is a prerequisite for Vk-Jk recombination (77). In line with the previously observed transcription defect, deletion of the3'Ek enhancer induces premature repositioning of Igk into PCH at the pre-B cell stage. Moreover, according to Park and colleagues, in pro-B cells, Igk seems to be distant from the IgH locus although both IgH and Igk appear to be located at the nuclear periphery (65). More strikingly, in 3'Ek deficient mice, Skok and colleagues also documented multiple defects in IgH decontraction, relocalization to PCH and IgH-Igk association (76). Altogether these results suggest that IgH decontraction is dependent on PCH repositioning and Igk-IgH colocalization. This particular model highlights an unexpected transacting effect upon deletion of an Ig gene enhancer. This observation supposes strong crosstalk between Ig loci in developing B cells and supports the hypothesis that regulatory regions are involved in interactions between loci. In parallel, crosstalk between Igk and IgH is a little more frequent in pre-B cells than in pro-B cells and becomes almost inexistent at immature stages (65).

NUCLEAR ORGANIZATION DRIVEN BY IG ENHANCERS IN LATE DEVELOPING B CELLS
In secondary lymphoid organs, mature B cells can encounter soluble or membrane antigens able to engage their BCR and induce proper B cell activation for secondary remodeling events. Within the germinal center (GC), B cells undergo to two major genetic rearrangements initiated by AID (78): SHM and CSR at IgH loci whereas only SHM occurs at IgL loci. By inducing frequent point mutations into the variable regions of Ig loci, SHM is a driving force for antibody affinity maturation. CSR results in µ heavy chain replacement by another subtype (IgG, IgA or IgE) after initiating DNA double strand breaks and recombination. In addition, locus suicide recombination (LSR) is another rearrangement which leads to deletion of all IgH constant genes and therefore induces B cell death by abrogating surface B cell receptor expression (1). In parallel to such secondary remodeling events, activated B cells differentiate into antibody secreting plasma cel or memory B cells (73).

IgH Nuclear Positioning and Loops in Mature Naive and Activated B Cells
Mature B cell differentiation and its accompanying late genetic remodeling events are also characterized by changes in nuclear organization and chromatin loops. Pioneering studies used 3D-FISH to show that IgH alleles were not located in similar positions within nuclei of resting naive and activated B cells. At the resting mature B cell stage, both IgH alleles are localized in euchromatin whereas their respective nuclear position upon in vitro activation is still debated. According to Skok's lab, the IgH allele colocalized with PCH and replicated later, suggesting that this allele is the unproductive allele (72) (Figure 4). These elements imply that the non-productive IgH allele is "tagged and maintained as excluded" by nuclear location. Although when using the same 3D-FISH approach in stimulated cells, the De Latt group showed that both IgH loci are sitting in an active compartment (79) ( Figure 4A). This observation is in agreement with another study in which IgH alleles relocalized to the nuclear periphery in proliferating splenic B cells (80). By using mouse models carrying IgM of "a" and "b" allotypes (respectively from C57Bl6 and SV129 backgrounds), Holwerda and colleagues also showed, by 4C-Seq, that tardive replication in splenic B cells was lymphoid specific but independent of nuclear location and topology of Ig loci (79). The fact that allelic exclusion might not be driven by nuclear location is in agreement with previous studies showing IgH biallelic expression in mature B cells (58,81). Evidence for loop formation was first provided by the Kenter group that described high frequencies of interaction between Eµ and 3'RR in mature resting B cells. Loop conformation within IgH changes upon in vitro stimulation by lipopolysaccharide (LPS) with or without interleukin (IL-4), which respectively induce CSR mostly towards IgG3 and IgG1; by acquiring additional contact between previously interacting enhancers and acceptor switch region (Sg 3 or Sg 1 ) involved in CSR (75) ( Figure 4B). The advantage of this architectural scaffolding, promoting synapsis between S regions, has been mechanistically demonstrated to facilitate CSR by the loop extrusion mechanism (74,82). Briefly, cohesin is loaded at the 3'RR end and the extrusion mechanism bring together 3'RR and Eµ regions. An additional internal loop is further formed to juxtapose the transcriptionally active S regions. This particular conformation first allows AID recruitment at both Sµ and S acceptor regions to induce DSB and second maintains S regions together for ligation by the NHEJ pathway [for review (83)].

Igk Nuclear Positioning in Mature Naive and Activated B Cells
Similar observations can be made for IgL loci (Figure 4). Igk monoallelic positioning in PCH is often observed in activated splenic B cells and this PCH-localized allele replicates later compared to the other one (64,84). The current model proposes that this monoallelic positioning drives both light chain allelic and isotypic exclusion. Effectively, both Igl alleles are located in repressive areas in the nuclear periphery of in vitro stimulated murine B cells, which mostly express Igk.When Igl expression is forced, one Igl allele is recruited to a central permissive compartment within the nucleus and both Igk alleles are repositioned to the nuclear periphery, thus inhibiting Igk expression (72). Moreover, colocalization of Igk with IgH loci is observed more frequently upon LPS stimulation of B cells (65).

Ig Nuclear Positioning and Chromosome Looping in Plasma Cells
The intense antibody secreting function of plasma cells requires high levels of immunoglobulin gene transcription. For this purpose, nuclear organization can now be considered as one important level of regulation as described by Garrard and colleagues (65). Although located on three different chromosomes, Ig genes (IgH, Igk and Igl) in plasma cells often undergo physical clustering by forming pairs or triplets. Such clusters, often composed of functional alleles, preferentially localize within the same transcription factory near the nuclear periphery ( Figure 4A). In this study, ChIP-3C-seq experiments performed with anti-RNA PolII antibodies indicate that physical interactions within transcription factories are mediated by Ig gene enhancers: cEµ and 3'RR for IgH, Eik and 3'Ek for Igk loci. Nuclear location also facilitates transport of Ig transcripts from the nucleus towards the endoplasmic reticulum (65). Moreover, 3C experiments performed by Birshtein's group underscored the importance of IgH chromatin conformation in a plasma cell line.
In this study, authors showed that physical interactions between V H genes and the 3'RR were involved in efficient IgH transcription, heavy chain expression and ensuing antibody synthesis (85). All of these elements demonstrate that nuclear organization contributes favorably to massive antibody synthesis (65).

Role of Ig Enhancers on B Cell Nuclear
Organization and Loops ( Table 1) While globally less documented, the implication of Ig gene enhancers in B cell nuclear organization (Ig positioning and loop formation) has been proposed in the context of the firstdescribed IgH locus loop bringing the 3'RR and Eµ regions into close contact before CSR. Upon LPS-stimulation, this configuration acquires additional contact between the acceptor S region involved in CSR and previous interacting IgH enhancers (75). In plasma cells, Ig loci "coordination" seems to be mediated by the 3'Ek regulatory element since its deletion leads to a decrease in cohabitation of all Ig loci with decreases in IgH-Igk, IgH-IgJ and Igk-IgJ communication. Likewise, nuclear localization of these loci seems to be modified with the relocation of IgH, Igk and IgJ alleles further from the nuclear periphery in 3'Ek -/plasma cells compared to wt plasma cells. Together, these results suggest that interactions between Ig genes (including interaction between IgH and IgJ) are mediated by Igk, especially by 3'Ek enhancer. Moreover, relocation and miscohabitation of Ig loci correlate with decreased transcription of each Ig gene, as observed in 3'Ek -/plasma cells in comparison with wt cells (65). IgH positioning within the nucleus might also be supported by the 3'RR. Complete 3'RR deletion leads to an increase in distance between both IgH loci in activated B cells (86). The pioneering study by Kenter's group showed that IgH looping requires an intact 3'RR. This statement is supported by the model devoid of its two last enhancers, hs3b and hs4, that led to a decrease in interactions between 3'RR and Eµ regions in resting and LPS ± IL4 stimulated splenic B cells (75). In contrast, partial deletion of 3'CBEs (hs5 to 7) does not impair loop formation at resting and activated B cell stages (26). However, Alt's group reported that total 3'CBEs deletion of led to significant decreases in interactions between Eµ and targeted switch regions in activated B cells (25). Deletion of the cEµ enhancer seems to have no impact on interactions between 3'RR and Eµ regions since the contact frequencies in cEµ KO B cells are comparable to wt B cells (75). Changes in IgH locus conformation mediated by interactions between promoters and enhancers, could impair CSR by limiting the activity of promoters located upstream from each constant gene, and therefore, their ability to initiate prerequisite germline transcription (87).

DISCUSSION
Nuclear positioning and chromosome looping have clearly been shown to display a functional role in early developing B cells and in B cells in general. Beyond some discrepancies, studies overall showed that positioning and conformation of Ig loci play a major role in ordered H and L chains rearrangements. Furthermore, at such early stages, nuclear repositioning of Ig loci to PCH is moreover integral to allelic exclusion mechanism while chromosome looping optimizes antibody repertoire constitution. Regulatory regions of Ig gene loci largely participate in positioning and looping. At both IgH and Igk, Eµ and 3'RR as well as Cer elements are essential for the proper loop formation in their respective loci. In contrast, Igk Sis element is supposed to ensure crosstalk between IgH and Igk loci since influencing cis-as well as trans-positioning. Similarly, 3'Ek also promotes, at early stages, temporal association between IgH and Igk.
In contrast, in mature B cells, the role of Ig loci position within the nucleus remains more elusive since studies unveil various differences. Nevertheless, IgH chromosome looping has been distinctly shown to play a role in CSR, especially by bringing donor Sµ and acceptor switch regions in close proximity to optimize switch recombination events. During SHM, primary transcription of the V exon, potentially in both sense and antisense direction, is an important prerequisite for AID-induced mutations. In KO mouse models in which the 3'RRpalindromic structure is disrupted, the observed SHM defects correlate to a decrease in V H primary transcription (29,35,36). As transcription is an essential step to initiate loop extrusion prior CSR, establishing a link between SHM and loop extrusion would be of critical interest. In addition, the function of ncRNA in chromosome topology during CSR (88) and SHM (89) has been elucidated. In plasma cells, only few studies reported that Ig loci are colocalized to transcription factories, surely to improve antibody production. Similarly, the role of Ig loci conformation throughout terminal B cell development is also of major interest.
IgH locus positioning within the nucleus during CSR seems to be important for legitimate maintenance. While IgH nuclear location in close proximity to an oncogene is a contributing factor for translocation (90); its distal 3'RR enhancers hs3b and hs4 have be shown to be only involved in oncogene activation but not in translocation (91). Given that AID activity initiates DNA modifications, it is mandatory that IgH rearrangements be tightly regulated to avoid any mutations or translocations within oncogenes (so called "AID off targets"). Several AID off target genes (ie: Pax5, Il4ra and Inf8) lie in close proximity to IgH during CSR and altogether are located in a chromosomal territory containing a high AID concentration (90). As a consequence, the non-random positioning and conformation of Ig loci could be widely considered as critical to maintain B cell genome integrity. In this way, their respective regulatory elements could play the role of B cell genome guardians thereby avoiding illegitimate events that contribute to lymphomagenesis.

AUTHOR CONTRIBUTIONS
CB wrote the manuscript and prepared the figures. SN wrote the manuscript. EP provided critical feedback. All authors contributed to the article and approved the submitted version.