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The Cohesin Complex Regulates Immunoglobulin Class Switch Recombination

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The Cohesin Complex Regulates Immunoglobulin Class Switch Recombination Brief Definitive Report The cohesin complex regulates immunoglobulin class switch recombination Anne-Sophie Thomas-Claudepierre, Ebe Schiavo, Vincent Heyer, Marjorie Fournier, Adeline Page, Isabelle Robert, and Bernardo Reina-San-Martin Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM) U964/Centre National de la Recherche Scientifique (CNRS) UMR 7104/Université de Strasbourg, 67404 Illkirch, France Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription- coupled recruitment of activation-induced cytidine deaminase (AID) to switch regions and by the subsequent generation of double-stranded DNA breaks (DSBs). These DNA breaks are ultimately resolved through the nonhomologous end joining (NHEJ) pathway. We show that during CSR, AID associates with subunits of cohesin, a complex previously implicated in Downloaded from sister chromatid cohesion, DNA repair, and the formation of DNA loops between enhancers and promoters. Furthermore, we implicate the cohesin complex in the mechanism of CSR by showing that cohesin is dynamically recruited to the S-C region of the IgH locus during CSR and that knockdown of cohesin or its regulatory subunits results in impaired CSR and increased usage of microhomology-based end joining. jem.rupress.org CORRESPONDENCE During immune responses, B cells diversify their of two S regions, which may be separated by Bernardo Reina-San-Martin: receptors through somatic hypermutation (SHM) several hundreds of kilobase pairs. For CSR to [email protected] and class switch recombination (CSR). SHM succeed, donor and acceptor S regions must Abbreviations used: 3RR, introduces mutations in Ig variable regions be brought into close proximity. This is be- 3 regulatory region; AID, acti- that modify the affinity of the receptor for its lieved to occur through three-dimensional con- on July 13, 2017 vation-induced cytidine de- cognate antigen (Di Noia and Neuberger, 2007). formational changes involving the generation of aminase; CSR, class switch recombination; DSB, double- CSR replaces the antibody isotype expressed transcription-coupled DNA loops (Kenter et al., stranded DNA break; IgH, Ig (from IgM to IgG, IgE, or IgA), providing novel 2012). Nevertheless, the precise mechanisms heavy chain; MudPIT, Multidi- antibody effector functions (Chaudhuri et al., controlling these conformational changes re- mensional Protein Identification Technology; NHEJ, nonho- 2007). Mechanistically, SHM and CSR are main to be elucidated. mologous end joining; SHM, initiated by activation-induced cytidine de- The cohesin complex has been described somatic hypermutation. aminase (AID), an enzyme which deaminates to play a prominent role in sister chromatid co- The Journal of Experimental Medicine cytosines in both strands of transcribed DNA hesion during cell division, in favoring DNA substrates (Petersen-Mahrt et al., 2002; Basu et al., repair by homologous recombination (Nasmyth 2011). AID-induced DNA deamination is then and Haering, 2009), in modulating gene ex- processed to trigger mutations in variable re- pression (Dorsett, 2009), and in promoting the gions during SHM or to generate double- transcription-coupled formation of long-range stranded DNA break (DSB) intermediates in DNA loop structures (Kagey et al., 2010). In switch (S) regions during CSR (Chaudhuri addition, cohesin and the transcriptional insulator et al., 2007; Di Noia and Neuberger, 2007). CTCF (Dorsett, 2009; Nasmyth and Haering, These breaks activate the DNA damage response 2009) have been shown to control the RAG1/ (Ramiro et al., 2007) and are resolved through 2-dependent rearrangement of antigen recep- classical and alternative nonhomologous end tor genes during early B and T lymphocyte devel- joining (NHEJ; Stavnezer et al., 2010). opment by mechanisms involving the regulation CSR is a transcription-dependent, long- of transcription and formation of long-range in range recombination that occurs at the Ig heavy chain (IgH) locus and that involves the joining © 2013 Thomas-Claudepierre et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Non- A.-S. Thomas-Claudepierre and E. Schiavo contributed commercial–Share Alike 3.0 Unported license, as described at http://creativecommons equally to this paper. .org/licenses/by-nc-sa/3.0/). Supplemental material can be found at: The Rockefeller University Press $30.00 http://doi.org/10.1084/jem.20130166 J. Exp. Med. 2013 Vol. 210 No. 12 2495-2502 2495 www.jem.org/cgi/doi/10.1084/jem.20130166 cis DNA interactions (Degner et al., 2011; Guo et al., 2011; on chromatin prepared from resting or activated splenic B cells Seitan et al., 2011). Here, we have examined the role of cohesin isolated from wild-type mice and using antibodies specific for in mature B cells undergoing CSR. Smc1, Smc3, and CTCF (Fig. 2). In resting B cells, we found that Smc1, Smc3, and CTCF are co-recruited to the 3 regu- RESULTS AND DISCUSSION latory region (3RR; Fig. 2 A). This is consistent with pub- Nuclear and chromatin-bound AID associate with cohesin lished ChIP data on CTCF (Chatterjee et al., 2011) in mature We have previously shown that nuclear AID exists in a large B cells and ChIP-Seq results for CTCF and cohesin (Rad21) molecular weight complex containing proteins that are re- in Rag1-deficient pro–B cells (Degner et al., 2011). A sharp quired for CSR (Jeevan-Raj et al., 2011). To further charac- peak of CTCF, Smc1, and Smc3 binding was observed at terize this complex and investigate the functional role of novel C. This peak occurred over a region containing a predicted AID partners in CSR, we have performed additional coimmu- DNaseI hypersensitive site and a CTCF consensus motif noprecipitation experiments coupled to identification by mass (Nakahashi et al., 2013). No significant enrichment was ob- spectrometry. Nuclear and chromatin extracts prepared from served at the E enhancer S or S1 (Fig. 2 A). After stimula- CH12 cells expressing a full-length N-terminally tagged AID tion, under conditions that induce CSR to IgG1, we found protein (AIDFlag-HA) or the epitope tags alone (Flag-HA) as that Smc1 and Smc3 are significantly co-recruited, indepen- negative controls were immunoprecipitated using an anti-Flag dently of CTCF, to a region spanning from the 5 end of the antibody. Eluted proteins were submitted for identification by donor switch region (S) to the 3 end of the C constant mass spectrometry. Among the proteins identified, we found region that did not comprise the E enhancer (Fig. 2 B). multiple AID partners previously implicated in CSR and/or Surprisingly, we failed to detect a reproducible recruitment Downloaded from SHM (Table S1). In addition, we found several proteins with of Smc1 or Smc3 over the S1 switch region (Fig. 2 B), sug- no known function in CSR (Table S2), including subunits of gesting that Smc1 and Smc3 are not recruited to the accep- the cohesin, condensin, Smc5/6 complex and Ino80 com- tor switch region upon activation. It is possible, however, plexes. Given the described role for cohesin in mediating long- that our cell culture conditions (in which 15–20% of the range recombination during B cell and T cell differentiation, cells switch to IgG1) are not robust enough to detect a spe- we focused on the potential role of cohesin in CSR. The asso- cific enrichment. Consistent with this, we were unable to re- jem.rupress.org ciation between AID and the cohesin complex subunits producibly detect a specific enrichment of AID at S1 by (Smc1, Smc3, Nipbl, and Wapal) was confirmed by reciprocal ChIP-qPCR (Fig. 2 E). coimmunoprecipitations and Western blotting in the nuclear The ChIP-Seq signal obtained in resting and activated (Fig. 1 A) and chromatin (Fig. 1 B) fractions and was specific, B cells for Smc1 and Smc3 (Fig. 2, A and B) is consistent with as they did not coprecipitate with an irrelevant tagged protein the fact that they are known to exist as a heterodimer and was on July 13, 2017 (EGFPFlag-HA; Fig. 1 C). Importantly, these interactions were reproducible and specific, as we did not observe any signifi- not mediated by nonspecific nucleic acid binding, as extracts cant enrichment at the IgH locus when using an IgG anti- and immunoprecipitations were done in the presence of the body as a negative control (Fig. 2, A and B). The recruitment benzonase nuclease. We conclude that endogenous subunits of of Smc1 and Smc3 at the IgH locus only partially correlated the cohesin complex associate with a fraction of nuclear and with that reported for AID (Yamane et al., 2011) and is con- chromatin-bound tagged AID through interactions that do sistent with the fact that only a fraction of chromatin-bound not involve nonspecific nucleic acid binding. AID associates with the cohesin complex (Fig. 1 B). This sug- gests that cohesin is not a targeting factor for AID. The re- Smc1 and Smc3 are dynamically recruited cruitment of Smc1, Smc3, and CTCF in resting and activated to the IgH locus during CSR B cells observed by ChIP-Seq (Fig. 2, A and B) was con- To determine whether cohesin is recruited to the IgH locus in firmed by additional independent analytical-scale ChIP-qPCR B cells undergoing CSR, we performed ChIP-Seq experiments experiments, using primer pairs at individual locations across Figure 1. Nuclear AID associates with cohesin subunits. Nuclear extracts (A and C) and chromatin fractions (B) prepared from CH12 cells expressing AIDFlag-HA (A and B) or EGFPFlag-HA (C) were immunoprecipitated and blotted with antibodies specific for Flag, AID, Smc1, Smc3, Wapal, and Nipbl. Note that the Nipbl antibody works only on immunoprecipi- tation. Input represents 1% of material used.
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