The 3 Require the Presence of the Hs4 Enhancer in Ig Synthesis and Class Switching Do

Ig Synthesis and Class Switching Do Not Require the Presence of the hs4 Enhancer in the 3′ IgH Regulatory Region This information is current as Christelle Vincent-Fabert, Véronique Truffinet, Remi of October 1, 2021. Fiancette, Nadine Cogné, Michel Cogné and Yves Denizot J Immunol 2009; 182:6926-6932; ; doi: 10.4049/jimmunol.0900214 http://www.jimmunol.org/content/182/11/6926 Downloaded from References This article cites 25 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/182/11/6926.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Ig Synthesis and Class Switching Do Not Require the Presence of the hs4 Enhancer in the 3؅ IgH Regulatory Region1 Christelle Vincent-Fabert,2 Ve´ronique Truffinet,2 Remi Fiancette, Nadine Cogne´, Michel Cogne´, and Yves Denizot3 Several studies have reported that regulatory elements located 3؅ of the IgH locus (namely hs3a, hs1,2, hs3b, and hs4) might play a role during class switch recombination (CSR) and Ig synthesis. While individual deletion of hs3a or hs1,2 had no effect, pairwise deletion of hs3b (an inverted copy of hs3a) and hs4 markedly affected CSR and Ig expression. Among these two elements, hs4 was tentatively presented with the master role due to its unique status within the 3؅ regulatory region: distal position outside repeated regions, early activation in pre-B cells, strong activity throughout B cell ontogeny. To clarify its role, we generated mice with a clean deletion of the hs4 after replacement with a floxed neoR cassette. Surprisingly, and as for previous deletion of hs3a or hs1,2, deletion of hs4 did not affect either in vivo CSR or the secretion level of any Ig isotype. Downloaded from In vitro CSR and Ig secretion in response to LPS and cytokines was not affected either. The only noticeable effects of the hs4 deletion were a decrease in the number of B splenocytes and a decreased membrane IgM expression. In conclusion, while dispensable for CSR and Ig transcription in plasma cells, hs4 mostly appears to contribute to Ig transcription in resting B lymphocytes. The Journal of Immunology, 2009, 182: 6926–6932. complex interplay of multiple regulatory elements is ments constitute a potent locus control region conferring position- responsible for the tissue-specific and stage-specific reg- independent and copy-dependent expression to transgenes (7). http://www.jimmunol.org/ A ulation of both transcription and rearrangement of the Studies with cell lines have suggested a role of the 3ЈRR on Ig IgH locus. Germline transcription of C␮, initiation of DJ and VDJ transcription. Thus, loss of the 3Ј IgH enhancers occurred con- rearrangement, expression of rearranged ␮ genes, and opening of comitantly with a dramatic decrease of Ig transcription in the the S␮ region to class switch recombination (CSR)4 mainly rely on LP1.2 IgA-secreting cell line (5, 8). Disruption of the 3ЈRR in the ␮ upstream regulatory elements such as VH promoters and the E 9921 cell line (which had an endogenous deletion of the E␮ ele- intronic enhancers (1, 2). In addition to upstream elements, a 3Ј ment) by inserting the neoR gene into hs1,2 abrogated IgH tran- regulatory region (3ЈRR) located downstream the locus has been scription (9). Transgenic mice bearing a bacterial artificial chro- shown to include four lymphoid-specific transcriptional enhancers: mosome that included a rearranged VDJ gene and the 3ЈRR by guest on October 1, 2021 hs3a, hs1,2, hs3b, and hs4 (3). A region of open chromatin con- showed that the 3Ј end of the IgH locus enhanced germline tran- Ј taining numerous CTCF sites is found 3 of hs4 and upstream of scription and switch recombination to the four ␥ genes (10). Fi- Ј the non-B specific gene hole, thus marking the 3 terminus of the nally, studies with IgH minilocus have highlighted the role of the IgH locus (4). The hs4 distal element is active from the pre-B cell hs3b/hs4 pair in sustaining IgH gene transcription in 9921 cells stage and throughout B cell ontogeny (5). A larger region with a (11). To elucidate the function of 3Ј IgH enhancers, mice with global “palindromic” structure encompassing the hs1,2 central en- genomic mutations were generated. Class switching and Ig syn- hancer flanked by inverted repeats including hs3a and hs3b ele- thesis were normal in mice lacking hs3a or hs1,2 (12). In contrast, ments is active at late B cell stages (6). Altogether, the four ele- joint deletion of the last two 3Ј enhancer hs3b and hs4 severely impaired germline transcription and class switching to most iso- types in mice (13). Whether these effects were due to the deletion Centre National de la Recherche Scientifique Unite´Mixte de Recherche 6101, Uni- of hs3b alone, to hs4 alone, or to both regulatory elements re- versite´de Limoges, France mained an open question. Three potent arguments argued for a role Received for publication January 21, 2009. Accepted for publication March 30, 2009. mostly carried by hs4. First, knockout deletion of hs3a (which The costs of publication of this article were defrayed in part by the payment of page shares 98% of sequence similarity with hs3b) had no effect on class charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. switching and Ig synthesis (12). Second, hs3b is absent in other 1 This work was supported by grants from Ligue Contre le Cancer (Comite´De´par- species than the mouse, so that humans could be considered as temental de la Haute-Vienne et de la Corre`ze), Conseil Re´gional du Limousin, and carrying a natural knockout of hs3b (14, 15). Third, the hs4 en- “Lions Club de la Corre`ze, Zone 33 District 103 Sud”. hancer was associated with active chromatin (demethylated DNA Y.D. and M.C. wrote the paper and performed and designed research. C.V.-F., V.T., and acetylated histone H3 and H4) from pro-B cells to plasma R.F., and N.C. performed research. cells, while hs3b has become associated with activated chromatin 2 C.V.-F. and V.T. contributed equally to this work. markers later in B cell differentiation (B cell and plasma cell 3 Address correspondence and reprint requests to Dr. Yves Denizot, Centre National de la Recherche Scientifique Unite´ Mixte de Recherche 6101, Laboratoire stages) (4, 16). In the present study, we have tested the hypothesis d’Immunologie, 2, rue du Dr Marcland, 87025 Limoges Cedex, France. E-mail that hs4 is of importance for CSR and Ig production. For this address: [email protected] purpose we therefore undertook gene targeting experiments where 4 Abbreviations used in this paper: CSR, class switch recombination; ES, embryonic hs4 was either replaced with a thymidine kinase (Tk)-neoR cassette stem; LCR, locus control region; 3ЈRR, 3Ј regulatory region; wt, wild type. or deleted by the cre/loxP system. Replacement of hs4 by Neo or Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 the clean deletion of hs4 from the 3ЈRR gave the surprising result www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900214 The Journal of Immunology 6927 that Ig synthesis and CSR were not affected as compared with 72 h of growth, cells were incubated for 15 min at 4°C with 7-aminoacti- wild-type (wt) animals. We conclude that the suggested role of hs4 nomycin D and FITC-labeled annexin V Abs (BD Biosciences) and ana- in driving CSR and/or Ig gene transcription can be largely sup- lyzed by flow cytometry. In a separate set of experiments, splenocytes were Ј treated 24 h with various concentration of etoposide before staining with planted in vivo by the activity of the remaining 3 IgH enhancers 7-aminoactinomycin D and annexin V Abs. or other elements within the IgH locus. Proliferation assay Materials and Methods ϫ 5 Vector construction, transfection, and embryonic stem (ES) cell Splenic B cells (2 10 cells/well) were cultured (in sextuplicates) in 96-well plates, either alone or in the presence of 20 ␮g/ml LPS from Sal- screening monella typhimurium,5␮g/ml anti-CD40, or 20 ␮g/ml anti-IgM for 72 h. The studies have been reviewed and approved by Centre National de la The number of viable cells in proliferation was assessed using the CellTiter Recherche Scientifique and the Universite´de Limoges review committee. 96 One Solution cell proliferation assay (Promega) according to the man- The hs4 targeting construct was similar to the one previously used for the ufacturer’s recommendations. joint deletion of hs3b and hs4 (13). The 3Ј arm was the same while the 5Ј arm was slightly longer to include hs3b and to generate an additional Spleen cell cultures for Ig production EcoR1 site facilitating the screening of recombinant clones.

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