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H3 Trimethyl K9 and H3 Acetyl K9 Chromatin Modifications Are Associated with Class Switch Recombination

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H3 Trimethyl K9 and H3 Acetyl K9 Chromatin Modifications Are Associated with Class Switch Recombination H3 trimethyl K9 and H3 acetyl K9 chromatin modifications are associated with class switch recombination Fei Li Kuanga, Zhonghui Luoa,b, and Matthew D. Scharffa,1 aDepartment of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461; and bDepartment of Ophthalmology, Massachusetts Eye and Ear Infirmary, 234 Charles Street, Boston, MA 02114 Contributed by Matthew D. Scharff, February 8, 2009 (sent for review October 15, 2008) Class switch recombination (CSR) involves a DNA rearrangement in pathways convert the single-strand breaks created by AID into the Ig heavy chain (IgH) gene that allows the same variable (V) double-strand breaks and form a new hybrid SR consisting of parts region to be expressed with any one of the downstream constant of the donor SR (S␮) and recipient SR (3). Unlike VDJ recombi- region (C) genes to encode antibodies with many different effector nation, CSR is a region-specific rather than a sequence-specific functions. One hypothesis for how CSR is targeted to different C event, which means that the junction site can be anywhere along the region genes is that histone modifications increase accessibility 1- to 10-kb tract that defines each SR (5). and/or recruit activation-induced cytosine deaminase (AID) and its Because AID is so mutagenic, it is critical that it be restricted to associated processes to particular donor and recipient switch the SRs, sparing the other parts of the IgH gene and the rest of the regions. In this work, we identified H3 acetyl K9 and H3 trimethyl genome. One hypothesis suggests that cytokine-induced sterile K9 as histone modifications that correlate with the recombining transcripts at recipient SRs provide the necessary specificity and pair of donor and recipient switch regions. The appearance of H3 create transcriptional accessibility (3, 4). AID, perhaps traveling in trimethyl K9 is surprising because usually it is thought to mark a complex with RNA pol II and other factors, accesses and mutates silent genes and heterochromatin. Nevertheless, the time course of the ssDNA regions in the transcribed SRs, triggering CSR (6, 7). appearance of these histone modifications, the regions in IgH they Although sterile transcription is necessary for CSR (3), transcrip- IMMUNOLOGY associate with, and their appearance independent of AID damage tion alone is not sufficient because it occurs in many other genes in suggest that both modifications play a role in targeting CSR. the B cell and at the unaffected downstream C region of the IgH gene, and thus additional elements must contribute to the targeting activation-induced cytoside deaminase ͉ B cells ͉ ChIP ͉ immunoglobulin of CSR to SRs. Another, not mutually exclusive, hypothesis is that histone mod- ntibodies are critical for the organism’s defense against the ifications in the SRs play a role in CSR targeting. The amino- Amany pathogens and toxins that it encounters. A diverse terminal tails of the four core histones are covalently modified in repertoire of antibodies is created by V(D)J rearrangement. Upon a variety of ways, such as acetylation, phosphorylation, and meth- activation by their cognate antigen and T cells, B cells enter ylation (reviewed in refs. 8 and 9). The ‘‘histone code’’ hypothesis germinal centers where they express activation-induced cytosine suggests that histone modifications direct or facilitate various deaminase (AID) that initiates somatic hypermutation (SHM) and biological processes through recognition of the modified residues by class switch recombination (CSR) (1). SHM results in point mu- DNA binding or regulatory proteins. These modifications may also tations in the Ig heavy chain and light chain variable region genes, result in relaxation of the nucleosome structure, increasing the which code for the antigen-binding site, and with selection, these accessibility of the targeted DNAs (8). The combinatorial possi- mutations lead to increased affinity for antigen (2). In CSR, the bilities provided by histone modifications offer a plausible mech- AID-induced mutations lead to double-stranded DNA breaks in anism to explain how targeting of specific regions of the IgH gene the switch regions (SR) that are upstream of the antibody constant is achieved. Similar hypotheses are proposed for VDJ recombina- regions, and DNA rearrangements result in the apposition of tion in both Ig and TCR genes (10), and histone modifications play different downstream constant region segments with the same a role in Rag2 recruitment to the IgH locus to carry out V(D)J heavy chain variable (V) region (3). Clonal progeny of an IgM- rearrangement (11, 12). expressing B cell can express a particular V region with any one of Previous studies have shown that donor and recipient SRs the four IgG subclasses, IgE, or IgA. These isotypes have different undergoing CSR are associated with histone modifications, includ- effector functions and tissue localizations facilitating an effective ing: H3 and H4 hyperacetylation, ␥-H2AX, and H2B Ser-14 response to a single antigenic determinant. phosphorylation (6, 13–16). In multiple studies, stimulation of naïve The rearranged and productive IgH locus in mice and humans is splenic B cells with LPS alone leads to the production of sterile organized in units consisting of a VDJ region, an intronic enhancer, transcripts from S␥3, but not S␥1, and switching to IgG3. Even followed by repeating units of intervening exons (I) and SRs that though stimulation with LPS ϩ IL4 leads to switching to IgG1 and are noncoding, and the constant region (C) coding exons (Fig. 1A). represses switching to IgG3, there is sterile transcription of both S␥3 Non-class-switched IgM- (IgD)-expressing B cells make the VDJ- ␥ ␮ ␦ and S 1, although the abundance of sterile transcripts is greater in C /C transcript that is spliced and translated into a protein, and S␥1 (14, 17). Because previous studies examined H3 or H4 hyper- a sterile transcript that is not encoded into a protein and begins 5Ј of the I␮ exon and continues through the ␮SR and ␮C regions. Upon receipt of the appropriate signals to begin CSR, sterile RNA Author contributions: F.L.K., Z.L., and M.D.S. designed research; F.L.K. and Z.L. performed transcripts originate 5Ј of one or more of the recipient downstream research; F.L.K. and Z.L. contributed new reagents/analytic tools; F.L.K. and M.D.S. analyzed ␥, ␧,or␣ I-SRs. Sterile transcription is required for CSR, perhaps data; and F.L.K. and M.D.S. wrote the paper. because it creates regions of single-stranded DNA that are the The authors declare no conflict of interest. substrate for AID (3, 4). Independently, AID is expressed and Freely available online through the PNAS open access option. introduces mutations at the donor ␮SR and recipient SRs by 1To whom correspondence should be addressed. E-mail: [email protected]. deamination of cytosines to uracil. Various enzymes from the base This article contains supporting information online at www.pnas.org/cgi/content/full/ excision repair, mismatch repair, and nonhomologous end-joining 0901368106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901368106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 2, 2021 A VDJ Eμ Sμ Cμ Sγ3 Cγ3 Sγ1 Cγ1 LPS donor SR recipient SR LPS + IL4 donor SR recipient SR B D H3 tri-methyl K9 H3 acetyl K9 0.7 7 INPUT H3 Tri-me H3K9 Ac-K9 H3 Tri-me totalK27 H3 rabbit IgG 0.6 6 0h Sγ3 0.5 5 LPS LPS 72h 0.4 4 Sγ1 LPS+IL4 72h Sμ 0.3 3 0.2 2 Relative to Input Relative Sγ3 INPUT to Relative 0.1 1 LPS + IL4 Sγ1 0 0 LPS 0.45 2.5 Sµ 0.4 0.35 2 LPS + IL4 0.3 1. 5 0.25 γ 0.2 S 3 1 Sγ2b Cγ2b-CH3 0.15 C to Input Relative Relative to INPUT to Relative 0.1 0.5 0.05 0 0 0 hr LPS LPS+IL4 0 hr LPS LPS+IL4 INPUT 0.25 2.5 0.2 2 H3 acetyl K9 Sγ1 0.15 1. 5 0.1 1 H3 tri-methyl K9 Relative to Input Relative Relative to INPUT 0.05 0.5 total H3 0 0 H3 tri-me K9 rIgG H3 AcK9 total H3 rIgG rIgG Fig. 1. H3 trimethyl K9 and H3 acetyl K9 associate with pairs of active SRs. (A) Diagram of the mouse IgH locus with a rearranged VDJ and the three SRs examined in this study (not to scale). Arrows, sterile transcription initiation sites; black boxes, I exons. (B) ChIP performed on chromatin derived from splenic B cells treated with LPS or LPS ϩ IL4 for 72 h, using rabbit antibodies against the indicated modified histones or control rabbit IgG. PCR was performed by using primers that identify S␥1 and S␥3, or S␮,or(C)S␥2b and C␥2b. These gels are representative of 2–6 independent experiments. (D) Real-time quantitative PCR was performed in triplicate, and error bars indicate the standard error. This figure is representative of 2 independent experiments. acetylation (6, 13, 14), this discordance of sterile transcription and to IgG1 [Fig. S1 in the supporting information (SI) Appendix]. histone modifications with the actual site of recombination, and the Recombination can be detected at 48 h (19) and continues through recent appreciation that combinations of modifications regulate 96 h. We selected 72 h to examine chromatin modifications because different DNA transactions (9), led us to examine individual lysine by then most cells have undergone at least the two divisions required acetylations and other histone modifications to determine whether to initiate switching, and CSR is still ongoing in a significant there were marks that would better predict which SR would percentage of them (20).
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