Deletion of the immunoglobulin heavy chain 3′ regulatory region super-enhancer affects somatic hypermutation in b1 b cells

Access through your institution Buy or subscribe Mouse B1 B cells originate in the embryonic liver and are the major B-cell population in the peritoneal and pleural cavities.1,2,3,4,5 By

contrast, mouse B2 B cells originate in the bone marrow and are the major B-cell population in the bone marrow, spleen, and blood. B1 and B2 B cells differ not only in their origin and

locations, but also in their antigen specificity, cell surface markers, capacities for class-switch recombination (CSR) and somatic hypermutation (SHM). IgH _cis_-regulatory regions and,

particularly, transcriptional super-enhancers are major locus regulators under both normal and pathological conditions.6,7 Important differences have been found regarding the ability of the

IgH 3′ regulatory region (3′RR) super-enhancer to control the B1 and B2 B-cell fate.8,9 Thus, the 3′RR super-enhancer controls the IgA CSR in B2 B cells,10,11 but not in B1 B cells.12 On the

other hand, the 3′RR super-enhancer controls the late VDJ repertoire diversity in B1 B cells,9 but not in B2 B cells.13,14 Taken together, these data indicate similarities and differences

in the 3′RR control of B1 and B2 B-cell maturation. Similar to CSR, SHM requires activation-induced cytidine deaminase (AID) activity.15 While AID targets the switch region during CSR,

generating double-strand DNA breaks to change the Ig heavy-chain isotype for new effector functions, the enzyme targets VDJ regions during SHM to improve the Ig affinity for antigens.15

Previous studies have highlighted the role of the 3′RR super-enhancer as the master control element of SHM in B2 B cells.16,17 We thus investigated whether the 3′RR super-enhancer also

controls SHM in B1 B cells. Our research was 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. The disruption of the 3′RR was carried out in the Sv/129 embryonic stem cell line.10 Mice were bred

and maintained under specific pathogen-free conditions. Sv/129 _wt_ mice were used as control mice. AID-deficient mice were used as a negative control (to correct for baseline noise due to

PCR errors and mutations in the VDJ segment, induced during the generation of the VDJ repertoire). B1 B cells were isolated from the peritoneal cavity of mice immunized orally with sheep red

blood cells for 2 weeks and intraperitoneally with 10 μg of LPS (Invivogen, Toulouse, France) for 3 days. B1 B cells were sorted using a BD FACSAria III sorter (BD Biosciences, Franklin

Lakes, NJ) (Flow cytometry and cell sorting platform of the University of Limoges, France). The following antibodies were used: anti-B220-BV510 (Biolegend, San Diego, CA), anti-CD23-PC 

(Biolegens) 7, anti-IgM-FITC (eBioscience, San Diego CA), anti-CD11b-eF780 (eBioscience), anti-IgD-BV421 (Biolegend), and anti-CD19-PE (Biolegend). Mouse B1 B cells are distinguished based

on cell membrane surface markers (CD19+B220lowIgMhighIgDlowCD23-CD11b+/low).9,12 RNA was extracted from sorted B1 B cells, and 250 ng of RNA was used for sequencing. Transcripts were

amplified by 5′ Rapid Amplification of cDNA-ends by polymerase chain reaction (5'RACE PCR) using a reverse primer hybridizing within the µ CH1 exon as described.18 Sequencing adapter

sequences were thus added by primer extension, and resulting amplicons were sequenced on a MiSeq system (Illumina, Evry, France). Sequence analysis was performed using the

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germ-line deletion of the IgH 3′ regulatory region. _J. Exp. Med._ 211, 975–985 (2014). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by grants

from Ligue Contre le Cancer (Equipe labellisée LIGUE 2018) and Agence Nationale de la Recherche (ANR: projet EpiSwitch-3′RR 2016). N.G. was supported by a grant from Association de

Spécialisation et d’Orientation Scientifique (Lebanon), the municipality of Khiam (Lebanon) and the Société Française d’Hématologie. H.I. is supported by a fellowship from the University of

Limoges. F.B. is supported by Fondation Partenariale de l′Université de Limoges and ALURAD. We thank the flow cytometry facility of the Limoges University for B-cell sorting. AUTHOR

INFORMATION Author notes * Saintamand Alexis Present address: INSERM U1236, Université Rennes 1, Rennes, France * These authors contributed equally: Issaoui Hussein, Ghazzaui Nour. AUTHORS

AND AFFILIATIONS * Centre National de la Recherche Scientifique Unité Mixte de Recherche 7276, Institut National de la Santé et de la Recherche Médicale U1262, CBRS, rue du Pr. Descottes,

87025, Limoges, France Issaoui Hussein, Ghazzaui Nour, Boyer François, Denizot Yves & Saintamand Alexis Authors * Issaoui Hussein View author publications You can also search for this

author inPubMed Google Scholar * Ghazzaui Nour View author publications You can also search for this author inPubMed Google Scholar * Boyer François View author publications You can also

search for this author inPubMed Google Scholar * Denizot Yves View author publications You can also search for this author inPubMed Google Scholar * Saintamand Alexis View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS H.I., N.G., A.S., F.B., and Y.D. designed and performed the experiments and wrote the manuscript. Y.D.

obtained financial grants. CORRESPONDING AUTHORS Correspondence to Denizot Yves or Saintamand Alexis. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests.

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Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Hussein, I., Nour, G., François, B. _et al._ Deletion of the immunoglobulin heavy chain 3′ regulatory region super-enhancer

affects somatic hypermutation in B1 B cells. _Cell Mol Immunol_ 16, 195–197 (2019). https://doi.org/10.1038/s41423-018-0091-2 Download citation * Received: 28 June 2018 * Accepted: 04 July

2018 * Published: 20 August 2018 * Issue Date: February 2019 * DOI: https://doi.org/10.1038/s41423-018-0091-2 SHARE THIS ARTICLE Anyone you share the following link with will be able to read

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