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, 32 (1), 107-17

Cross Talk Between Immunoglobulin Heavy-Chain Transcription and RNA Surveillance During B Cell Development

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Cross Talk Between Immunoglobulin Heavy-Chain Transcription and RNA Surveillance During B Cell Development

Aurélien Tinguely et al. Mol Cell Biol.

Abstract

Immunoglobulin (Ig) genes naturally acquire frequent premature termination codons during the error-prone V(D)J recombination process. Although B cell differentiation is linked to the expression of productive Ig alleles, the transcriptional status of nonfunctionally recombined alleles remains unclear. Here, we tracked transcription and posttranscriptional regulation for both Ig heavy-chain (IgH) alleles in mice carrying a nonfunctional knock-in allele. We show that productively and nonproductively VDJ-rearranged alleles are transcribed throughout B cell development, carry similar active chromatin marks, and even display equivalent RNA polymerase II (RNAPII) loading after B cell stimulation. Hence, these results challenge the idea that the repositioning of one allele to heterochromatin could promote the silencing of nonproductive alleles. Interestingly, the efficiency of downstream RNA surveillance mechanisms fluctuates according to B cell activation and terminal differentiation: unspliced nonfunctional transcripts accumulate in primary B cells, while B cell activation promotes IgH transcription, RNA splicing, and nonsense-mediated mRNA decay (NMD). Altogether, IgH transcription and RNA splicing rates determine by which RNA surveillance mechanisms a B cell can get rid of nonproductive IgH mRNAs.

Figures

Fig 1
Fig 1
Normal VDJ rearrangements in B cells from heterozygous IgHfrVκ/wt mice. (A) Schematic representation of wild-type and targeted IgH loci. Primers are represented by black arrows and referenced using letters in italics (a to k). Primers and probes used for allele-specific qPCR assays are represented by light gray (IgHwt) (set A) and gray (IgHfrVκ) (set B) arrows and short rectangles. The probe used for Northern blot experiments is shown below the frVκ exon (black rectangle). (B) Analysis of VDJ junctions, performed as described in Materials and Methods, after RT-PCR using the a/j PCR primer set. After VDJ recombination, PTCs appeared in VDJ, frVκ, and CH1μ exons with equal frequencies in splenic B cells from IgHfrVκ/wt mice. ORFs, open reading frames. (C) V(D)J recombination was analyzed by quantifying the copy number of Adam6a by qPCR (primer set b/c) after normalization to the DNA copy number of Eμ (primer set e/f). DNA amounts of Adam6a were determined for spleen B cells isolated from wild-type and IgHfrVκ/wt animals and compared to reference nonlymphoid DNA from tail tissues (empty bar). Results from 3 to 4 independent mice are shown.
Fig 2
Fig 2
Equivalent transcription and accessibility of functional and nonfunctional IgH alleles. ChIP assays were performed with spleen B cells isolated from heterozygous IgHfrVκ/wt mice and stimulated with LPS for 4 days. Immunoprecipitations were done by using antibodies to RNA polymerase II (RNAPII) (A and B) and acetylated histone H3 (Ac-H3) (C and D). (A and C) The relative enrichments (percent input) were analyzed by quantitative PCR using the allele-specific primers described in the legend of Fig. 1A (sets A and B) and were compared to those of negative controls obtained without Ab (mock) (A) or using a control IgG Ab (ctrl) (C). (B and D) Nonfunctional/functional (NF/F) ratios were obtained from these data, and the mean of ratios obtained for chromatin inputs (NF/Finput) was set to 1. Results from 5 (A and B) or 4 (C and D) independent ChIP experiments are shown. (ns [not significant], P > 0.05; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001).
Fig 3
Fig 3
FACS analysis of B cell populations. (A to C) Primary B cells were isolated from heterozygous IgHfrVk/wt mice and sorted after staining with anti-B220, anti-CD43, and anti-CD138 Abs. (A) Percentages of B220+ CD43+ and B220+ CD43 cells and gates used for cell sorting. The B220+ CD43+ population (solid rectangle) includes pro-B and large pre-B cells (precursor B cells before allelic exclusion), and the B220+ CD43 population (dotted rectangle) represents B cells after pre-BCR-mediated allelic exclusion (including small pre-B and immature and mature B cells). (B) Representative dot plot indicating the gates used for cell sorting and the percentages of total bone marrow B cells (solid rectangle) (B220+ CD138) and plasma cells (dotted rectangle) (B220 CD138+). (C) Dot plot indicating the frequency of activated B cells (B220+ CD138) and plasmablasts (B220+ CD138+) upon LPS stimulation (day 4) and gates used for cell sorting. (D) Spleen cells were isolated from B6/129 F1 mice (IgHa/b), and IgMa-positive cells (solid rectangle) were sorted after staining with total and a-allotype-specific anti-IgM Abs. Representative results from 5 to 7 (A to C) or 2 (D) independent cell sorting experiments are shown. The purity of sorted populations was above 90%.
Fig 4
Fig 4
Correlation between IgH transcription and accumulation of nonfunctional pre-mRNAs. B cell populations were sorted from heterozygous IgHfrVκ/wt mice as described in the legend of Fig. 3. (A to C) NF/F IgH pre-mRNA ratios were determined for cDNAs by qPCR using allele-specific primers (sets A and B in Fig. 1A) after normalization to the NF/F ratio obtained by using DNA from IgHfrVκ/wt mice (NF/FDNA, set to 1). Dotted lines corresponds to the mean of NF/F values obtained for RNAPII-bound fractions (NF/F ratio=0.68) (Fig. 2B). This value was used as a threshold reference and reflects the RNAPII loading on both VDJ-rearranged IgH alleles in B cell populations harboring either biallelic (VDJ+/VDJ) or monoallelic (VDJ+/DJ) VDJ rearrangements. (D) Relative levels of functional IgH pre-mRNAs (IgHwt) in B and plasma cells were determined by using allele-specific primers (set A in Fig. 1A) after normalization to β-actin gene transcripts. Values obtained for resting B cells were set to 1. Results from 5 to 7 independent cell sorting experiments are shown. (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001).
Fig 5
Fig 5
Nonfunctional VDJ- and DJ-rearranged IgH mRNAs are efficiently degraded by NMD. mRNA levels from nonproductively rearranged IgHfrVκ or IgHwt alleles in LPS-stimulated B cells treated or not with CHX on day 4 were determined. (A) Northern blotting was performed by using the frVκ probe that is specific for Vκ6 segments and allows the simultaneous identification of IgHfrVκ and Igκ (normalization) mRNAs. The stimulation of B cells with LPS in the absence of additional cytokines induces preferential class switch recombination (CSR) to γ2b and γ3 isotypes. (B) Nonproductive mRNAs from switched IgHfrVκ alleles were assessed by semiquantitative PCR using primers frVκ-for and Cγ3-rev (primer set k/h in Fig. 1A). (C) IgMa-positive B cells were sorted from 129/B6 F1 (IgHa/b) mice (as described in the legend to Fig. 3D) and stimulated with LPS for 4 days. Nonproductive VDJ-rearranged mRNAs from switched IgHb alleles were then analyzed by using a consensus VH7183 forward primer (VH7183-for) and allotype-specific Cγ2b reverse primers (primer set a/i in Fig. 1A). (D) Degradation of DJ-Cμ mRNAs by NMD was assayed with wt mice by semiquantitative RT-PCR using a consensus forward primer (DHL-for) located at the 5′ end of most DH segments (primer set d/g in Fig. 1A). Representative results from 3 to 4 different mice (A, B, and D) or from 2 independent cell sorting experiments (C) are shown.
Fig 6
Fig 6
Correlation between the RNA splicing rate and the extent of degradation by NMD. The degradation of nonproductive Igμ mRNAs was assessed with pro-B cells (dark gray bar) isolated from homozygous IgHfrVκ/frVκ mice and with the B cell populations (empty and light gray bars) described in the legend for Fig. 3A to C. (A to D) RT-qPCRs were performed with cells treated or not with CHX (A to C) or wortmannin (Wort) (D) using primers frVκ-for and Cμ-rev (primer set k/g in Fig. 1A). Relative mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts, and the NMD efficiency corresponds to the fold increase in levels of nonproductive Igμ mRNAs upon treatment with inhibitors (see Fig. S2A in the supplemental material). (E) Relative expression levels of Upf1, Upf2, Upf3a, Upf3b, and Smg1 were determined for resting B cells (with a mean set to 1) and LPS-stimulated B cells after normalization to GAPDH gene transcripts. (F) Splicing rates correspond to the nonproductive mRNA/pre-mRNA ratio obtained for CHX-treated cells. Values obtained for resting B cells were set to 1. (G) The NMD efficiency was plotted against the splicing rate using data from resting and LPS-stimulated B cells (B and F) and those from activated B cells and plasmablasts (C; also see Fig. S2B in the supplemental material). Results from 3 (A, pro-B cells) or 5 to 7 independent cell sorting experiments are shown. (ns, P > 0.05; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001).
Fig 7
Fig 7
Regulation of RNA surveillance mechanisms in undivided and divided cells. (A) Resting B cells from IgHfrVκ/wt mice were stained with CFSE and then stimulated with LPS for 2 days. A representative dot plot indicates the frequency of divided (CFSElow) and nondivided (CFSEhigh) B cells and the gates used for cell sorting. (B and C) In those sorted cells, NF/F pre-mRNA ratios (B) and relative levels of functional IgH pre-mRNA (C) were obtained as described in the legend to Fig. 4. (D) The NMD efficiency was assessed as described in the legend to Fig. 6 and corresponds to the fold increase in levels of nonproductive Igμ mRNAs upon treatment with CHX. (E) For each sorted population, the splicing efficiency of nonfunctional IgHfrVκ transcripts was calculated for CHX-treated cells by dividing mRNA by pre-mRNA levels, as described in the legend to Fig. 6F. Values obtained for CFSEhigh cells were set to 1. (F) Relative levels of nonproductive Igμ mRNAs were calculated for cells treated (+) or not treated (−) with CHX after normalization to GAPDH gene transcripts. Values obtained for CFSEhigh cells (without CHX) were set to 1. Results from 7 independent cell sorting experiments are shown. (ns, P > 0.05; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001).
Fig 8
Fig 8
Model for the regulation of IgH transcription and RNA surveillance during B cell differentiation. Although the transcriptional pattern of IgH alleles is mainly biallelic during B cell development, RNA surveillance mechanisms strongly decrease the mRNA amount of nonproductively rearranged alleles (empty rectangle) and thus ensure the monoallelic expression of functional alleles. This model shows that the improvement in levels of IgH transcription, upon B cell activation and during plasma cell differentiation, is associated with a low level of NMUP and a strong degradation by NMD. Similar RNA splicing rates and NMD efficiencies in plasma cells and activated B cells are also depicted. Dotted lines indicate that our analysis accounts only for the splicing of full-length nonproductive Igμ mRNAs and hence might minimize the overall splicing rate by excluding alternatively spliced mRNAs (see also Discussion).

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