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_HD_H region, located between the most distal D_H segment, DFL16, and the most proximal V_H segment, V_{H 7183a.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 Cγ1 and Cγ2b constant genes, two transcriptional enhancers hRE1 and hRE2 (19) form the γ1E 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 quasi-palindrome (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).

Our group and others developed numerous mouse models carrying deletions within the IgH regulatory elements that helped elucidate Eµ and 3’RR super-enhancer functions during early and late B cell development (20, 27–37).

The 3.2 Mb Igκ locus contains regulatory elements within the Vκ segments ( Figure 2B ) which are partially homologous to those within the IgH: κRE1 (19) and E88 (38). Six DNase I hypersensitive sites (hs), hs1 to 6, are located in the intervening Vκ-Jκ 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 Eκ enhancer (MiEκ) (41–43), located downstream from the last Jκ segment, is composed of a 5’flanking matrix attachment region (MARκ) and an intronic enhancer. The 3’Eκ enhancer region is situated downstream from the unique Cκ gene (44). A third regulatory region, called Ed (due to its downstream location within the Igκ locus), is located distal to 3’Eκ (45).

In comparison to IgH and Igκ loci, the Igλ locus is smaller (200kb) and uniquely organized. It comprises four families which contain a pair of J _λ and C _λ segments with only three V _λ segments (46) ( Figure 2C ). The Igλ locus contains two main enhancers called E_{λ2- 4,} located between Cλ4 and Vλ1, and E_λ3-1 located downstream from Cλ1 (47). Three supplementary elements featuring enhancer activity have been described: λRE3, λRE2 and λRE1 (19). λRE3 lies between E_λ2-4 and Vλ1 while λRE1 and λRE2 are located close to the E_λ3-1 enhancer. Both Igλ enhancers, E_λ2-4 and E_λ3-1 are involved in transcription and VJ rearrangement regulation (47). λRE elements, especially λRE1 and λRE3, have been shown to potentiate the enhancer activity of E_λ3-1 and E_λ2-4 , in pro-B and plasma cells respectively (19).

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.

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 ).

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 Igκ loci. In CLP and pre-pro B cells, both Igκ 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 Igκ allele becomes more closely associated with active chromatin and the other Igκ allele stays in the PCH (64) ( Figure 3A ). This chronology is still debated. Rother et al. described relocation of both Igκ loci only at pre-B cell stages (60). In small pre-B cells, contraction occurs at the Igκ locus, in preparation for Vκ-Jκ recombination and this contracted status persists until the immature B cell stage (39, 57) ( Figure 3B ). While some controversy still remains concerning contraction of Igκ 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 Igκ looping through ordered coordination between MiEκ, 3’Eκ and Sis regulatory elements spread throughout the locus (67).

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 (Sγ₃ or Sγ₁ ) 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)].

Similar observations can be made for IgL loci ( Figure 4 ). Igκ 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 Igλ alleles are located in repressive areas in the nuclear periphery of in vitro stimulated murine B cells, which mostly express Igκ.When Igλ expression is forced, one Igλ allele is recruited to a central permissive compartment within the nucleus and both Igk alleles are repositioned to the nuclear periphery, thus inhibiting Igκ expression (72). Moreover, colocalization of Igκ with IgH loci is observed more frequently upon LPS stimulation of B cells (65).

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, Igκ and Igλ) 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, Eiκ and 3’Eκ for Igκ 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).

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 first-described 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’Eκ 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’Eκ ^-/- 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 mis-cohabitation of Ig loci correlate with decreased transcription of each Ig gene, as observed in 3’Eκ ^-/- 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).