Specifically to Iga Increases Class Switch Recombination the Histone

The Histone Methyltransferase Suv39h1 Increases Class Switch Recombination Specifically to IgA

This information is current as Sean P. Bradley, Denise A. Kaminski, Antoine H. F. M. of September 27, 2021. Peters, Thomas Jenuwein and Janet Stavnezer J Immunol 2006; 177:1179-1188; ; doi: 10.4049/jimmunol.177.2.1179 http://www.jimmunol.org/content/177/2/1179 Downloaded from

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Histone Methyltransferase Suv39h1 Increases Class Switch Recombination Speciﬁcally to IgA1

Sean P. Bradley,2* Denise A. Kaminski,* Antoine H. F. M. Peters,3† Thomas Jenuwein,† and Janet Stavnezer4*

Ab class (isotype) switching allows the humoral immune system to adaptively respond to different infectious organisms. Isotype switching occurs by intrachromosomal DNA recombination between switch (S) region sequences associated with CH region genes.

Although isotype-specific transcription of unrearranged (germline) CH genes is required for switching, recent results suggest that isotype specificity is also determined by the sequences of downstream (acceptor) S regions. In the current study, we identify the histone methyltransferase Suv39h1 as a novel S␣-specific factor that specifically increases IgA switching (S␮-S␣ recombination) in a transiently transfected plasmid S substrate, and demonstrate that this effect requires the histone methyltransferase activity of Suv39h1. Additionally, B cells from Suv39h1-deficient mice have an isotype-specific reduction in IgA switching with no effect Downloaded from on the level of germline I␣-C␣ transcripts. Taken together, our results suggest that Suv39h1 activity inhibits the activity of a sequence-specific DNA-binding protein that represses switch recombination to IgA. The Journal of Immunology, 2006, 177: 1179–1188.

pon activation by Ag and accessory signals, naive Together with B cell activators, the cytokines IL-4, IFN-␥, and ϩ ϩ IgM IgD B cells undergo Ig class switching, resulting TGF-␤ regulate GL transcription, which in-turn regulates isotype http://www.jimmunol.org/ in expression of a different C gene, while maintaining specificity. IL-4 induces GLTs from the unrearranged C␥1 and C␧ U H expression of the same V region gene. Because the CH region genes, and directs switching to IgG1 and IgE in LPS- or CD40L- determines the Ab effector function, class switching allows opti- activated mouse splenic B cells. TGF-␤ induces ␣ and ␥2b GLTs and mization of the Ab for elimination of different pathogens. Class directs switching to IgA and to IgG2b. IFN-␥ induces GL ␥3 and ␥2a switching is mediated by DNA recombination between switch (S)5 transcripts, and directs switching to IgG3 and to IgG2a (1). Ј ␦ ␤ ␣ region sequences located 5 to each CH gene, except C . The iso- TGF- is required for induction of GLTs, but very poor type to which the B cells will S is regulated by Th cells, by the switching to IgA is induced by treatment of cells with LPS ϩ particular APCs involved, and by the anatomical site of Ag pre- TGF-␤ alone. Although a mixture of activators (LPS, or CD40L, ϩ ␦ ϩ ϩ ϩ ␤ by guest on September 27, 2021 sentation (1). Transcripts from unrearranged CH genes, termed anti-IgD ( ) dextran IL-4 IL-5 TGF- ) increases IgA germline (GL) transcripts (GLTs), are essential for regulating iso- switching in splenic B cells, the frequencies are still quite low type specificity of class switch recombination (CSR) (2, 3). Recent compared with most isotypes (11). The poor switching in culture evidence indicates that one function of GL transcription is to create is especially puzzling because IgA is the most abundant isotype in a transcription bubble and an R-loop, resulting in S region ssDNA the body, although mostly limited to mucosal areas (12). This re- (4). ssDNA is the substrate for activation-induced deaminase stricted expression suggests that something special in the mucosal (AID), the enzyme that initiates CSR by deamination of dC resi- environment supports IgA switching and expression. dues within S regions (5–10). Additional regulation of CSR isotype specificity

*Immunology and Virology Program, Department of Molecular Genetics and Micro- A plasmid assay for switch recombination (SR) has been devel- biology, University of Massachusetts Medical School, Worcester, MA 01655; and oped to examine the regulation of isotype speciﬁcity (13, 14). In † Research Institute of Molecular Pathology, Vienna Biocenter, Vienna, Austria this assay, plasmids containing both the S␮ segment and a down- Received for publication March 24, 2006. Accepted for publication May 1, 2006. stream S region segment are transiently transfected into B cell The costs of publication of this article were defrayed in part by the payment of page lines or splenic B cells. Such plasmid substrates undergo AID- charges. This article must therefore be hereby marked advertisement in accordance ␮ with 18 U.S.C. Section 1734 solely to indicate this fact. dependent S -Sx recombination in B cells capable of undergoing CSR, but not in cell types that do not undergo CSR (14, 15). 1 This work was supported by grants from the National Institutes of Health: R01 AI 23283 and R21 AI 57463 (to J.S.) and F32 AI 56806 (to D.A.K.). Importantly, these plasmids show isotype speciﬁcity. The tran- ␮ ␣ 2 Current address: Department of Surgery, University of Massachusetts Medical siently transfected plasmid p273 containing S and S segments School, Worcester, MA 01655. undergoes S␮-S␣ recombination in LPS-activated splenic B cells 3 Current address: Friedrich Miescher Institute for Biomedical Research, Novartis and in three B cell lines (I.29␮, CH12.LX, and 1B4.B6) capable of Research Foundation, CH-4058 Basel, Switzerland. undergoing CSR to IgA on their endogenous chromosomes. The 4 Address correspondence and reprint requests to Dr. Janet Stavnezer, Immunology pG3.1 plasmid, in which the S␥3 segment is substituted for the S␣ and Virology Program, Department of Molecular Genetics and Microbiology, Uni- ␮ ␥ versity of Massachusetts Medical School, Worcester, MA 01655. E-mail address: segment, also undergoes S -S 3 recombination in LPS-activated [email protected] splenic B cells, but only recombines in one (1B4.B6) of the three 5 Abbreviations used in this paper: S, switch; AID, activation-induced deaminase; B cell lines, the only one that is capable of switching to IgG3 on CSR, class switch recombination; DC-PCR, digestion-circularization PCR; GL, germ- its endogenous chromosome. Additional results obtained using line; GLT, GL transcript; HMT, histone methyltransferase; HP-1, heterochromatin protein-1; LSF, late SV40 factor; me3, trimethyl; Sbf-1, SET-binding factor-1; SR, S other plasmids that differ only in the downstream acceptor S region recombination; WT, wild type. provided evidence for factors that discriminate between the S␣,

S␥3, S␥1, and S␧ segments (16). These data have led to the hy- K9-H3 and HP-1␥ are associated with transcribed segments of pothesis that, in addition to GLTs, differentially expressed factors active genes in an erythroid cell line and in primary T cells (44). bind different acceptor S regions and regulate isotype-specific CSR The specific HMT that performs this modification at actively tran- by regulating chromatin accessibility. It has also been suggested scribed genes is unknown. Thus, although me3-K9-H3 is generally that such factors might directly bind AID to recruit it to specific an inhibitory modification, it is possible that very low levels of this downstream S regions (15). modification, perhaps if combined with certain additional modifications, can be activating. Histone modification and Suv39h1 In this study, we investigate whether Suv39h1 has an effect on Enzymatic modification of histones can either increase or decrease CSR. We find that overexpression of Suv39h1 increases SR spe- chromatin accessibility to transcription, DNA repair, and recom- cifically to the S␣ segment in a transiently transfected plasmid, and bination machinery. Acetylation of the N-terminal tails of histone that this enhancement requires the HMT activity of Suv39h1. Fur- H3 or H4 generally correlates with an increase in chromatin ac- thermore, B cells from mice deficient in Suv39h1 or both Suv39h1 cessibility. Methylation of H3 or H4 N-terminal tails inhibits or and Suv39h2 have a reduced ability to switch to IgA, with no effect increases gene activity, depending on the specific amino acid res- on the levels of ␣ GLTs. We suggest possible models for how idue that is methylated and the level of methylation (17–19). Sev- Suv39h1 specifically stimulates IgA CSR. eral R and K residues within the H3 and H4 N-terminal tails can be acetylated or methylated, singly or in combination, determined Materials and Methods by the specificity of the enzymes involved. In addition, some K S plasmid construction

residues can be ubiquitinated, and some S residues can be phos- Downloaded from S plasmids p273, p218, pG3.1, and pE.1 were previously described (13, phorylated, but very little is known about the roles of these latter 16). pG3.1 and pE.1 were gifts from A. Kenter (University of Illinois, two modifications. Chicago, IL). The S␥2a and S␥2b segments were obtained by PCR am- Histone modifications at specific genomic sites can be measured plification using genomic DNA from the B cell line I.29␮ (clone 22D). by the chromatin immunoprecipitation assay. Using this assay, it PCR primers were designed with incorporated restriction sites (underlined) for cloning into a p218 BamHI-NotI vector backbone. Primers were de- has been found that specific domains within the IgH locus undergo signed from National Center for Biotechnology Information-Accession

histone acetylation or methylation at defined stages during B cell D78344, which contains both S␥2b and S␥2a sequences from a BALB/c http://www.jimmunol.org/ development, correlating with the rearrangement potential of the mouse. Primers Sga-up, 5Ј-CGGATCCTACAGCTTGCGTGGGAAT genes (20–24). Furthermore, during activation of B cells, histones CAG-3Ј and Sga-down, 5Ј-ATAAGAATGCGGCCGCAGAGTGTCCTG Ј ␥ on specific acceptor S regions undergoing switching are acetylated GTC-3 were used to amplify a 1.9-kb S 2a fragment. PCR products were cloned into the TOPO-pCR-4 T/A cloning vector (Invitrogen Life Tech- (25–27). However, H3 acetylation alone does not substitute for GL nologies) and then subcloned into the p218 backbone with BamHI-NotI transcription (25). directional sites. To amplify a 1.4-kb S␥2b fragment, primers G2b5Ј, One of the best-studied histone-modifying enzymes is the ubiq- 5Ј-CGGGATCCGCGAGCTCTTAGGGACTACTCCTAGCAGCCTG-3Ј Ј Ј uitously expressed histone methyltransferase (HMT) Suv39h1, the and G2b3 ,5-ATATAGCGGCCGCCAATACTGGATGCTCACCTATA CTCCAAAG-3Ј were used and cloned as described for S␥2a. Deletion of mammalian ortholog of the Drosophila suppressor of position-ef- an 83-bp segment containing Pax5 binding sites at the 5Ј end of the S␣ fect variegation, Su(var)3-9 (28). Suv39h1 trimethylates the K9 segment in p218 was performed by overlap-extension PCR. by guest on September 27, 2021 residue of H3 (29). The closely related HMT, Suv39h2, also performs the same methylation, but shows much more limited expres- Switch plasmid digestion-circularization PCR (DC-PCR) assay sion in adult mice (17, 30). The HMT activity of these proteins The switch plasmid DC-PCR assay was performed, as described (14), with resides in their conserved SET domain, which is also found in ϳ70 modifications. The I.29␮ cells (22D subclone) (1 ϫ 107 cells) in exponen- ␮ other mammalian genes. Suv39h1 accumulates at the pericentro- tial growth phase were cotransfected with 10 g of switch plasmid and either 10 ␮g of empty expression-vector control (pcDEF3) or 10 ␮gof meric regions of chromosomes during metaphase, where it is es- pcdef3-Suv39h1 expression vector by electroporation (300 v, 1250 ␮F). sential for the trimethyl (me3)-K9-H3 modification and for het- Other cell lines and LPS-stimulated splenic B cells were similarly trans- erochromatin formation. During interphase, Suv39h1 is broadly fected. I.29␮ cells were cultured in medium described previously (45), but distributed (31–36). all other cell lines were cultured in medium identical with that used for ␤ splenic B cells (see below). T-depleted splenic B cells were purified and Suv39h1 forms a complex with heterochromatin protein-1 activated with LPS (50 ␮g/ml, Escherichia coli 055:B5; Sigma-Aldrich). (HP-1␤/M31), which binds the me3-K9-H3 residue with high af- On day 3, 1 ϫ 107 cells were transfected and cultured with LPS for another finity (33). HP-1␤ can self-associate and bind to a series of adja- 48 h. Although the frequency of transfection of B cell lines and splenic B cent nucleosomes to form heterochromatin (17). Suv39h1 is also cells is very low (Յ1% of the cells are transfected; our unpublished data), found in complexes with histone deacetylases 1, 2, 4, and 5 (37). both the S plasmid and the expression plasmids enter the same cells. Due to this very low transfection efficiency, it was not possible to examine the This interaction should allow a concerted process whereby histone levels of Suv39h1 protein in the transfected cells. Genomic DNA was deacetylases remove acetyl groups from histone tails, leaving K9 purified from nuclei prepared by Nonidet P-40 treatment (final concentra- residues available for methylation by the associated Suv39h1. tion 0.63%) and lysis with SDS (final concentration 0.4%), RNase (100 In addition to initiating the formation of large heterochromatin ␮g/ml), and proteinase K (100 ␮g/ml). A total of 2 ␮g of DNA was used for the S plasmid DC-PCR assay. Samples were digested with SacI and regions, Suv39h1 is involved in repressing transcription of specific EcoRI, sequentially, purifying with a spin column (QIAquik; Qiagen) after genes. It can specifically target certain promoters due to the ability each digestion. Ligation reactions were performed at 0.4 Ϫ 2.0 ng/␮l with of the Suv39h1-HP-1␤ complex to bind Rb, p107, p130, and hu- T4 DNA ligase (5 U; MBI Fermentas) in 200 ␮l overnight at 16°C. man T cell leukemia virus-1 Tax (38–41). For example, Suv39h1 PCR to detect the recircularized vector backbone 510-bp product was can be recruited to the cyclin A and E promoters by the interaction conducted on all S plasmids with the primer set P1/P4 for 30 cycles, as described previously (14). Hotstar Taq (Qiagen) was used for all experi- of Rb and the E2F transcription factor, resulting in methylation of ments. The PCR product indicating S-S recombination varies in length H3 and binding of HP-1␤ to a single nucleosome (42, 43). By this among the different isotype S plasmids: 180-bp PCR for p273S and p218, mechanism, Suv39h1 represses transcription of cyclins A and E in using primer set P2/P3␣ (14). A 180-bp product is obtained from pG3.1 HeLa cells, resulting in G cell cycle arrest. using primers P2/P3␥ (14). A 163-bp product is obtained from pG2a, using 1 primer set P2/PG2a: PG2a 5Ј-AGGCAGAGGAAACAAACAAAG Although most data indicate that the me3-K9-H3 modification is GACCTACACAGGG-3Ј. A 171-bp product is obtained from pG2b, using associated with heterochromatin and with the promoters of a few primers P2/P3G2b: P3G2b 5Ј-GGAGTATTAGGGACCAGTCCTAT inactive genes, it was recently shown that very low levels of me3- CAGCTGTGG-3Ј. A 160-bp product is obtained from pE.1, using primers The Journal of Immunology 1181

P2/P3␧:P3␧ 5Ј-CCTCTCTCAGGAGAATGTCCTGGGCATG TGGAC-3Ј. Thermocycler conditions for the S-S products were as for the 510-bp band, except 36 cycles were performed. [32P]dCTP was included in the reactions to label the PCR products, and all results shown are from autoradiography or phosphor imaging of dried gels. Two different template inputs were used for each PCR, and each transfection was performed in duplicate. The intensities of the signals due to SR (generally the 180-bp band) and for the control band (510 bp) were determined by densitometry of autoradiograph ﬁlms or by phosphor imaging of dried gels. Only data from signals in the linear range were included, and the SR results for each template dose were normalized to the 510-bp band. Cloning of Suv39h1 cDNA into pcDEF3 and creation of mutants Mouse Suv39h1 cDNA cloned into pBluescript (46) was excised with BamHI/EcoRI, cloned using BamHI/NotI adaptors into NotI/EcoRI sites of the expression plasmid pcDEF3 (47). The H324L mutation was introduced into this plasmid with the Stratagene Quikchange kit. Internal deletions ⌬124–229 and ⌬41–81 were created by PCR overlap extension, cloned into pGEM, and then cloned into the KpnI site of pcDEF3. The internal deletion ⌬42–229 was a natural variant of Suv39h1 cloned from the I.29␮ B lymphoma cell line, and inserted into the KpnI site of pcDEF3. The Pax5

binding site was removed as a 83-bp deletion by overlap extension PCR, Downloaded from and the S␣ segment was cloned in pGEM and then excised with BamHI/ NotI and cloned into these sites in p218. B cell cultures for induction of CSR and isolation of Peyer’s patch cells T-depleted splenic B cells were puriﬁed and cultured under conditions to

induce CSR, and switching was assayed by ﬂow cytometry, as previously http://www.jimmunol.org/ described (48, 49). To induce SR to IgG3, LPS (50 ␮g/ml; Sigma-Aldrich) and anti-␦-dextran (0.3 ng/ml) (gift from C. Snapper, Uniformed Services University of the Health Sciences, Bethesda, MD) were added at the ini- tiation of culture. To induce SR to IgG1, LPS and IL-4 (800 U/ml; from W. Paul, National Institutes of Health, Bethesda, MD) were added. LPS and IFN-␥ (10 U/ml) were used to induce IgG2a switching, and LPS and dextran sulfate (30 ␮g/ml; Pharmacia) were used to induce IgG2b switching. LPS, TGF-␤1 (2 ng/ml), IL-4, IL-5 (1.5 ng/ml; BD Pharmingen), and anti- ␦ dextran were used to induce IgA CSR. Cells were isolated from excised Peyer’s patches by using forceps to tease apart the isolated patches, fol- lowed by straining through a cotton wool column to isolate single cell by guest on September 27, 2021 suspensions. Peyer’s patch cells were analyzed by ﬂow cytometry, as for cultured B cells.

RNA isolation; semiquantitative RT-PCR FIGURE 1. S plasmids and DC-PCR assay for plasmid SR activity. A, Maps of six S plasmids used in these experiments (13, 14). Neo, neomycin- Total RNA was isolated from splenic B cell cultures using RNAwiz solu- tion (Ambion). First strand cDNA synthesis used 2 ␮g of RNA, oligo(dT) resistance gene; EP, IgH enhancer-VH promoter cassette; TK, herpes virus ␣ ␣ primer, and Moloney murine leukemia virus-reverse transcriptase (Pro- thymidine kinase gene; I , GLT promoter; RI, EcoRI site. p273S is iden- mega). PCR primers were previously described (50, 51). tical with previously described p273 (13), except for the addition of a SacI site at the 3Ј end of the S␮ segment to increase the specificity of the assay. Mice pG3.1 also has SacI sites arranged as shown in p273S in B. B, DC-PCR AID-deficient mice (aidϪ/Ϫ) were obtained from T. Honjo (5). They were assay for plasmid S-S recombination. The 510-bp PCR product measures backcrossed for several generations with C57BL/6 mice. Wild-type (WT) the amount of circularized vector backbone. The 180-bp product measures mice were aidϩ/ϩ littermates of aidϪ/Ϫ mice. Suv39h1- and Suv39h2- the S-S recombination. deficient mice were previously described (32). Suv39h1-deficient mice were backcrossed to C57BL/6. Mice were used under protocols approved by the University of Massachusetts Medical School Institutional Animal cassette placed upstream of S␮, and the promoter for ␣ GLTs (I␣) Care and Use Committee. placed 5Ј to the acceptor S region (Fig. 1A), whereas the other plasmids do not contain these elements. Although endogenous Results CSR requires transcription across the S regions, SR in our tran- ␮ ␣ Regulation of S -S SR by Suv39h1 siently transfected S plasmids does not require the transcriptional To determine whether histone modification might regulate CSR, activator elements (13, 14). This is most likely due to the fact that we examined the effect of Suv39h1 on SR in plasmid substrates in both strands of supercoiled plasmids such as these have been dem- transient transfection assays. In this assay, plasmids containing S␮ onstrated to be AID targets in the absence of transcription (52, 53). and one of several different downstream acceptor S regions (Fig. Supercoiling apparently creates DNA of sufficient single-stranded 1A) are transiently transfected into lymphoid cells. SR between the character to serve as an AID substrate, whereas linearized plasmids two S regions in the plasmids is assayed after 48 h by DC-PCR are not attacked by AID. (Fig. 1B). The 180-bp PCR product indicates the amount of S␮-S␣ Because Suv39h1 trimethylates histone H3 at K9, a modification recombination, whereas the 510-bp product, due to circularization that recruits the repressive HP-1, we hypothesized that cotransfec- of the vector backbone, is an internal control for transfection ef- tion of an expression plasmid for Suv39h1 along with a S plasmid ficiency, plasmid recovery, restriction enzyme digestion, and liga- might inhibit plasmid recombination. Instead, we found that tion efficiency. The plasmids, p273S and pG3.1, contain transcrip- Suv39h1 stimulates S␮-S␣ plasmid switching (ϳ10-fold) in LPS- tional activator elements: the Ig H chain enhancer-VH promoter activated splenic B cells from WT mice, but not in AID-deficient 1182 Suv39h1 IS AN IgA-SPECIFIC SWITCH FACTOR

B cells (Fig. 2A). Shown above the graph are the lanes of the endogenous switching and have very low plasmid switch activity corresponding DC-PCR assay for plasmid SR, performed in du- (Fig. 2B). However, Suv39h1 was unable to stimulate plasmid plicate with 2-fold doses of input template. The results shown in switching in two cell lines (J558L plasmacytoma cells and EL-4 T Fig. 2A demonstrate that whereas the recombined vector band (510 cells) (Fig. 2B and data not shown) that lack AID. bp) was recovered and amplified from aidϪ/Ϫ cells as well as from ␣ WT B cells, the 180-bp band, which indicates S␮-S␣ recombina- S -specific effect of Suv39h1 tion, could not be amplified from aidϪ/Ϫ cells. This experiment To test whether Suv39h1 might be isotype specific, we tested the was performed with p218, a plasmid that lacks transcriptional pro- effect of Suv39h1 on SR in I.29␮ cells of four additional plasmids moters and enhancers upstream of the S␮ and S␣ segments (Fig. in which S␥3, S␥2a, S␥2b, or S␧ segments replace the S␣ segment 1A). Thus, the effect of Suv39h1 is not due to effects on transcrip- (Fig. 1A). Cotransfected Suv39h1 had no effect on SR of any of tional elements. Suv39h1 also stimulates S␮-S␣ recombination in these plasmids, except perhaps a small stimulatory effect on the S␧ p273S in a variety of B cell lines, even those that rarely undergo plasmid, pE.1 (Fig. 2C). However, because I.29␮ cells can only Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Suv39h1 stimulates plasmid S␮-S␣ recombination. A,SR(S␮-S␣) of transiently transfected S plasmid p218 in WT, but not aidϪ/Ϫ splenic B cells. The expression plasmid pSuv39h1 and the empty vector control plasmid (pcDEF3) were cotransfected along with p218 into WT or aidϪ/Ϫ splenic B cells. Duplicate transfections were performed 3 days after LPS activation, and cells were harvested 2 days after transfection. Nuclear DNA was isolated and assayed for recombination, as in Fig. 1B. Two-fold doses of each template were assayed, and the data were quantitated by phosphor imaging. The graph shows the average signal (ϩSEM) of the 180-bp product band normalized to the 510-bp band for each template dose (relative recombination activity) (14). In this experiment only, the relative recombination activity for WT cells transfected with the empty vector were set ϭ 1, and the other data were normalized to this value. Shown above the graph is an autoradiograph of a polyacrylamide gel containing the ␣-32P-labeled PCR products: 180 bp (S-S recombination) and 510 bp (vector control). B, Suv39h1 stimulates S␮-S␣ recombination in p273S in four different B cell lines, but not in J558L plasmacytoma cells. Relative recombination activity is shown for cells transfected with the control vector (pcDEF3) or the expression plasmid pSuv39h1. Cells were not stimulated. N, Indicates the number of independent transfections/cultures, each of which was analyzed by DC-PCR at least twice. C, Cotransfection of Suv39h1 into I.29␮ cells stimulates SR in S plasmids that contain S␣ sequences, but not in plasmids containing other S regions (except perhaps S␧ slightly). Fold stimulation by pSuv39h1 is shown relative to the recombination in cells transfected with pcDEF3 (vector control plasmid). D, Cotransfection of Suv39h1 does not stimulate S␮-S␥3 SR in pG3.1 in 1B4.B6, I.29␮, or BFO.3 cells. The Journal of Immunology 1183 switch to IgA and IgE (54), and do not support plasmid SR to IgG3 rather than heterochromatin (36, 56). Overexpression of G9a had or IgG1 (14, 16), it is possible that the inability of Suv39h1 to no effect on plasmid SR (our unpublished data). stimulate plasmid SR to other isotypes is due to the lack of a required isotype-specific activity in I.29␮. Therefore, we tested the Suv39h1 regulates endogenous IgA CSR ability of Suv39h1 to increase plasmid S␮-S␥3 SR (pG3.1) in To determine whether Suv39h1 regulates SR of endogenous IgH 1B4.B6, a B cell line with IgG3 switching activity (14). As shown genes, B cells from Suv39h1-deficient mice (32) were examined in Fig. 2D, Suv39h1 does not stimulate SR in pG3.1 in 1B4.B6 for their ability to undergo CSR. We used flow cytometry to con- cells. These data indicate that Suv39h1 specifically stimulates firm that the spleen cell populations from Suv39h1-deficient mice S␮-S␣ recombination. did not differ from those of WT mice. In addition to equivalent numbers of CD3ϩ T cells and B220ϩ B cells, there was no dif- ␣ HMT activity of Suv39h1 is required to stimulate S SR ference in the marginal zone (CD23low, CD21high) or follicular To determine whether Suv39h1 stimulation of S␮-S␣ recombina- (CD23high, CD21low) B cell numbers (our unpublished data). To tion requires the HMT activity of this enzyme, we created a single examine CSR, splenic B cells were isolated and cultured for 4 days amino acid mutation in the SET domain (H324L) of the Suv39h1 under conditions that induce switching to the isotypes shown in gene, which should completely abolish the HMT activity of Fig. 4A, then stained with Abs to various IgH chain isotypes (48). Suv39h1, according to studies of the human protein (29). This B cells from Suv39h1-deficient mice showed a 2-fold reduction in mutation eliminated the stimulatory effect of Suv39h1 on plasmid switching to IgA compared with the control mice, but unchanged S␮-S␣ recombination, indicating that the HMT activity is essential levels of switching to all other isotypes (Fig. 4A). The results from for enhancing switching (Fig. 3A). Additionally, a naturally occur- the individual cultures for IgA CSR are shown in Fig. 4B. The Downloaded from ring Suv39h1 splice variant in I.29␮ cells (⌬42–229) (data not difference in percentage of IgAϩ cells between WT and Suv39h1- shown), which lacks the nuclear localization domain, chromodo- deficient cultures is highly significant ( p ϭ 0.003), as determined main, and cys-rich domain, had almost no stimulatory activity (Fig. by a paired t test. We also examined whether mice doubly deficient 3B). Smaller internal deletions were created and resulted in inter- in Suv39h1 and Suv39h2 (32) would have a further reduction in mediate activity, consistent with the involvement of these domains IgA switching and found they did not. Splenic B cells from two in nuclear localization, interaction with HP-1 and H3 (46, 55). mice deficient in both Suv39h1 and Suv39h2 switched 60% as well http://www.jimmunol.org/ To determine whether the effect on plasmid S␮-S␣ recombina- to IgA as WT B cells (Fig. 4C and data not shown). These data tion is specific to Suv39h1, in similar experiments we overex- show that Suv39h1, but probably not Suv39h2, enhances IgA CSR. pressed G9a, another repressive HMT that performs mono- and CSR is strongly correlated with cell division, and is ongoing as dimethylation at H3-K9, but which is associated with euchromatin B cells proliferate in LPS cultures (57). Although no differences in by guest on September 27, 2021

FIGURE 3. Mutations within Suv39h1 reduce its ability to stimulate plasmid SR. A, Mutation of the catalytic domain abolishes Suv39h1 stimulation of plasmid S␮-S␣ recombination. Results of DC-PCR assays of S␮-S␣ recombination in p273S cotransfected into I.29␮ (22D) cells with either WT or mutant Suv39h1 (H324L) or with empty expression plasmid pcDEF3, as indicated. B, Deletions of the indicated segments of Suv39h1 reduce p273S SR stimulatory activity. 1184 Suv39h1 IS AN IgA-SPECIFIC SWITCH FACTOR

FIGURE 4. Suv39h1-deficient B cells show reduced switching to IgA. A, Splenic B cells from Suv39h1-deficient mice show reduced switching to IgA in culture. T-depleted B cells were induced to switch to each of the indicated isotypes for 4 days. Switching was assayed by flow cytometric analysis of surface-expressed isotypes (48). Data are re- ported as the percentage of switching of Suv39h1- deficient cells relative to WT cells. Shown are the mean and SEM of FACS results from three to four mice. Switching to IgE was not reliably detected and is not shown. Three of the Suv39h1-deficient mice assayed for IgA were on a mixed C57BL/6 ϫ 129/Sc background, and the fourth was backcrossed to C57BL/6 for five generations. The results did not differ between strains. B, The percentage of IgAϩ cells in the individual cultures and Downloaded from their means (shown by the bars) are given for the results presented in A. The significance of the difference between the cultures was calculated by a paired t test. C, FACS analysis of surface IgA and IgM expression in a WT littermate and Suv39h1/ Suv39h2-deficient splenic B cells induced to

switch to IgA. D, Expression of surface IgA on http://www.jimmunol.org/ freshly isolated Peyer’s patch cells from WT and Suv39h1-deﬁcient mice. Data from three 4- to 9-mo-old mice are presented as the percentage of B220ϩ cells expressing surface IgA (ϩSEM).

proliferation of cells from Suv39h1-deficient mice have been re- Suv39h1 does not regulate GL␣ transcripts by guest on September 27, 2021 ported, and the body weights are normal (32), it was important to The S plasmids p273 and pG3.1 both have a GL␣ promoter that determine whether Suv39h1-deficient B cells had normal levels of drives transcription across the S␣ segment (13) (Fig. 1A). Thus, the proliferation during induction of CSR in culture. We found that ability of Suv39h1 to stimulate S␮-S␣ recombination cannot be B cell proliferation was similar in mutant and WT cells, as due to stimulation of transcription from this promoter, as both assayed by DNA content analysis and [3H]thymidine incorpo- p273 and pG3.1 have the identical GL␣ promoter, but only p273 ration (Fig. 5). switching is stimulated by Suv39h1. Additionally, cotransfection Lymphoid cell clusters along the small intestine, called Peyer’s of Suv39h1 increases SR in plasmid p218 as well as it does in patches, are a major site for CSR to IgA in vivo (58). To determine p273, although p218 has no transcriptional elements upstream of whether Suv39h1 regulates expression of IgA in vivo, we exam- either the S␮ or S␣ segments (Figs. 1A and 2C). In agreement with ined freshly isolated Peyer’s patch cells from Suv39h1-deficient these results, we found no difference in ␣ GLT levels in Suv39h1- mice, and found that the frequency of IgA-expressing B cells in the Peyer’s patches was ϳ50% of their frequency in WT mice (Fig. deficient and WT splenic B cells stimulated to switch to IgA (Fig. 4D). Suv39h1 deficiency did not affect the recovery of total lym- 6). Therefore, Suv39h1 does not increase CSR to IgA by regulat- ␣ phocytes nor B cells from Peyer’s patches (data not shown). ing GLTs.

The effects of Suv39h1 on endogenous vs plasmid SR The effect of Suv39h1 on plasmid CSR does not require the S␣ The above results suggest that either overexpression of Suv39h1 Pax5 binding site has a more marked effect on IgA CSR than loss of Suv39h1, or alternatively, that endogenous CSR is less dependent upon To reconcile the finding that Suv39h1 specifically increases CSR Suv39h1 than plasmid SR. We examined this question by trans- involving the S␣ segment, but not with other acceptor S regions, fecting p218 into LPS-activated splenic B cells from WT and and the fact that Suv39h1 is a repressor of gene activity, we hy- Suv39h1-deficient B cells. p218 recombination was reduced by pothesize that Suv39h1 activity inhibits a repressor that has spec- 40% in the mutant B cells relative to WT cells (our unpublished ificity for S␣ sequences. One known repressor of IgA CSR that data). Thus, both plasmid and endogenous CSR are similarly re- binds to S␣ is Pax5 (59). In the p273 and p218 plasmids, there are duced in the absence of Suv39h1. It is possible that the normal two binding sites for Pax5, one at the 5Ј end of S␮ and another at levels of Suv39h1 are not sufficient for maximal IgA CSR, and the 5Ј end of S␣ (13). We have tested the effect of deleting the thus, loss of Suv39h1 has a smaller effect than does increased Pax5 binding site from S␣ in p218, the S␮-S␣ plasmid lacking levels of Suv39h1. It is also possible that another HMT, e.g., Eset, transcriptional regulatory elements (Fig. 1A). Deletion of the which also performs trimethylation of K9-H3, substitutes for known Pax5 binding site from the S␣ segment did not alter plas- Suv39h1 (19). mid recombination or its ability to respond to Suv39h1 (Fig. 7). The Journal of Immunology 1185 Downloaded from

FIGURE 6. ␣ GLTs are not regulated by Suv39h1. ␣ GLTs were as-

sayed by semiquantitative RT-PCR in splenic B cells from WT and http://www.jimmunol.org/ Suv39h1-deficient mice after 4 days of culture with the indicated inducers of CSR. ␣ GLTs are induced in cells during induction of switching to IgA, but not in cells induced to switch with LPS ϩ IL-4. Two-fold doses of input template were amplified. Amplification of GAPDH cDNA served as an internal control for template dose. Similar results were obtained in two experiments.

FIGURE 5. Suv39h1 does not affect B cell cycle or proliferation during CSR. A, DNA content analysis of splenic B cells 48 h after induction to gions, it is possible that Suv39h1 HMT activity inhibits binding of by guest on September 27, 2021 switch to each of the indicated isotypes. The percentage of cells in each a S repressor protein to S␣. ϭ ϭ phase of the cell cycle is indicated. G1 G0/G1 and G2 G2/M. Similar A candidate for a repressor that might be inhibited by Suv39h1 results were obtained in two experiments. B, Proliferation ([3H]TdR in- activity is LSF (late SV40 factor/CP2), a protein that binds S␣ and corp) of splenic B cells on day 3 after addition of inducers of switching of S␮ segments in EMSA and has been shown to inhibit IgA CSR ϩ the indicated isotypes. Shown are means of two experiments ( SEM), (45). There are numerous binding sites in each of the S␣ tandem each performed in triplicate.

Thus, Suv39h1 does not stimulate CSR to IgA by inhibiting the binding of Pax5 to S␣ segments.

Discussion In this study, we show that the HMT Suv39h1 specifically increases S␮-S␣ SR in plasmid S substrates by ϳ10-fold, and that Suv39h1-deficient B cells have reduced abilities (ϳ2-fold) to undergo CSR to IgA in culture, but switch at normal levels to all the IgG subclasses. Furthermore, we found a 50% reduction in the proportion of freshly isolated Peyer’s patch B cells that express IgA in Suv39h1-deficient mice. The finding that Suv39h1 increases IgA CSR is surprising because Suv39h1 trimethylates the K9 residue of H3, a repressive modification associated with pericentromeric and centromeric heterochromatin (35, 60). Addition- ally, Suv39h1 binds Rb and is involved in repression of euchro- matic genes that are dependent upon E2F transcription factors (39, 40, 42). Because Suv39h1 is known to be a transcriptional repressor, these results suggest that Suv39h1 stimulates CSR specifically FIGURE 7. Deletion of the Pax5 binding site from the S␣ segment in to IgA by inhibiting a repressor specific for IgA switching. Inter- p218 does not affect the ability of Suv39h1 to stimulate S␮-S␣ recombi- estingly, the regulation of IgA CSR by Suv39h1 does not occur by nation. Either p218 or p218-Pax5⌬ was cotransfected into I.29␮ cells with regulating ␣ GLTs, as their levels are unaffected by Suv39h1 de- the Suv39h1 expression plasmid or the empty vector, and plasmid SR was ficiency. As we have found that Suv39h1 stimulates plasmid assayed 2 days later, as described in Fig. 2 legend and Materials and S␮-S␣ recombination activity, but not SR to other acceptor S re- Methods. 1186 Suv39h1 IS AN IgA-SPECIFIC SWITCH FACTOR

repeats and also in the S␮ tandem repeats, but very few LSF bind- the nucleus during the remainder of the cell cycle (34). Several ing sites in the other mouse S regions. LSF binds to the SV40 studies suggest that the activity of Suv39h1 might be regulated promoter, HIV long-terminal repeat, and ␣-globin promoter, where during the cell cycle by phosphorylation, rather than at the protein it stimulates or inhibits transcription, depending on its binding site level (34, 40, 65, 66). Suv39h1 undergoes phosphorylation in ␮ and interacting proteins (61). In the I.29 B cell line, which un- HeLa cells at the G1/S phase transition, and this phosphorylation dergoes CSR from IgM to IgA, stable expression of two different appears to be regulated by the anti-phosphatase SET-binding fac- dominant-negative forms of LSF increased IgA CSR ϳ2-fold (45). tor 1 (Sbf1) (65), although the kinase(s) involved is unknown. In Therefore, an attractive hypothesis is that by an unknown mech- cotransfection experiments, Sbf1 inhibited the transcriptional re- anism, Suv39h1 activity inhibits the binding of LSF to S␣ regions pression activity of Suv39h1 (65). The activity of Suv39h1 might or inhibits its activity when bound to S␣. be stimulated upon activation of B cells to undergo proliferation One possible mechanism might be that Suv39h1 inhibits the and isotype switching, thereby repressing an inhibitor of IgA CSR. putative IgA CSR repressor protein by inhibiting its transcription One possibility is that Sbf1 is degraded or inhibited in activated B by methylating H3 on a nucleosome(s) at its promoter. Alterna- cells, thereby activating Suv39h1. tively, Suv39h1 might interact with the putative repressor protein, perhaps methylating it and thereby sequestering it from S␣ re- Regulation of IgA CSR by S region-specific factors gions. Although the only substrate identified to date for Suv39h1 Isotype specificity of switching is regulated by GL transcription, is histone H3, it was shown recently that another HMT, SET9, which is necessary for CSR. However, the sequences of the ac- methylates histone H3 and also TAF10, a component of the gen- ceptor S regions themselves can contribute to regulation of isotype eral transcription machinery, and that this methylation stimulates specificity (this study) (14–16). Each S region consists of tandem Downloaded from transcription in a promoter-specific manner (62). We considered repeats of sequence elements ranging from 20 to 80 bp in length, the possibility that Suv39h1 might affect the DNA-binding activity up to a total length of 1–10 kb. As each S region is unique, se- of LSF, but found no difference between LSF-binding activity in quence-specific DNA-binding proteins might regulate SR to each WT and Suv39h1-deficient B cells by EMSA (data not shown). isotype, possibly by regulating accessibility and/or AID recruit- Experiments performed by Drouin et al. (45) suggested that LSF is ment (14–16). Kenter et al. (15) have examined SR in plasmids

modified in an unknown way upon induction of CSR, and this containing minimal-length S regions to study the importance of http://www.jimmunol.org/ reduces its ability to bind S regions. It is possible that methylation specific S␥3 and S␥1 sequences. They analyzed a specific se- of the S␣ region by Suv39h1 might inhibit the binding of LSF, but quence motif within the S␥3 consensus repeat that differs between this model would require that transiently transfected nonreplicat- the S␥3 and S␥1 consensus repeats to ask why plasmids with the ing plasmids could associate with nucleosomes. S␥3 region can switch in LPS-activated cells, whereas plasmids Another possibility is suggested by previous findings that over- with the S␥1 region switch only if IL-4 is also added. Mutation of expression of Suv39h1 in a variety of cell types alters the intranu- the element showed that the S␥1 sequence element prevents plas- clear localization of proteins associated with heterochromatin (the mid S␮-S␥1 recombination in splenic B cells treated with LPS polychrome group protein HPC2 and HP-1␤), resulting in concen- alone, but if this motif is replaced by the S␥3 sequence element, by guest on September 27, 2021 tration of these proteins into large heterochromatin domains away plasmid SR occurs in cells treated with LPS alone. This result from euchromatin (46, 63). This relocalization depends on the suggests that a specific factor required for SR binds this Sg3 ele- Suv39h1 SET domain, which contains the HMT catalytic site, and ment. The Kenter studies (16) also suggest that there are S␣- and also on its chromodomain, and is associated with increased H3-K9 S␧-specific factors, but none of the factors have been identified. In methylation in these heterochromatin domains (63). Both HMT the current study, we identify Suv39h1 as an S␣-specific factor. activity and the chromodomain are required for Suv39h1 to stim- In conclusion, Suv39h1 specifically stimulates IgA CSR, most ulate plasmid switching. Perhaps the relocalization of HPC2 and likely by inhibiting an inhibitor that binds S␣, as the effect was HP1␤ to pericentromeric heterochromatin domains results in the specific to S plasmid constructs containing the S␣ segment. Fur- sequestration of an isotype-specific inhibitory protein away from thermore, mice deficient in Suv39h1 show reduced frequency of the transiently transfected plasmids. In Suv39h1-deficient cells, IgAϩ B cells in Peyer’s patches, and splenic B cells from these one could speculate that these same HPs are more widely distrib- mice show specific reductions in IgA class switching. Although uted in euchromatin regions than in WT cells, and thus associate how Suv39h1 inhibits such an inhibitor is unclear, there are several with the S␣ segment. possibilities that can be investigated. It is unclear why the effect of deleting the suv39h1 gene is so small relative to the large stimulation of plasmid SR by overex- Acknowledgments pression of Suv39h1. Although it could be due to an artifact of We thank Dr. Makoto Tachibana for the mouse cDNA clone for G9a; Dr. overexpression, it is also possible that the endogenous level of Wenyue Bai for assaying whether G9a stimulates plasmid SR; and Dr. Suv39h1 is low and limiting in WT B cells, and thus its loss has Amy Kenter for plasmids pE.1 and pG3.1 and for helpful discussions. a smaller effect than its overexpression. Another possibility is that Eset, another HMT that trimethylates K9-H3 (19) and is inducibly Disclosures expressed in splenic B cells (64), is partially redundant with The authors have no financial conflict of interest. Suv39h1. Is Suv39h1 regulated during B cell activation? References 1. Stavnezer, J. 2000. Molecular processes that regulate class switching. Curr. Top. Suv39h1 is ubiquitously expressed (55). We have examined Microbiol. Immunol. 245: 127–168. whether expression of Suv39h1 mRNA differs among cell lines 2. Bottaro, A., R. Lansford, L. Xu, J. Zhang, P. Rothman, and F. Alt. 1994. I region transcription (per se) promotes basal IgE class switch recombination but addi- that can or cannot switch to IgA and found it does not (data not tional factors regulate the efficiency of the process. EMBO J. 13: 665–674. shown). Furthermore, we observed no consistent change in 3. Lorenz, M., S. Jung, and A. Radbruch. 1995. Switch transcripts in immunoglob- Suv39h1 mRNA levels when splenic B cells are induced to un- ulin class switching. Science 267: 1825–1828. 4. Yu, K., F. Chedin, C. L. Hsieh, T. E. Wilson, and M. R. Lieber. 2003. R-loops dergo CSR. Suv39h1 associates with centromeric regions during at immunoglobulin class switch regions in the chromosomes of stimulated B prometaphase and metaphase, but is more broadly distributed over cells. Nat. Immunol. 4: 442–451. The Journal of Immunology 1187

5. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, and 31. Peters, A. H., J. E. Mermoud, D. O’Carroll, M. Pagani, D. Schweizer, T. Honjo. 2000. Class switch recombination and hypermutation require activa- N. Brockdorff, and T. Jenuwein. 2001. Histone H3 lysine 9 methylation is an tion-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell epigenetic imprint of facultative heterochromatin. Nat. Genet. 30: 77–80. 102: 553–563. 32. Peters, A. H., D. O’Carroll, H. Scherthan, K. Mechtler, S. Sauer, C. Schofer, 6. Petersen-Mahrt, S. K., R. S. Harris, and M. S. Neuberger. 2002. AID mutates E. K. Weipoltshammer, M. Pagani, M. Lachner, A. Kohlmaier, et al. 2001. Loss of coli suggesting a DNA deamination mechanism for antibody diversification. Na- the Suv39h histone methyltransferases impairs mammalian heterochromatin and ture 418: 99–104. genome stability. Cell 107: 323–337. 7. Chaudhuri, J., M. Tian, C. Khuong, K. Chua, E. Pinaud, and F. W. Alt. 2003. 33. Lachner, M., D. O’Carroll, S. Rea, K. Mechtler, and T. Jenuwein. 2001. Meth- Transcription-targeted DNA deamination by the AID antibody diversification en- ylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410: zyme. Nature 422: 726–730. 116–120. 8. Ramiro, A. R., P. Stavropoulos, M. Jankovic, and M. C. Nussenzweig. 2003. 34. Aagaard, L., M. Schmid, P. Warburton, and T. Jenuwein. 2000. Mitotic phos- Transcription enhances AID-mediated cytidine deamination by exposing single- phorylation of SUV39H1, a novel component of active centromeres, coincides stranded DNA on the nontemplate strand. Nat. Immunol. 4: 452–456. with transient accumulation at mammalian centromeres. J Cell Sci. 113: 9. Dickerson, S. K., E. Market, E. Besmer, and F. N. Papavasiliou. 2003. AID 817–829. mediates hypermutation by deaminating single stranded DNA. J. Exp. Med. 197: 35. Schotta, G., M. Lachner, K. Sarma, A. Ebert, R. Sengupta, G. Reuter, 1291–1296. D. Reinberg, and T. Jenuwein. 2004. A silencing pathway to induce H3–K9 and 10. Bransteitter, R., P. Pham, M. D. Scharff, and M. F. Goodman. 2003. Activation- H4–K20 trimethylation at constitutive heterochromatin. Genes Dev. 18: induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA 1251–1262. Proc. Natl. Acad. Sci. USA but requires the action of RNase. 100: 4102–4107. 36. Rice, J. C., S. D. Briggs, B. Ueberheide, C. M. Barber, J. Shabanowitz, 11. McIntyre, T. M., M. R. Kehry, and C. M. Snapper. 1995. Novel in vitro model D. F. Hunt, Y. Shinkai, and C. D. Allis. 2003. Histone methyltransferases direct for high-rate IgA class switching. J. Immunol. 154: 3156–3161. different degrees of methylation to define distinct chromatin domains. Mol. Cell 12. Brandtzaeg, P., E. S. Baekkevold, I. N. Farstad, F. L. Jahnsen, F. E. Johansen, 12: 1591–1598. E. M. Nilsen, and T. Yamanaka. 1999. Regional specialization in the mucosal 37. Vaute, O., E. Nicolas, L. Vandel, and D. Trouche. 2002. Functional and physical immune system: what happens in the microcompartments? Immunol. Today 20: interaction between the histone methyl transferase Suv39H1 and histone deacety-

141–151. Downloaded from lases. Nucleic Acids Res. 30: 475–481. 13. Stavnezer, J., S. P. Bradley, N. Rousseau, T. Pearson, A. Shanmugam, 38. Nielsen, A. L., M. Oulad-Abdelghani, J. A. Ortiz, E. Remboutsika, P. Chambon, D. J. Waite, P. R. Rogers, and A. L. Kenter. 1999. Switch recombination in a and R. Losson. 2001. Heterochromatin formation in mammalian cells: interaction transfected plasmid occurs preferentially in a B cell line that undergoes switch between histones and HP1 proteins. Mol. Cell 7: 729–739. recombination of its chromosomal Ig heavy chain genes. J. Immunol. 163: 2028–2040. 39. Vandel, L., E. Nicolas, O. Vaute, R. Ferreira, S. Ait-Si-Ali, and D. Trouche. 14. Shanmugam, A., M.-J. Shi, L. Yauch, J. Stavnezer, and A. L. Kenter. 2000. 2001. Transcriptional repression by the retinoblastoma protein through the re- Evidence for class specific factors in immunoglobulin isotype switching. J. Exp. cruitment of a histone methyltransferase. Mol. Cell. Biol. 21: 6484–6494. Med. 191: 1365–1380. 40. Nicolas, E., C. Roumillac, and D. Trouche. 2003. Balance between acetylation 15. Kenter, A. L., R. Wuerffel, C. Dominguez, A. Shanmugam, and H. Zhang. 2004. and methylation of histone H3 lysine 9 on the E2F-responsive dihydrofolate http://www.jimmunol.org/ Mapping of a functional recombination motif that defines isotype specificity for reductase promoter. Mol. Cell. Biol. 23: 1614–1622. ␮3␥3 switch recombination implicates NF-␬B p50 as the isotype-specific 41. Kamoi, K., K. Yamamoto, A. Misawa, A. Miyake, T. Ishida, Y. Tanaka, switching factor. J. Exp. Med. 199: 617–627. M. Mochizuki, and T. Watanabe. 2006. SUV39H1 interacts with HTLV-1 Tax 16. Ma, L., H. H. Wortis, and A. L. Kenter. 2002. Two new isotype-specific switch- and abrogates Tax transactivation of HTLV-1 LTR. Retrovirology 3: 5. ing activities detected for Ig class switching. J. Immunol. 168: 2835–2846. 42. Nielsen, S. J., R. Schneider, U. M. Bauer, A. J. Bannister, A. Morrison, D. 17. Zhang, Y., and D. Reinberg. 2001. Transcription regulation by histone methyl- O’Carroll, R. Firestein, M. Cleary, T. Jenuwein, R. E. Herrera, and ation: interplay between different covalent modifications of the core histone tails. T. Kouzarides. 2001. Rb targets histone H3 methylation and HP1 to promoters. Genes Dev. 15: 2343–2360. Nature 412: 561–565. 18. Lachner, M., and T. Jenuwein. 2002. The many faces of histone lysine methyl- 43. Narita, M., S. Nunez, E. Heard, A. W. Lin, S. A. Hearn, D. L. Spector, ation. Curr. Opin. Cell Biol. 14: 286–298. G. J. Hannon, and S. W. Lowe. 2003. Rb-mediated heterochromatin formation

19. Wang, H., W. An, R. Cao, L. Xia, H. Erdjument-Bromage, B. Chatton, and silencing of E2F target genes during cellular senescence. Cell 113: 703–716. by guest on September 27, 2021 P. Tempst, R. G. Roeder, and Y. Zhang. 2003. mAM facilitates conversion by 44. Vakoc, C. R., S. A. Mandat, B. A. Olenchock, and G. A. Blobel. 2005. Histone ESET of dimethyl to trimethyl lysine 9 of histone H3 to cause transcriptional H3 lysine 9 methylation and HP1␥ are associated with transcription elongation repression. Mol. Cell 12: 475–487. through mammalian chromatin. Mol. Cell 19: 381–391. 20. Chowdhury, D., and R. Sen. 2001. Stepwise activation of the immunoglobulin ␮ 45. Drouin, E. E., C. E. Schrader, J. Stavnezer, and U. Hansen. 2002. The ubiqui- heavy chain gene locus. EMBO J. 20: 6394–6403. tously expressed mammalian transcription factor LSF binds Ig switch regions and 21. Johnson, K., C. Angelin-Duclos, S. Park, and K. L. Calame. 2003. Changes in represses class switching to IgA. J. Immunol. 168: 2847–2856. histone acetylation are associated with differences in accessibility of VH gene 46. Melcher, M., M. Schmid, L. Aagaard, P. Selenko, G. Laible, and T. Jenuwein. segments to V-DJ recombination during B-cell ontogeny and development. Mol. 2000. Structure-function analysis of SUV39H1 reveals a dominant role in het- Cell. Biol. 23: 2438–2450. erochromatin organization, chromosome segregation, and mitotic progression. 22. Morshead, K. B., D. N. Ciccone, S. D. Taverna, C. D. Allis, and M. A. Oettinger. Mol. Cell. Biol. 20: 3728–3741. 2003. Antigen receptor loci poised for V(D)J rearrangement are broadly associ- 47. Goldman, L. A., E. C. Cutrone, S. V. Kotenko, C. D. Krause, and J. A. Langer. ated with BRG1 and flanked by peaks of histone H3 dimethylated at lysine 4. 1996. Modifications of vectors pEF-BOS, pcDNA1 and pcDNA3 result in im- Proc. Natl. Acad. Sci. USA 100: 11577–11582. proved convenience and expression. BioTechniques 21: 1013–1015. 23. Johnson, K., D. L. Pflugh, D. Yu, D. G. Hesslein, K. I. Lin, A. L. Bothwell, 48. Schrader, C. E., W. Edelmann, R. Kucherlapati, and J. Stavnezer. 1999. Reduced A. Thomas-Tikhonenko, D. G. Schatz, and K. Calame. 2004. B cell-specific loss isotype switching in splenic B cells from mice deficient in mismatch repair en- of histone 3 lysine 9 methylation in the VH locus depends on Pax5. Nat. Immunol. zymes. J. Exp. Med. 190: 323–330. 5: 853–861. 49. Schrader, C. E., J. Vardo, and J. Stavnezer. 2002. Role for mismatch repair 24. Espinoza, C. R., and A. J. Feeney. 2005. The extent of histone acetylation cor- proteins Msh2, Mlh1, and Pms2 in immunoglobulin class switching shown by relates with the differential rearrangement frequency of individual V genes in H sequence analysis of recombination junctions. J. Exp. Med. 195: 367–373. pro-B cells. J. Immunol. 175: 6668–6675. ␣ 25. Nambu, Y., M. Sugai, H. Gonda, C. G. Lee, T. Katakai, Y. Agata, Y. Yokota, and 50. Qiu, G., G. R. Harriman, and J. Stavnezer. 1999. I exon-replacement mice A. Shimizu. 2003. Transcription-coupled events associating with immunoglobu- synthesize a spliced HPRT-Ca transcript which may explain their ability to switch lin switch region chromatin. Science 302: 2137–2140. to IgA: inhibition of switching to IgG in these mice. Int. Immunol. 11: 37–46. 26. Li, Z., Z. Luo, and M. D. Scharff. 2004. Differential regulation of histone acet- 51. Boekhoudt, G. H., Z. Guo, G. W. Beresford, and J. M. Boss. 2003. Communi- ␬ ylation and generation of mutations in switch regions is associated with Ig class cation between NF- B and Sp1 controls histone acetylation within the proximal switching. Proc. Natl. Acad. Sci. USA 101: 15428–15433. promoter of the monocyte chemoattractant protein 1 gene. J. Immunol. 170: 27. Wang, L., N. Whang, R. Wuerffel, and A. L. Kenter. 2006. AID-dependent hi- 4139–4147. stone acetylation is detected in immunoglobulin S regions. J. Exp. Med. 203: 52. Shen, H. M., and U. Storb. 2004. Activation-induced cytidine deaminase (AID) 215–226. can target both DNA strands when the DNA is supercoiled. Proc. Natl. Acad. Sci. 28. Tschiersch, B., A. Hofmann, V. Krauss, R. Dorn, G. Korge, and G. Reuter. 1994. USA 101: 12997–13002. The protein encoded by the Drosophila position-effect variegation suppressor 53. Shen, H. M., S. Ratnam, and U. Storb. 2005. Targeting of the activation-induced gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene cytosine deaminase is strongly influenced by the sequence and structure of the complexes. EMBO J. 13: 3822–3831. targeted DNA. Mol. Cell. Biol. 25: 10815–10821. 29. Rea, S., F. Eisenhaber, D. O’Carroll, B. D. Strahl, Z. W. Sun, M. Schmid, 54. Stavnezer, J., S. Sirlin, and J. Abbott. 1985. Induction of immunoglobulin isotype S. Opravil, K. Mechtler, C. P. Ponting, C. D. Allis, and T. Jenuwein. 2000. switching in cultured I.29 B lymphoma cells: characterization of the accompa- Regulation of chromatin structure by site-specific histone H3 methyltransferases. nying rearrangements of heavy chain genes. J. Exp. Med. 161: 577–601. Nature 406: 593–599. 55. Aagaard, L., G. Laible, P. Selenko, M. Schmid, R. Dorn, G. Schotta, S. Kuhfittig, 30. O’Carroll, D., H. Scherthan, A. H. Peters, S. Opravil, A. R. Haynes, G. Laible, A. Wolf, A. Lebersorger, P. B. Singh, et al. 1999. Functional mammalian ho- S. Rea, M. Schmid, A. Lebersorger, M. Jerratsch, et al. 2000. Isolation and char- mologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-asso- acterization of Suv39h2, a second histone H3 methyltransferase gene that dis- ciated proteins which complex with the heterochromatin component M31. EMBO plays testis-specific expression. Mol. Cell. Biol. 20: 9423–9433. J. 18: 1923–1938. 1188 Suv39h1 IS AN IgA-SPECIFIC SWITCH FACTOR

56. Tachibana, M., K. Sugimoto, T. Fukushima, and Y. Shinkai. 2001. Set domain- 62. Kouskouti, A., E. Scheer, A. Staub, L. Tora, and I. Talianidis. 2004. Gene-spe- containing protein, G9a, is a novel lysine-preferring mammalian histone meth- cific modulation of TAF10 function by SET9-mediated methylation. Mol. Cell yltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of 14: 175–182. histone H3. J. Biol. Chem. 276: 25309–25317. 63. Sewalt, R. G., M. Lachner, M. Vargas, K. M. Hamer, J. L. den Blaauwen, 57. Hodgkin, P. D., J.-H. Lee, and A. B. Lyons. 1996. B cell differentiation and T. Hendrix, M. Melcher, D. Schweizer, T. Jenuwein, and A. P. Otte. 2002. Se- lective interactions between vertebrate polycomb homologs and the SUV39H1 isotype switching is related to division cycle number. J. Exp. Med. 184: 277–281. histone lysine methyltransferase suggest that histone H3–K9 methylation con- 58. Craig, S. W., and J. J. Cebra. 1971. Peyer’s patches: an enriched source of pre- tributes to chromosomal targeting of polycomb group proteins. Mol. Cell. Biol. cursors for IgA-producing immunocytes in the rabbit. J. Exp. Med. 134: 22: 5539–5553. 188–200. 64. Baxter, J., S. Sauer, A. Peters, R. John, R. Williams, M. L. Caparros, K. Arney, 59. Qiu, G., and J. Stavnezer. 1998. Overexpression of BSAP/Pax-5 inhibits switch- A. Otte, T. Jenuwein, M. Merkenschlager, and A. G. Fisher. 2004. Histone hy- ing to IgA and enhances switching to IgE in the I.29␮ B cell line. J. Immunol. pomethylation is an indicator of epigenetic plasticity in quiescent lymphocytes. 161: 2906–2918. EMBO J. 23: 4462–4472. 65. Firestein, R., X. Cui, P. Huie, M. L. Cleary, and S. Fagarasan. 2000. Set domain- 60. Guenatri, M., D. Bailly, C. Maison, and G. Almouzni. 2004. Mouse centric and dependent regulation of transcriptional silencing and growth control by pericentric satellite repeats form distinct functional heterochromatin. J. Cell Biol. SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9. Mol. Cell. Biol. 20: 166: 493–505. 4900–4909. 61. Veljkovic, J., and U. Hansen. 2004. Lineage-specific and ubiquitous biological 66. Jenuwein, T. 2001. Re-SET-ting heterochromatin by histone methyltransferases. roles of the mammalian transcription factor LSF. Gene 343: 23–40. Trends Cell Biol. 11: 266–273. Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021