RETRACTED ARTICLE: Trans-silencing effect of the 3′RR immunoglobulin heavy chain enhancer on Igκ transcription at the pro-B cell stage

Due to their impact on nuclear organization, enhancers are master regulators of cell fate.^1,2 The immunoglobulin heavy chain (IgH) locus undergoes numerous changes (such as transcription, accessibility, DNA breaks, and mutations) throughout B-cell differentiation. Several of these events are controlled by the IgH 3′ regulatory region (3′RR). The 3′RR is the master control element of mature B-cell IgH transcription,³ somatic hypermutation (SHM),^4,5 conventional class switch recombination (CSR),^4,6,7,8,9,10 and locus suicide recombination (LSR).¹¹ In contrast, the 3′RR is expected to be dispensable for V(D)J recombination.^12,13 During B-cell development, the heavy and light chain loci are poised for their VDJ and VJ rearrangements, respectively. The IgH locus rearranges first, with D-J joining at the pro-B-cell stages, followed by V-DJ joining at the pre-B-cell stage. The Igk locus is poised for VJ rearrangements at the pre-B cell stage. A transient association (trans-mediated by Igκ enhancer elements) between IgH and Igk loci has been demonstrated at the pre-B cell stage.^14,15 Recently, unexpected and novel findings have shown that the 3′RR acts as a cis transcriptional silencer of sense and antisense germinal V, D, and J transcription at the pro-B cell stage.^16,17 In light of these intriguing 3′RR features, we undertook the current study to determine if such a trans silencing effect could also be found for Igk transcription.

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 out according to the European guidelines for animal experimentation. Pro-B cell experiments were performed with RAG-deficient (RAG^−/−) and double 3′RR⁶-RAG-deficient (Δ3′RR–RAG^−/−) mice developed in our animal facility. Femoral pro-B cells were recovered with the EasySep^TM Mouse B-cell Isolation Kit (STEMCELL Technologies, France). RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) according to manufacturer’s instructions. Two pooled RNA samples (from four to six mice) were obtained for each genotype. RNA libraries were obtained using TruSeq stranded total RNA with Ribo-Zero Gold (Illumina) according to the manufacturer’s instructions. RNAseq experiments were performed using the Nice Sophia Antipolis genomics platform as previously reported.^7,8,17 Data were deposited in Gene Expression Omnibus under the accession number GSE117449. Mature B-cells (CD43⁻ splenocytes) were obtained from four 129 wt mice (Charles Rivers Laboratories, France) and four Δ3′RR mice (in a 129 background) before and 48 h after in vitro stimulation (1 × 10⁶ cells per ml in RPMI 1640 with 10% fetal calf serum) with 5 μg/ml LPS. Two pooled RNA samples (from two mice each) were obtained for each genotype. RNAseq experiments were performed as above and RNAseq data were deposited under the accession number GSE90760.

Femoral pro-B cells were isolated from RAG^−/− and Δ3′RR-RAG^−/− mice to explore potential transcriptional cross-talk between IgH and Igκ loci in immature B-cells. A schematic representation of these two loci is reported in Fig. 1a. RNAseq experiments led to an unexpected novel finding: deletion of the IgH 3′RR enhancer markedly enhanced sense and antisense transcription of the Igk locus in trans in pro-B cells (Fig. 1b). This effect was not found in mice deficient for the IgH Eμ enhancer (ΔEμ-RAG^−/− mice); the Eμ enhancer (located in blue in Fig. 1a) being the major control element for IgH VDJ recombination (data not shown).² As a positive control, we found no such trans effect on the Igλ locus. We next examined if this effect could be detected in mature B-cells. Deletion of the 3′RR had no trans silencer (nor activator) effect on Igk (and Igλ) transcription in resting and LPS-stimulated splenocytes (Fig. 1c, d). These results are expected since a close association between the Igk and IgH loci has not been reported in mature B cells.

Fig. 1

Influence of the 3′RR enhancer on Igκ transcription in pro-B cells. a Schematic representation of the IgH, Igκ, and Igλ loci (not to scale). b Igκ and Igλ sense (top) and antisense (bottom) transcription in pro-B cells of RAG^−/− and RAG^−/−Δ3’RR mice. c Igκ and Igλ sense and antisense transcription in resting splenocytes of 129 wt mice and Δ3′RR mice. d Igκ and Igλ sense and antisense transcription in LPS-stimulated splenocytes of 129 wt mice and Δ3′RR mice. The same mice as in c. e Quantitative representation for Cκ and Cλ transcription (in reads per million). The error bars show the extreme values of two independent experiments. The same samples as in c and d

The concept of pro-B 3′RR cis-mediated transcriptional silencing activity was first reported (using RT-QPCR) by Braikia and colleagues¹⁶ and recently confirmed (using RNAseq analysis) by our group.¹⁷ The current study is the first report of a trans silencing effect of the 3′RR. This effect is found at the Igk locus known to have a feedback inhibition effect on the establishment of allelic exclusion of the IgH locus in pre-B cells. The present study reinforces the concept of mutual crosstalk through enhancer/silencer effects between the IgH and Igk loci during immature B-cell stages. It is possible that the trans silencer effect of the 3′RR on Igκ transcription would use the same mechanism as that of its cis silencing effect on the transcription of the V, D, and J segments of the IgH locus. The 3′RR trans silencing on the Igκ locus would be of interest to prevent its usage until the end of IgH D–J recombination. Clearly, the resolution of how the 3′RR mediates trans transcriptional silencing on the Igκ locus is an exciting challenge to meet.