Eμ and 3′RR IgH enhancers show hierarchic unilateral dependence in mature B-cells

Enhancer and super-enhancers are master regulators of cell fate. While they act at long-distances on adjacent genes, it is unclear whether they also act on one another. The immunoglobulin heavy chain (IgH) locus is unique in carrying two super-enhancers at both ends of the constant gene cluster: the 5′Eμ super-enhancer promotes VDJ recombination during the earliest steps of B-cell ontogeny while the 3′ regulatory region (3′RR) is essential for late differentiation. Since they carry functional synergies in mature B-cells and physically interact during IgH locus DNA looping, we investigated if they were independent engines of locus remodelling or if their function was more intimately intermingled, their optimal activation then requiring physical contact with each other. Analysis of chromatin marks, enhancer RNA transcription and accessibility in Eμ- and 3′RR-deficient mice show, in mature activated B-cells, an unilateral dependence of this pair of enhancers: while the 3′RR acts in autonomy, Eμ in contrast likely falls under control of the 3′RR.

. E μ and CSR. (a) Schematic 3D conformation of the IgH locus during LPS-induced CSR. 3C experiments indicated that E μ and the 3′RR are in close proximity in resting B cells. After appropriated stimulation the activated S acceptor region gets closer the S μ donor region. Adapted from Wuerffel et al. 4

. (b)
Flow cytometry analysis of IgG 3 and IgG 2b CSR in E μ -deficient B-cells. Cells were stimulated 4 days with LPS. Cells were labelled with anti-B220-BV421, anti-IgG 3 -FITC, anti-IgG 2b -PE and anti-IgM-PC7 antibodies. Cells were gated on B220 + B-cells. One representative experiment out of four is shown. (c) Quantitative analysis of IgG 1 , IgG 3 and IgG 2b CSR. Results are reported as mean ± SEM of 4 independent experiments. *p < 0.05, Mann-Whitney U-test. (d) Flow cytometry analysis of IgG 1 CSR in E μ -deficient B-cells. Cells were stimulated 4 days with LPS + IL-4. Cells were labelled with anti-B220-BV421, anti-IgG 1 -PE and anti-IgM-PC7 antibodies. Cells were gated on B220 + B-cells. One representative experiment out of four is shown. (e) H3K4me3 epigenetic mark in S μ and S γ1 during IgG 1 CSR ChIP assays were performed with CD43 − splenic B-cells from E μ -deficient and wt mice. Cells were stimulated with LPS + IL-4 for 2 days. Background signals from mock samples with irrelevant antibody were subtracted. Values were normalized to the total input DNA. Data are the mean ± SEM of 3 independent experiments with 2 mice. (f) H3K4me3 epigenetic mark in S μ and S γ3 during IgG 3 CSR. Cells were stimulated with LPS for 2 days. Same protocol as in part E.
B-cells previously reported in this model 7 . These results are in accordance with the lowered serum IgG 1 levels but normal IgG 3 /IgG 2b levels in this model 7 . Specific epigenetic mark enrichment in S regions is a prerequisite for CSR 8,9 . During CSR, the 3′RR SE fosters H3K4me3 histone modifications in the S acceptor but not S μ donor region 10 , suggesting another cis-transcriptional enhancer for this role. This role is not devolved to E μ since its deletion did not affect H3K4me3 histone modifications in S μ and S γ1 in response to LPS + IL-4 stimulation (Fig. 1e) nor in S μ and S γ3 in response to LPS stimulation (Fig. 1f). Altogether, these data indicate that the E μ enhancer is mostly dispensable for CSR and did not poise S μ for CSR. The cis-transcriptional enhancer that poised S μ for CSR is currently unknown. One might suggest the enigmatic transcriptional enhancer located between C γ1 and C γ2b found to interact with both E μ and 3′RR in pro-B cells 2, 11, 12 . E μ , 3′RR and respective epigenetic marks. Epigenetic changes in E μ and 3′RR are of importance during CSR 13 . In this study we have focussed on H3K4me3, H3K9ac and H3K27ac epigenetic marks that are associated with active regulatory regions 14 . Lack of the E μ enhancer did not affect H3K4me3, H3K9ac and H3K27ac epigenetic marks in the four 3′RR transcriptional enhancers (hs3a; hs1,2; hs3b; hs4) during LPS-induced B-cell CSR (Fig. 2a). In turn, if deletion of the 3′RR SE had no effect on H3K4me3 and H3K27ac marks of E μ (Fig. 2b), a significant decrease was found for H3K9ac. This difference between epigenetic marks may be explained by the proposed model of sequential histone modifications. Indeed, several studies suggest that tri-methylation of H3K4 is the earliest modification, and that H3K4me3 then facilitate H3 acetylation and thus establishment of chromatin openness. All these marks together may then positively regulate transcription and enhancer activation 15,16 . At the mature B-cell stage, data suggest the lack of E μ -dependence for the 3′RR SE during CSR, and the influence of the 3′RR on the E μ enhancer, a result that fits well with their different kinetics of activation, i.e., at the immature and mature B-cell stages for Eμ and 3′RR, respectively 3 .

Mutual activation of transcription of E μ and 3′RR
SEs. The 3′RR SE controls CSR by acting on germline transcription and histone modifications 8,17 , that are hallmarks of CSR accessibility. We investigated the effect of the lack of the E μ enhancer or of the 3′RR SE on IgH constant gene transcription units (C H ) in response to LPS-induced stimulation in vitro. RNAseq experiments showed that, except for C μ , C H sense and antisense transcripts were dramatically reduced in 3′RR-deficient mice ( Fig. 3a and Supplementary Figs 1-3). In contrast, E μ deletion had no effect on C H transcription at the IgH locus. RNASeq data are presented in a quantitative way with statistics in the Supplementary Fig. 4. Non coding RNAs (ncRNAs) play an important role in the targeting of the CSR machinery and contribute to chromosomal looping 10,18 . Among these ncRNAs, enhancer RNA (eRNA) are transcribed from DNA sequences of enhancer including the 3′RR and contribute to their enhancer function 19,20 , and chromosomal looping 4 . RNAseq experiments did not highlight any effect of E μ deletion on both sense and antisense 3′RR eRNA levels (Fig. 2a). In contrast both sense and antisense transcription around the E μ enhancer were consistently lowered in 3′RR-deficient activated B-cells. This decrease is reminiscent of the notable effect of the 3′RR SE deletion on μ transcription evidenced with quantitative PCR in resting mature B-cells 21 . Physical E μ -3′RR interactions were also documented in resting B-cells 4 . RNAseq data comparing splenic RNAseq experiments were done with CD43-depleted splenocytes after depletion of rRNA. Data are the mean of two independent experiments with 3 mice per genotype. Precise locations of I μ , S μ , C μ , C δ , I γ3 , S γ3 , Iγ 2b and S γ2b are reported in Supplementary Figs 1-3. resting B-cells indicate that enhancer activity was, at this stage, already dominated by the 3′RR: while E μ deletion showed no obvious effect on C H transcription and 3′RR eRNAs, the 3′RR deletion does not modifies E μ eRNA but still decreases μ transcripts and abrogates basal transcription of all downstream C H (Fig. 2b). This confirmed a passive role for E μ at the resting stage while H chain production originated from pV H mostly rely on 3′RR, and more precisely on hs4 22 .

Discussion
Studies highlighted different roles and kinetics of activation for IgH SEs during B-cell development. The E μ SE regulates V(D)J recombination in pro-B cells 6, 7 , but is not crucial for SHM and CSR in mature B-cells 23 . The 3′RR SE regulates SHM 24 , and conventional CSR in mature B-cells 10,25,26 , but is dispensable for V(D)J recombination 27 . GFP transgenic mice reported that the E μ SE is active at pro-B/pre-B cell stages 28 , while the 3′RR SE is active at immature/mature B-cell stages 29 . Despite different roles and kinetics, the IgH locus assumes in resting B-cells and CSR an enigmatic loop conformation with the E μ enhancer and the 3′RR SE in close proximity 4 , suggesting a potential transcriptional cross-talk between these two enhancer entities. Present results strongly suggest that cross-talk is unidirectional. The 3′RR SE stands as a fully autonomous module in mature B-cells. The E μ enhancer by contrast appears mostly dispensable at this stage, its deletion neither abrogating CSR-required sense/antisense germline transcription or 3′RR SE eRNA expression and activation-associated epigenetic mark enrichment. If the E μ enhancer has no role on 3′RR activation during CSR, it itself shows at least partial 3′RR-dependence. Deletion of the 3′RR SE impacts E μ H3K9ac activation-associated epigenetic marks and sense/anti-sense eRNA transcription.
During CSR the IgH locus assumes a loop conformation. While this conformation has obviously a major interest to simultaneously bring enhancer, SE, S regions and promoters into close proximity, our data suggest another unknown functional role, which is to place E μ under the authority of the 3′RR SE. The E μ /S μ /S x /3′RR hub might have several successive purposes to facilitate CSR. This hub brings the S x acceptor region in close proximity to the 3′RR SE, allowing for its efficient activation by acting notably on transcription and AID targeting 10 . It support physical/functional interactions between 3′RR elements hereby building their synergy 3 , and finally facilitates S μ -S x synapsis, increasing the probability of a legitimate junction between donor and acceptor S regions and, in the meantime, reducing the risk of potentially oncogenic translocation [30][31][32] . Previously reported phenotypes of E μ -and 3′RR-deficient mice, now completed by the present study, show that both IgH enhancers can behave as completely autonomous elements with regards to chromatin marks, eRNA transcription and accessibility, with E μ solely controlling early rearrangements of immature B-cells, and the 3′RR being both necessary and sufficient for late B-cell remodelling events. However in mature B-cells, this ends with E μ falling under the control of the 3′RR SE and then undergoing 3′RR-dependent transcription and chromatin remodelling as almost all basic promoters of the locus. Finally, three SEs have been reported in pro-B cells: Eμ, 3′RR and an enigmatic region between C γ1 and C γ2 2, 11, 12 . Despite that 3′RR has little role on V(D)J recombination except for silencing early transcription in pro-B cells 33 , investigation of the cross-talk between these three SEs in pro-B cells would be of interest to reinforce our knowledge of their role at the immature B-cell maturation stage.

Material and Methods
Mice. 129 wt mice (from Charles Rivers Laboratories, France), E μ MAR-deficient mice 7 , and 3′RR-deficient mice 25 were used. E μ MAR-deficient mice and 3′RR-deficient mice were in a 129 background. Our research has been approved by our local ethics committee review board (Comité Régional d'Ethique sur l′Expérimentation Animale du Limousin, Limoges, France) and carried according the European guidelines for animal experimentation.